Hot off the presses! Sep 01 Nucleic Acids Symp Ser (Oxf)

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Latest Articles Include:

• Characterization and Use of Tricyclic Fluorescent Nucleic Acid Base Analogues
  - Nucleic Acids Symp Ser (Oxf) 52(1):3-4 (2008)
  The two recently developed nucleic acid probe molecules tC and tCO both have unique properties compared to other molecules in the family of fluorescent base analogues.1-5 These tricyclic base analogues both form very stable base pairs with guanine and give minimal perturbations to the native structure of DNA.2 We have found that tCO is the brightest fluorescent base analogue reported4 and that tC also is very bright and has a fluorescence quantum yield that is virtually insensitive to its surrounding microenvironment within the nucleic acid3. These base analogues have so far been used in FRET-studies of a DNA-polymerase system6 and in initial anisotropy-studies of DNA-containing systems4.
• Nucleic acid duplexes with zippers of additional nucleobases and aromatics in minor or major groove
  - Nucleic Acids Symp Ser (Oxf) 52(1):5-6 (2008)
  Two series of thymidine derivatives with additional nucleobases/aromatics attached to either the 5'(S)-C- or the 5-position were prepared by epoxide opening and/or "click chemistry" cycloaddition protocols and introduced into DNA duplexes. Interstrand base-base communication in the minor groove and intrastrand stacking interactions in the major groove were detected.
• DNA and RNA Quadruplex ligands
  - Nucleic Acids Symp Ser (Oxf) 52(1):7-8 (2008)
  Guanine-rich nucleic acids can adopt unusual structures called guanine quadruplexes (G4) based on stacked guanine quartets. Both RNA and DNA backbones are compatible with G4 formation. As RNA and DNA quadruplexes may be recognized by ligands, it is important to understand the rules that govern the stability and specificity of these complexes. We explore the binding of a pyridine dicarboxamide derivative to various oligoribo- and oligodeoxyribo-nucleotides.
• G-quadruplex forming oligonucleotides as finely tunable aptamers: towards better DNA mimics
  - Nucleic Acids Symp Ser (Oxf) 52(1):9-10 (2008)
  The intense search for oligonucleotides (ODNs) endowed with pharmacological activities has led, in the past decade, to the identification of tens of candidate drugs, now being evaluated in preclinical or clinical trials. Based on G-rich DNA sequences, several aptamers, adopting G-quadruplex structures with different topologies, have been selected as potent in vitro antiviral and/or antitumoral agents. In order to develop novel therapeutically relevant Gquadruplex-based aptamers, we have investigated - as a model compound - the 5'd(TGGGAG)3' sequence, known to be anti-HIV-1 active if 5'-modified with bulky aromatic residues. A set of 5'-conjugated analogues has been analyzed by integrated CD, DSC and molecular modelling studies, allowing a detailed biophysical characterization of the resulting G-quadruplexes. Following the assumption that the kinetically and thermodynamically favoured formation of the quadruplex complexes is a pre-requisite for their efficient antivira! l activity, novel hybrid ODNs, carrying diverse terminal modifications, were prepared via a fully automated, on-line phosphoramidite-based strategy and evaluated for anti-HIV activity.
• The use of light to investigate and modulate DNA and RNA conformations
  - Nucleic Acids Symp Ser (Oxf) 52(1):11-12 (2008)
  We report the use of light for two distinct kinds of investigation of the conformational changes of, respectively, DNA and RNA. First, Deoxyribosensors' are a class of DNA constructs that incorporate aptamers, and which report the binding of a ligand to the aptamer by attenuating the conformational/stacking relationship of two constituent DNA double helices within the sensor. Such attenuations can be monitored as electrical outputs via a light irradiation protocol. Second, RNA aptamers were selected for the specific binding of one but not the other isomer of different photochromic switch compounds. One such RNA aptamer, specific for binding the closed' isomer of a dihydropyrene compound, was used to create a highly effective, light-sensitive, RNA-cleaving ribozyme. Such ribozymes and riboswitches should find broad utility for the light-mediated control of gene expression within living cells and organisms.
• Manipulation of the peptide-binding specificity of an RNA in a rational manner by combinations of specificity-altering mutations
  - Nucleic Acids Symp Ser (Oxf) 52(1):13-14 (2008)
  In this study, the potential to manipulate the peptide-binding specificity of an RNA in a rational manner was investigated. First, variants of the Rev-response element (RRE) RNA with different specificities towards the natural binding partner, Rev, and two RRE-binding aptamers, the RSG-1.2 and K1 peptides, were identified. Next, hybrid RRE mutants with combinations of two sets of specificity-altering substitutions were tested for peptide-binding specificity. It was shown that, in most cases, the results of the combination of individual mutations were of an additive nature, therefore providing a way to manipulate the peptide-binding specificity of an RNA in a predictable manner.
• Quantitative, Sensitive Analysis of DNA and RNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):15 (2008)
  DNA or RNA sequences that contain useful information about disease risk, disease occurrence, therapeutic response, and probable prognosis are potentially valuable biomarkers. They can be accessed by biopsy, or in ideal cases non-invasively from easily accessible fluids like blood or urine. Tolls are available for biomarker discovery, validation, and clinical use. Discovery usually requires whole genome analysis, and currently this is done with either nucleic acid arrays (DNA chips) or sequencing. Validation requires high quality data scalability to large numbers of samples. Clinical utility normally needs a high degree of automation, and for non-invasive approaches, great experimental sensitivity and specificity but on small numbers of samples. No one platform can efficiently span the broad range of requirements and project sizes. SEQUENOM uses an automated mass spectrometry platform for the quantitative analysis of DNA and RNA in a variety of settings including genot! yping, genecopy number measurements, gene expression, epigenetics, and automated bacterial and viral identification. In collaboration with Amit Meller at Boston University, SEQUENOM is developing optically detected nanospores as a companion platform for its mass spectrometry offering. Both platform use similar homogeneous solution biochemistry to prepare samples. Both platforms depend on vary rapid digital signal processing for real time data analysis and interpretation. The nanopore method uses pores large enough to pass single-stranded DNA but too small to pass double strands. Hence pore passage strips off one of the DNA stands, and optical method are used to detect changes in fluorophores on this strand or attached to reporter probes, as it is stripped. We expect that, when mature, the nanopore method will be extremely const effective for whole genome analysis of genotypes, gene expression, epigenetics, and whole genome sequencing. A key aspect of the nanopore method is! its speed. Post sample preparation, many analyses may require! only seconds of instrument time.
• Artificial molecular switches made from DNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):17-18 (2008)
  The unique biochemical and biophysical properties of DNA can be utilized to construct nanoscale machines and switches, among them devices which can stretch and rotate, translocate, or perform computations. Switchable devices based on aptamers can controllably bind or release enzymes, which can be used to control biochemical reactions. DNA can also be used as a component for switchable materials, e.g. for the realization of switchable gels. These might find applications in the controlled release of particles or substances. An exciting possibility lies in the interaction of DNA nanodevices with RNA molecules. For instance, the behavior of DNA nanodevices may be controlled by natural or artificial gene regulatory mechanisms.
• Construction of a photo-switchable gene for turning on and off gene expression with light irradiation
  - Nucleic Acids Symp Ser (Oxf) 52(1):19-20 (2008)
  A photoresponsive GFP gene was constructed by attaching a T7 promoter that involves two azobenzene moieties as the photoswitch. The azobenzene moieties tethered on D-threoninol were inserted precisely into the sequence of T7 promoter at two positions in the nontemplate strand. By using azobenzene-tethered DNA as one primer, azobenzene was attached to GFP gene after PCR amplification. However, a single-stranded overhang involving azobenzene was formed because primer extension stopped at the position of azobenzene moiety. Interestingly we found that oligonucleotide complementary to the overhang could be ligated by T4 DNA ligase at the stopped position, and the intact photoresponsive T7 promoter was attached onto GFP gene. Furthermore, the in vitro expression of the constructed photoresponsive GFP gene was successfully switched on and off with light irradiation.
• DNA Controlled Assembly of Liposomes
  - Nucleic Acids Symp Ser (Oxf) 52(1):21-22 (2008)
  DNA-encoding of solid nanoparticles requires surfacechemistry, which is often tedious and not generally applicable. In the present study non-covalently attached DNA are used to assemble soft nanoparticles (liposomes) in solution. This process displays remarkably sharp thermal transitions from assembled to disassembled state for which reason this method allows easy and fast detection of polynucleotides (e.g. DNA or RNA), including single nucleotide polymorphisms as well as insertions and deletions.
• Zinc finger protein-based detection system of PCR products for pathogen diagnosis
  - Nucleic Acids Symp Ser (Oxf) 52(1):23-24 (2008)
  A novel detection system of PCR products from bacterial genomes using Zinc finger proteins was developed. Zinc finger proteins are DNA-binding proteins that can bind to dsDNA with high affinity and specificity. Since Zinc finger proteins can directly detect PCR products and can double-check the specific PCR amplification and sequence specificity of the PCR products, this novel method would be quick and highly accurate. In this study, we tried to construct the detection system for three pathogen, Legionella pneumophila, Salmonella spp. and Influenza A virus using well-characterized Zinc finger proteins. As a result, we succeeded in detecting the PCR products from Legionella pneumophila, Salmonella spp. and Influenza A virus using Sp1 and Zif268. Therefore, this methodology can be applied to the detection of most pathogen using various Zinc finger proteins.
• LNA(R) incorporated siRNAs exhibit lower off-target effects compared to 2'-OMethoxy in Cell Phenotypic Assays and Microarray Analysis
  - Nucleic Acids Symp Ser (Oxf) 52(1):25-26 (2008)
  Despite the promise of short interfering RNAs (siRNA), contending with off-target is a challenge for RNAi users. To alleviate these problems, we have developed locked nucleic acid (LNA(R)) modified siRNAs and optimized performance using cellular phenotypic assays as well as microarray analysis. During development, we compared LNA(R) and 2'OMethoxy (2'OMe) chemistries placed strategically throughout the siRNA molecule and found a novel pattern of LNA(R) placement that greatly improved the specificity of the siRNA and reduced it's toxicity in culture while preserving the potency of the siRNA. The improvements in specificity made by LNA(R)-modified siRNAs were developed and validated by measuring the phenotypic signatures in a high content cell-based screening assay as well as comparison of the level of differentially expressed genes observed in microarray analysis between modified and unmodified siRNAs. HT screening of a collection of genes demonstrated that the LNA(R)-m! odified siRNAs exhibits the best overall rate to elicit the expected phenotype, reduced toxicity and achieved an improved coherence of phenotype compared to 2'OMe-modified or unmodified siRNAs.
• Building Biologically Active Nucleic Acid Nanocomplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):27-28 (2008)
  The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.
• Recognizing and Controlling Biomolecules with "Smart" Hybridization-based Switches
  - Nucleic Acids Symp Ser (Oxf) 52(1):29-30 (2008)
  The rules that govern the formation of DNA duplex structures are well known. On one hand, this process is used in the design of probe molecules that report the presence of target nucleic acids by responding to changes of structure and reactivity. On the other hand, molecules may be developed that transduce changes of nucleic acid structure to changes of peptide structure, and vice versa. Applications in the fields of bioanalytical chemistry and synthetic biology are discussed.
• PNA-peptide conjugates as intracellular gene control agents
  - Nucleic Acids Symp Ser (Oxf) 52(1):31-32 (2008)
  Serum-stabilized PNA-internalization peptides (Pip) conjugated to PNA complementary to the 705 aberrant {beta}-globin splice site are able to correct splicing and increase luciferase production in Hela pLuc705 cells with sub {micro}M EC50 in the absence of a transfection agent. Inhibition of microRNA-122 in liver cells is achieved by treatment with complementary PNA containing just a few attached Lys residues, again without need of a transfection agent.
• Structural and functional prerequisites for ribosomal nascentpeptide acceptors: Attempts to decipher the nature of the ribosome's catalysis of peptide bond formation.
  - Nucleic Acids Symp Ser (Oxf) 52(1):33-34 (2008)
  Aminoacyl ribonucleoside analogues that are capable of binding to the acceptor site of ribosomes and taking over the nascent peptide bear, if properly designed, thepotential of antibiotic and cytostatic activity. Here wepresent a study on the intrinsic conformations of natural and synthetic peptide acceptors and the basicities of their peptide accepting amino groups. The conformations and thermodynamic parameters of several synthetic puromycin analogues have been elucidated through ab intio calculations as well as temperature and pH dependent 1H NMR experiments. The intrinsic basicities of their peptide accepting amino groups were determined through 1H NMR and compared to the effective basici-ties of the peptide accepting amino groups of aminoacyl transfer RNAs with the same amino acid side chains, as estimated from the pH dependent kinetics of mRNAprogrammed ribosomal peptidyl transfer.
• The acetal levulinyl ester (ALE) group for the 2'-hydroxyl protection of ribonucleosides and the synthesis of oligoribonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):35-36 (2008)
  A novel 2'-O-ALE (Acetal Levulinyl Ester) uridine phosphoramidite derivative has been efficiently prepared and used for the solid-phase synthesis of a chimeric oligonucleotide strand, 5'-rU11-dT-3'. The average coupling yield of the phosphoramidite was comparable to one obtained with a 2'-TBDMS rU phosphoramidite reagent. Upon completion of the RNA chain assembly, the cyanoethyl phosphate protecting groups were removed using a solution of triethylamine in acetonitrile (2:3 v/v). The 2'-O-ALE protecting groups were cleaved under hydrazinolysis conditions. Finally, the RNA oligonucleotide was released from the Q-CPG support when treated with 1 molar TBAF in THF. When the TBAF step was carried out prior to hydrazinolysis, an oligonucleotide with its intact 2'-OALE groups was obtained.
• Triplex glue by synthesizing conjugated flexible intercalators
  - Nucleic Acids Symp Ser (Oxf) 52(1):37-38 (2008)
  Bulge insertions of conjugated intercalators into the DNA triplex structure are found to give a dramatic contribution to the triplex stability. On the other hand insertions of conjugated intercalators are found to diminish quadruplex structures and in this way breaking down the self association of G-rich oligonucleotides under physiologically potassium ion conditions. A large number of intercalators are described here and they all result in dramatic increases of thermal stability of the corresponding triplexes. Another interesting aspect of conjugated intercalators is their use for assembling alternate strand triplexes. Targeting of neighbouring purine sequences on each their strand in the duplex DNA is a challenge for the 5'- 5' connectivity of the TFOs because of a large distance between the 5'-ends. The intercalator approach offers a linkage with the proper combination of flexibility and rigidity to produce alternate strand triplexes with higher stability than a sim! ilar wild type triplex of the same total length.
• New strategies for the synthesis of unmodified and modified oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):39-40 (2008)
  In this paper, our recent studies on the synthesis of unmodified and modified oligonucleotides are comprehensively reviewed. We have developed a new synthetic strategy using TrS, a new 5'-protecting group, that enabled us to reduce one of the steps previously required for DNA synthesis. The "activated phosphite method" without base protection could provide a new tool for the synthesis of DNA oligomers involving baselabile functional groups such as N-acylated nucleobases capable of Watson-Crick base pairing. This new method was successfully applied to the synthesis of oligodeoxynucleotides incorporating cytosine N-oxide or adenine N-oxide that could not be synthesized by the current methods. Furthermore, several approaches we recently developed for the synthesis of several kinds of 2'-O-modified RNA oligomers will be reported.
• RNA interference in silencing of genes of Alzheimer's disease in cellular and rat brain models
  - Nucleic Acids Symp Ser (Oxf) 52(1):41-42 (2008)
  Accumulation of insoluble aggregates of {beta}-amyloid peptide, a cleavage product of amyloid precursor protein, is thought to be a central step in the pathogenesis of Alzheimer's disease. The major enzymes required for the generation of toxic amyloidbeta peptide are {beta}- (BACE1) and {gamma}-secretases. Here, we present the rational design and the application of synthetic and lentivirus vector-encoded siRNAs for specific and efficient knockdown of overexpressed and endogenous BACE1, both in dividing and neural stem cells and in a rat brain. We also tested an approach to anti-amyloid therapy by the use of the allele-specific siRNAs to silence the mutant presenilin 1 (L392V PS-1), the main component of {gamma}-secretase, responsible for development of Familial Alzheimer's disease. Reducing the level of {beta}-amyloid accumulation in the brain could be beneficial for metabolic studies as well as potential therapeutic approach for prevention and treatment of Alzheimer's! disease.
• Synthesis of the new nucleoside analogue connecting 2-amino-6-vinylpurine to the 2'-deoxyribose skeleton via the methylene linker
  - Nucleic Acids Symp Ser (Oxf) 52(1):43-44 (2008)
  We have previously reported that the 2-amino-6- vinylpurine nucleoside exhibits the highly efficient and selective cross-linking reaction toward the cytosine base at the target site in the duplex DNA. The nucleoside analogues that connect the 2-amino-6-vinylpurine to the 2'-deoxyribose skeleton through the ethylene or the butylene linker formed the cross-link selectively to the adenine base of the TA pair or the cytosine base of the GC pair in the triplex DNA, respectively. They did not form cross-link in the duplex DNA. These results lead us to study in detail the relationship between the linker length and the cross-linking ability. In this study, we describe the synthesis of the new nucleoside analogue that connects 2-amino-6-vinylpurine to the 2'- deoxyribose unit via the methylene linker.
• Potent single stranded RNA inhibition
  - Nucleic Acids Symp Ser (Oxf) 52(1):45 (2008)
  The high affinity provided by LNA is the basis for its high antisense potency. This property offers furthermore the opportunity to make shorter than usual antisense oligonucleotides exhibiting very high in vivo potency and efficacy. The potency of short-stranded LNA is comparable to the very best lipid formulated siRNA's. But in contrast to siRNA short-stranded LNA provides a common platform for highly potent mRNA and microRNA inhibitors. All these features of LNA will be comprehensively illustrated in the presentation. Inhibition of coding RNA will be illustrated using ApoB-100 as the target, and inhibition of miR-122 will be used to illustrate inhibition of non-coding RNA. The conclusions in the presentation are all based on comprehensive rodent and non-human primate data.
• Click chemistry and Oligonucleotides: How a simple reaction can do so much
  - Nucleic Acids Symp Ser (Oxf) 52(1):47-48 (2008)
  Copper catalyzed Alkyne Azide 1,3-dipolar cycloaddition (CuAAC) "click reaction" was applied for the construction of oligonucleotide conjugates, circular objects and phosphodiester glyco-clusters. To this end, several strategies were developed to introduce either alkyne or azide functions into an oligonucleotide.
• Endogenous cytosine methylation and the formation of carcinogen carcinogen-DNA adducts
  - Nucleic Acids Symp Ser (Oxf) 52(1):49-50 (2008)
  All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC, X = Me in Scheme 1). The same sites (e.g. p53 codons 157, 158, 245, 248, and 273) are mutational hotspots in smoking induced lung cancer, suggesting that methylated CG dinucleotides may be preferentially targeted by the reactive metabolites of tobacco carcinogens. We employed a stable isotope labeling HPLC-ESI-MS/MS approach to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diolepoxide metabolites of bay region polycyclic aromatic hydrocarbons, e.g. benzo[a]pyrene diol epoxide (BPDE). In contrast, cytosine methylation was protective against O6-guanine alkylation by tobacco tobacco-specific nitrosamines, e.g. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), To investigate the mechanisms behind these effects, a series of structural analogs of MeC were prepared, and their effects on reactivity of base th! e paired dG towards BPDE was examined. We found that the presence of the C-5 substituent on cytosine influences the reactivity of its partner guanine towards BPDE and modifies the stereoisomeric composition of the resulting N2-BPDEdG adducts.
• A Whole Base-Labile Strategy for RNA Synthesis with 2'-O-acetalester Protections
  - Nucleic Acids Symp Ser (Oxf) 52(1):51-52 (2008)
  The use of base-labile groups for 2'-OH protection is a major challenge for RNA synthesis. A new method with acyloxymethyl groups has been developed. These groups were fully compatible with standard base-labile protections for nucleobases and phosphates and were removed in a short two-step all-base deprotection. Oligoribonucleotides were synthesized efficiently, rapidly and in high purity without chain rupture or isomerisation via this new whole base-labile strategy.
• Novel base-functionalized DNA. Efficient methodology for construction and bioanalytical applications
  - Nucleic Acids Symp Ser (Oxf) 52(1):53-54 (2008)
  A novel efficient two-step methodology for the construction of base-functionalized DNA is based on direct aqueous cross-coupling reactions of unprotected nucleoside triphosphates followed by polymerase incorporation. Preliminary applications of the modified DNA in electrochemical detection and bioanalysis are outlined.
• Recent Developments in the Synthesis of RNA Oligonucleotides for Potential Therapeutic Applications.
  - Nucleic Acids Symp Ser (Oxf) 52(1):55-56 (2008)
  The 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl group for 2'-hydroxyl protection has been successfully employed in the solid-phase synthesis of AUCCGUAGCUAACGUCAUGG. The use of the 2'-Omethylthiomethyl (2'-O-MTM) protecting group in the development of a cost effective approach to RNA synthesis has been evaluated through the solid-phase synthesis of [2'-O-MTM U]19dT. Given the sensitivity of 2'-thioacetals to acidic conditions, the base-labile [2-(9- fluorenyl)propyl-2-oxy]carbonyl group for 5'-Oprotection of ribonucleosides has been designed to accommodate the synthesis of oligoribonucleotides functionalized with 2'-thioacetal groups.
• Repair of DNA-protein crosslink damage: Coordinated actions of nucleotide excision repair and homologous recombination
  - Nucleic Acids Symp Ser (Oxf) 52(1):57-58 (2008)
  DNA-protein crosslinks (DPCs) are extremely bulky DNA lesions, and steric hindrance imposed by covalently trapped proteins would hamper the transaction of DNA such as replication, transcription, and repair. However, it has been largely elusive how cells mitigate the genotoxic effect of DPCs. We have recently shown that nucleotide excision repair (NER) and homologous recombination (HR) differentially contribute to the repair of DPCs in E. coli cells. Several lines of genetic and biochemical evidence indicate that NER repairs DPCs with crosslinked proteins (CLPs) of sizes less than 12-14 kDa, whereas DPCs with oversized CLPs are processed exclusively by RecBCD-dependent HR. The present result shows that cells use the coordinated actions of NER and HR to deal with unusually bulky DNA lesions like DPCs.
• Characterization of endogenous human Argonautes and their miRNA partners in RNA silencing
  - Nucleic Acids Symp Ser (Oxf) 52(1):59-60 (2008)
  Small RNAs triggering RNA silencing are loaded onto Argonautes and then sequence-specifically guide them to target transcripts. Epitope-tagged human Argonautes (hAgo1, hAgo2, hAgo3, and hAgo4) are associated with siRNAs and miRNAs, but only epitope-tagged hAgo2 has been shown to have Slicer activity. Contrarily, how endogenous hAgos behave in respect to small RNA association and target RNA destruction has remained unclear. Recently, we produced monoclonal antibodies for individual hAgos and characterized small RNAs specifically associated with hAgo2 and hAgo3 endogenously expressed in Jurkat cells.
• Physicochemical Stability of NOX-E36, a 40mer L-RNA (Spiegelmer) for Therapeutic Applications
  - Nucleic Acids Symp Ser (Oxf) 52(1):61-62 (2008)
  Spiegelmers are structured mirror-image oligonucleotides that are designed to bind and inhibit pharmacologically relevant target molecules. The synthesis and purification of mirror-image oligonucleotides is comparable to the manufacturing of standard oligonucleotides that consist of naturally configured nucleotides. Due to the use of the nonnatural L-nucleotides in Spiegelmers, these oligonucleotides show an exceptional biostability. Further, they also display a high physicochemical stability in solution. These properties make them interesting substances for drug development.
• Inhibition of picornaviruses by means of RNA interference
  - Nucleic Acids Symp Ser (Oxf) 52(1):63-64 (2008)
  Picornaviruses are a class of RNA viruses with a single-stranded genome in positive orientation. Since the prospects of treatment are limited, we employ RNA interference (RNAi) as an antiviral tool to inhibit different picornaviruses. We identified small interfering RNAs (siRNAs) against the 3D RNA dependent RNA polymerase of coxsackievirus B3 that were capable of efficiently inhibiting the virus. Targeting of the conserved 5' UTR of the virus turned out to be a challenging task since stable structures of this region are detrimental to silencing. We developed a rational strategy to solve this problem and found an siRNA containing locked nucleic acids (LNAs) to possess high antiviral potency. To analyse the mechanism of virus inhibition in more detail, LNAs were incorporated into the siRNA to inactivate either of the siRNA strands. These experiments clearly revealed that only the genomic plus-strand but not the intermediary synthesised minus-strand can be targeted by si! RNAs. Furthermore, siRNAs were employed to silence the virus receptor on the host cell and thus prevent viral spread.
• Protein-Facilitated Ribozyme Folding and Catalysis
  - Nucleic Acids Symp Ser (Oxf) 52(1):67-68 (2008)
  In vivo, large RNAs rely on proteins to fold to their native conformation. In the case of the S. cerevisiae group II intron ai5{gamma}, the DEAD-box protein Mss116 has been shown to promote the formation of the catalytically active structure. However, it is a matter of debate whether it does this by stabilizing on-pathway intermediates or by disrupting misfolded structures. Here we present the available experimental evidence to distinguish between those mechanisms and discuss the possible interpretations.
• 2'-O-Methoxyethyl/2'-Fluoro Modified Oligonucleotides Result in More Potent Inhibition of micro RNA-122 in Vivo: A Target implicatedin HCV Replication
  - Nucleic Acids Symp Ser (Oxf) 52(1):69 (2008)
  MicroRNAs are endogenous 20-24 nt small non-codingRNAs that have profound roles in multiple developmental and cellular processes. Dysregulation of microRNAs can lead to a host of pathologies suggesting that microRNAs could be important for therapeutic intervention in cancer, metabolic diseases, autoimmune disorders and viral diseases. Through recent studies,mir-122 has emerged as a potential target for metabolic diseases and HCY. Chemical modifications are essential to achieve clinically relevant potency and efficacy andtherapeutic index of anti-miRNA oligonucleotides. Wehave evaluated more than 65 chemically-modified ASOs for their ability to inhibit the activity of miR-122 inmice. Inhibition of miR-122 with ASOs resulted inincreased levels of miR-122 target gene mRNAs in theliver, as well as lowering of plasma cholesterol in a dose dependant manner. The current investigation led to the identification of a chimeric 2'Fluoro/2'-O-methoxyethyI(2'OME) modified motif with! improved efficacy and 5-10 fold improvement in potency compared to LNA/DNAmodified and uniform 2'-MOE-PS compounds. Theseefforts have identified significantly improved anti-miR-122 ASOs for further evaluation as anti-HCV therapeutic agents.
• An efficient fluorescent method for selective detection of mature miRNA species
  - Nucleic Acids Symp Ser (Oxf) 52(1):71-72 (2008)
  Methods for specific detection of RNA molecules have been widely developed in biotechnology and diagnostic field. The diagnostic target is recently expanded to non-coding RNA, including microRNAs (miRNAs). miRNAs are initially synthesized as precursor, and then processed to their physiologically active species, miRNAs. Detection of only the active mature miRNA without interference of the precursor forms is extremely important for their functional and physiological significance. We developed a novel fluorescent DNA probe to detect the mature miRNA with high specificity. Since the probe discriminates the mature miRNA from the precursor without electrophoretic separation, it should be useful for real-time detection of the mature miRNAs.
• In vitro selection of a DNAzyme with three modified nucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):73-74 (2008)
  Three modified nucleosides have been used in the in vitro selection of the self-cleaving DNAzyme 9-33. This DNAzyme operates in the absence of divalent metal cations with a first-order constant of {approx} 0.05 min-1.
• Transition state analogues in quorum sensing and SAM recycling
  - Nucleic Acids Symp Ser (Oxf) 52(1):75-76 (2008)
  Transition state structures can be derived from kinetic isotope effects and computational chemistry. Molecular electrostatic potential maps of transition states serve as blueprints to guide synthesis of transition state analogue inhibitors of target enzymes. 5'- Methylthioadenosine phosphorylase (MTAP) functions in the polyamine pathway by recycling methylthioadenosine (MTA) and maintaining cellular Sadenosylmethionine (SAM). Its transition state structure was used to guide synthesis of MT-DADMe-ImmA, a picomolar inhibitor that shows anticancer effects against solid tumors. Biochemical and genomic analysis suggests that MTAP inhibition acts by altered DNA methylation and gene expression patterns. A related bacterial enzyme, 5'-methylthioadenosine nucleosidase (MTAN), functions in pathways of quorum sensing involving AI-1 and AI-2 molecules. Transition states have been solved for several bacterial MTANs and used to guide synthesis of powerful inhibitors with dissociatio! n constants in the femtomolar to picomolar range. BuT-DADMe-ImmA blocks quorum sensing in Vibrio cholerae without changing bacterial growth rates. Transition state analogue inhibitors show promise as anticancer and antibacterial agents.
• Chemistry and Structure-Activity Relationship of Antibacterial Nucleoside Natural Products
  - Nucleic Acids Symp Ser (Oxf) 52(1):77-78 (2008)
  Synthetic methodology of the caprazamycins, which are promising antibacterial nucleoside natural products, was developed. Palmitoylcaprazol, which possesses a simple fatty acyl side chain at the diazepanone moiety of caprazamycins, has been synthesized and exhibited antibacterial activity against pathogens threatening a public health. Simplification of the caprazamycins was further pursued to develop diketopiperazine analogs.
• Development of A3 Adenosine Receptor Ligands
  - Nucleic Acids Symp Ser (Oxf) 52(1):79-80 (2008)
  4'-Thioadenosines have been discovered as novel templates for A3 adenosine ligands. Among these, 4'-thioadenosine-5'-monoalkyluronamides were discovered as novel potent and selective A3 adenosine receptor agonists, while 4'-thioadenosine-5'-dialkyluronamides and truncated 4'-thioadenosine derivatives exhibited potent and selective antagonism at the A3 adenosine receptor.
• Synthesis and properties of ({alpha}-P-borano)-nucleoside 5'-triphosphate analogues as potential antiviral agents
  - Nucleic Acids Symp Ser (Oxf) 52(1):81-82 (2008)
  The {alpha}-P-borano modification, where one of the {alpha}-phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the {alpha}-P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl {alpha}-P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions. These analogues will be used for future structure-activity relationship (SAR) studies.
• Nucleoside Diphosphate Prodrugs
  - Nucleic Acids Symp Ser (Oxf) 52(1):83-84 (2008)
  Nucleoside analogs are widely applied in antiviral and antitumor therapy. A severe limitation of these compounds arises from the need of biotransformation to the eventually active nucleoside triphosphates by stepwise addition of phosphate by kinases. This problem can be circumvented by employing reversibly masked nucleotides (prodrugs). However, the known concepts to bypass enzyme activation have almost exclusively been applied to nucleoside monophosphates. Here, we report on the transfer of the bis- (acyloxybenzyl)-concept (BAB-concept) from nucleoside monophosphates to nucleoside diphosphates (NDP) of the anti HIV drugs AZT and d4T. After successful synthesis and isolation of the compounds (BAB-NDPs), it was shown that these compounds exhibit promising hydrolytic properties ranging from high stability at physiological pH to very low stability in cellular extracts. Furthermore, we demonstrated that for some of the compounds a selective cleavage mechanism resulted in t! he exclusive delivery of the corresponding nucleoside diphosphate within 15 minutes without any degradation of the pyrophosphate unit, which is a unique result in the field of NDP-prodrugs.
• Inhibition of Orotidine-5'-monophosphate decarboxylase - Discoveries and lessons
  - Nucleic Acids Symp Ser (Oxf) 52(1):85-86 (2008)
  Orotidine-5'-monophosphate decarboxylase (ODCase) is one of most proficient enzymes, and this enzyme catalyzes the decarboxylation of orotidine-5'- monophosphate (OMP) to uridine-5'-monophosphate (UMP). A number of C6-substituted uridine derivatives are designed to investigate the mechanism of decarboxylation by this enzyme. In this process, novel reactions and mechanisms were uncovered for this decarboxylase. This led to the discovery of novel ODCase inhibitors and their biological activities. Medicinal chemistry of these novel inhibitors of ODCase in the context of its catalytic mechanism, and therapeutics development will be discussed.
• Preparation of Mannosylated Oligoribonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):89-90 (2008)
  Multivalent mannosides were synthesized for attachment to oligoribonucleotides via 3,4-diethoxy-3-cyclobutene-1,2-dione (squarate). The conjugates have potential to be used in receptor-mediated endocytosis of small interference RNAs into dendritic cells through interactions with mannose-binding lectins.
• Tyrosine-modified PEI: A novel and highly efficient vector for siRNA delivery in mammalian cells
  - Nucleic Acids Symp Ser (Oxf) 52(1):91-92 (2008)
  Delivery of synthetic small interfering RNA (siRNA) into cell remains the major obstacle to its biological activity. Cationic polymers, and in particular the "proton sponge" polyethylenimine (PEI), has shown promise for cancer gene therapy but appears inefficient for siRNA delivery. Here we report that modifying branched PEI with amino acids led to efficient siRNA delivery into mammalian cell lines, even in the presence of serum, and at dose as low as 1 nM.
• DNA electron transfer mechanism and dynamics
  - Nucleic Acids Symp Ser (Oxf) 52(1):95-96 (2008)
  The dynamics and efficiency of photoinduced charge separation in synthetic DNA hairpins and dumbbells possessing stilbenedicarboxamide (Sa) and stilbene-diether (Sd) linkers separated by short poly(dA)-poly(dT) base pair sequences have been investigated by means of time resolved fluorescence and transient absorption spectroscopy. Charge separation occurs via a single step superexchange mechanism at short distances and by a multistep hole transport mechanism when the linkers are separated by two or more base pairs. The dynamics and efficiency of hole transport are strongly distance dependent over the first few base pairs, but are relatively insensitive to distance at larger separations. The distance dependence is attributed to a Coulomb cage effect. Hole transport across alternating AT base sequences is less efficient than across poly(dA) sequences. Introduction of guanine into the poly(dA) sequence can either enhance or diminish the efficiency of charge separation, dep! ending upon the location of G. Our experimental results are interpreted using several theoretical models.
• Addressable Molecular Node Assembly - Functional DNA Nanostructures
  - Nucleic Acids Symp Ser (Oxf) 52(1):97-98 (2008)
  The use of nucleic acids as a nanomaterial is becoming increasingly widespread due to the suitability of the hydrogen-bonding patterns and sequence specificity inherent to the double-helix. As minimisation of size becomes ever more important it is imperative to employ nucleic acids in the most efficient and functional manner possible. To this end we have constructed DNA nanostructures on what may be the smallest possible scale (basic components of just 10 bp) that not only reliably self-assemble but also where each unit of a 2-dimensional DNA network can be uniquely identified and selectively functionalised.1,2.3 On this length scale and using full addressability of the network to engrave specific pathways on the scaffold, energy and electron transfer become efficient for potential information storage applications.4
• Synthesis of 2',3'-Cyclohexene Bicyclic Nucleoside Analogues as Antiviral Compounds
  - Nucleic Acids Symp Ser (Oxf) 52(1):99-100 (2008)
  Chiral syntheses of a series of hexahydroisobenzofuran (HIBF) nucleosides have been accomplished via glycosylation of a stereo-defined (syn-isomer) sugar motif with the appropriate silylated bases. All nucleoside analogues were obtained in 52-71% yield as a mixture of {alpha}- and {beta}-anomeric products increasing the breadth of the novel nucleosides available for screening. Nucleoside derivatives were tested as inhibitor of HIV-1 in human peripheral blood mononuclear (PBM) cells.
• Preparation of Nucleoside-Carbohydrate Phosphodiester Prodrug Analogues by Chemoenzymatic Procedure
  - Nucleic Acids Symp Ser (Oxf) 52(1):101-102 (2008)
  An efficient synthesis protocol for the glucosylnucleoside phosphodiester derivatives has been developed. These mononucleotides were designed to act as pronucleotides with potential to deliver the parent compound as its monophosphate. Key step of the synthesis is the regioselective hydrolysis of peracetylated {alpha}-D-glucose catalyzed by Candida rugosa lipase.
• RNA Synthesis: Phosphoramidites for RNA synthesis in the reverse direction. Highly efficient synthesis and application to convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3'- end.
  - Nucleic Acids Symp Ser (Oxf) 52(1):103-104 (2008)
  Defined sequence RNA synthesis by 3'[->]5' direction is now well established and currently in use for synthesis and development of vast variety of therapeutic grade RNA and Si RNA etc. A number of such synthetic RNA requires a modification or labeling of 3'- end of an oligonucleotide. The synthesis of 3'- end modified RNA requiring lipophilic, long chain ligands or chromophores, using 3' [->] 5' synthesis methodology is challenging, requires corresponding solid support and generally results in low coupling efficiency and lower purity of the final oligonucleotide in general because of large amount of truncated sequences containing desired hydrophobic modification. We have approached this problem by developing reverse RNA monomer phosphoramidites for RNA synthesis in 5' [->] 3'- direction. They lead to very clean oligonucleotide synthesis allowing for introduction of various modifications at the 3'- end.
• Studies of sequence-specific recognition and interaction of bishairpin polyamide minor groove binders with target DNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):105-106 (2008)
  The binding of bis-MGB to target DNA was studied by DNase footprint, native gel shift, circular dichroism, thermal dissociation, electrospray mass-spectrometry, and molecular modelling methods. A new method for the determination of the relative affinity of ligands against various dsDNA sequences was elaborated by using ESI-QTOF mass spectrometry. Information about affinity, sequence preferences, conformation and mode of interaction between bis-MGB and target DNA was obtained. Our experiments demonstrated that MGB have different affinity for similar cognate target sequences depending on the sequence context of the target region and other structural factors.
• Binding of two bis-bipyridine minor groove binders to a DNA template in the presence of Cu2+ ions
  - Nucleic Acids Symp Ser (Oxf) 52(1):107-108 (2008)
  Some diseases are associated with abnormally extended regions of triplet repeats. These repeating regions are an attractive target for both diagnostic and therapeutic goals. In an attempt to approach to this goal, we have focused on establishment of an allosteric binding mechanism, in which the binding of the ligand promotes the next ligand binding. In the previous study, we already reported that the ligand having the bipyridine unit for binding with Cu2+ and the Hoechst33258 for binding to A3T3 site displayed Cu2+- mediated assembly on the DNA with two A3T3 sites. In this study, we synthesized the new ligands containing two bipyridine units attached to Hoechst33258 by different length linkers. It was expected that the bipyridine-Cu2+ complexation would enhance assembly of a number of the lingand on the DNA sequence with repeating regions. UV spectroscopy has been used to demonstrate the binding of these ligands to a DNA template mediated by the complexation of Cu2+ io! ns.
• Recognition of Homopyrimidine Mismatches by Distance-Constrained Macrocyclic bisintercalators.
  - Nucleic Acids Symp Ser (Oxf) 52(1):109-110 (2008)
  Binding of three macrocyclic bisintercalators to mismatch-containing duplexes was analyzed by thermal denaturation experiments, electrospray mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bisintercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched one and affinity in the submicromolar range (Kd). The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine-pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch.
• The interaction between the purine motif triplex and the triplex DNA-binding domain of Saccharomyces cerevisiae Stm1 protein
  - Nucleic Acids Symp Ser (Oxf) 52(1):111-112 (2008)
  Saccharomyces cerevisiae Stm1 protein (273 amino acids) is a purine motif triplex DNA-binding protein. We have previously found that Stm(1-113) (amino acids 1-113) is the minimal domain to specifically bind with the purine motif triplex. Here, to reveal the triplex recognition mechanism of Stm(1-113), we have examined the interaction between Stm(1-113) and each of the purine motif triplexes with various lengths and base sequences. As the length of the target triplex was increased, the binding affinity of Stm(1-113) to the target triplex was increased. Stm(1-113) had the ability to bind to the purine motif triplexes with various base sequences. We conclude that Stm(1-113) may recognize the shape of the triplex rather than the base sequence of the triplex.
• Effect of cationic comb-type copolymer on the B- Z transition of poly(dG-dC){middle dot}poly(dG-dC)
  - Nucleic Acids Symp Ser (Oxf) 52(1):113-114 (2008)
  The structural transition of a double helical DNA from B-DNA to Z-DNA, B-Z transition, has been received growing attention because of its potential roles in biological systems and its applicability to nanobiotechnology. It was known that the B-Z transition is induced by cationic molecules. However, there are only a few reports describing cationic polymers as potential B-Z inducers. In this study, we demonstrated that cationic comb-type copolymer, poly(L-lysine)-graft-dextran, induced the B-Z transition in which distinct intermediate formation was involved. Furthermore, we suggested that the grafted-dextran plays an important role in the B-Z transition.
• A pteridine derivative with electron-withdrawing groups for binding and sensing of nucleobases in AP site-containing DNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):115-116 (2008)
  A pteridine derivative having electron-withdrawing CF3 groups, 2-amino-6,7-bis(trifluoromethyl)-4-hydoroxypteridine (2CF3-pteridine), is presented as a candidate for multi-functional fluorescent ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 8.0 (I = 0.1 M, at 5{degrees}C), 2CF3- pteridine can bind to guanine, cytosine and thymine opposite an abasic site in DNA duplexes (5'-TCTGC GTCCA GXG CAACGCACAC-3'/3'-AGACG CAGGT CNC GTTGCGTGTG-5', X = abasic site; Spacer-C3, N = G, C, A, T). For these three nucleobases, the binding of 2CF3-pteridine is explained by 1:1 complexation, and the binding affinities are comparable (K11 / 105 M-1: G: 3.0; C: 1.6; T: 3.3). Binding-induced fluorescence responses are effectively different between guanine and pyrimidines (C, T): the binding to pyrimidines is accompanied by a significant change in the shape of fluorescence spectra. These binding and sensing properties allow a detection of G/T or G/C muta! tion based on a single fluorescence ligand.
• Effect of an alkyl amino group on the binding of 1,8-naphthyridines to AP site-containing DNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):117-118 (2008)
  A 1,8-naphthyridine derivative having a positively charged side-chain, N-(3-aminopropyl)-5,6,7-trimethyl-1,8-naphthyridin-2-amine (APATMND), is synthesized, and its binding to AP site-containing DNA duplexes (5'- GCA GCT CCC GXG GTC TCC TCG-3'/ 5'-CGA GGA GAC CNC GGG AGC TGC-3', X = AP site; dSpacer, N = C, T) is examined in solutions buffered to pH 7.0 (I = 0.11 M, at 20{degrees}C). Fluorescence titration experiments reveal that, as compared to a parent ligand, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), capable of selectively binding C over T opposite an AP site in the duplex (Kd/nM: C: 56, T: 100), APATMND shows a stronger binding affinity for T, while an affinity for C is reduced (Kd/nM: C: 135, T: 37). An examination of salt dependence of binding constants reveals that a polyelectrolyte contribution ({Delta}Gpe) is indeed increased for C- and T-bindings of APATMND, but a loss of non-polyelectrolyte contribution ({Delta}Gt) is significant when binding to C. ! These binding properties of APATMND are discussed with a view towards further development of DNA-binding ligands suitable for gene detection.
• Competitive binding of small ligands to nucleobases in AP site-containing DNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):119-120 (2008)
  By using lumichrome (Lch) as a masking ligand, we successfully control the binding selectivity of 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) when binding to nucleobases in AP site-containing DNA duplexes (5'-TCT GCG TCC AGX GCA ACG CAC AC-3'/3'-AGA CGC AGG TCN CGT TGC GTG TG-5', X = AP site; Spacer C3, N = C or T). In solutions buffered to pH 7.0 (I = 0.11 M, at 5{degrees}C), ATMND binds to cytosine and thymine with a comparable binding affinity (Kd / nM: C: 7.7, T: 15). By contrast, in the presence of Lch, ATMND shows a clear binding selectivity for cytosine over thymine (Kd/nM: C: 17, T: 204). Such competitive binding events are discussed with a view towards development of ligand-based fluorescence assay for single-nucleotide polymorphisms (SNPs) typing.
• Development of label-free molecular beacons based on abasic sitebinding fluorescence molecules
  - Nucleic Acids Symp Ser (Oxf) 52(1):121-122 (2008)
  Here we report on a class of label-free molecular beacons (MBs) based on a non-covalent interaction with abasic site (AP site)-binding fluorescence molecules. In contrast to conventional MBs that require the chemical labelling with fluorophores and quenchers, our MB simply contains the AP site in the stem moiety, so that a small molecule specifically binds to the AP site. This binding event is accompanied by a significant quenching of its fluorescence, and thus a closed state of the AP site-containing MB (APMB) shows almost no fluorescence. Upon hybridization with a complementary DNA, APMB undergoes a conformational change to take an open state, resulting in an effective fluorescence enhancement due to a release of the molecule from the AP site. These sensing functions of APMB are discussed with a view towards further development of gene detection chemistry based on DNA-binding small molecules.
• A surface plasmon resonance sensor based on 3,5-diaminopyrazine with a high selectivity for thymine in AP site-containing DNA duplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):123-124 (2008)
  We here report on a surface plasmon resonance (SPR) sensor carrying small organic ligands for the detection of single-nucleotide polymorphisms (SNPs). Two kinds of ligands are prepared, both of which have a hydrogen-bond forming site suitable for nucleobase recognition, and have an active amino group for the immobilization to the sensor chip. While the sensor immobilized flavin does not show any useful responses, the sensor based on 3,5-diaminopyrazine shows a highly selective response to thymine over other nucleobases opposite an abasic site in DNA duplexes (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5', X = AP site, N = target; G, C, A, T). In PBS buffer (pH 6.4, 0.25 M NaCl, at 5{degrees}C), the sensor can detect 10 nM of the sample solution, and the SPR signal for thymine is linear in the concentration range from 10 nM to 100 nM. These sensing functions of the present sensor are discussed for the development of SNPs detection chemistry based o! n DNA-binding small molecules.
• Mismatch recognition in PNA double-duplex invasion
  - Nucleic Acids Symp Ser (Oxf) 52(1):125-126 (2008)
  Double-duplex invasion of pseudo-complementary PNA (pcPNA) to double-stranded DNA is promising for recognition of a specific sequence in double-stranded DNA. In order to apply this process for various purposes such as gene suppression, one base-pair change at the target site in DNA must be strictly distinguished by the pcPNA additives. In this study, mismatch-recognizing activity of double-duplex invasion was investigated under various salt conditions. It has been found that the mismatch-recognition of the invasion is strict enough to distinguish one base-pair alternation, as long as the invasion is achieved in the media of appropriate ionic strength (e.g., [NaCl] = 20 mM).
• Crystal structures of DNA duplexes stabilized by bicyclic-C residues
  - Nucleic Acids Symp Ser (Oxf) 52(1):127-128 (2008)
  Chemical modification of nucleic acids is being studied extensively as an approach for the development of nucleic acid-based therapies. We found that a nucleotide carrying 7,8-dihydropyrido[2,3-d]pyrimidin-2-one (bicyclic-C or X), which is a cytosine derivative with a propene attached at the N4 and C5 atoms, increases the stability of DNA duplexes. To establish the conformational effects of X on DNA and to obtain insight into the correlation between the structure and stability of X-containing DNA duplexes, the crystal structures of [d(CGCGAATT-X-GCG)]2 and [d(CGCGAAT-X-CGCG)]2 have been determined at 2.9 A resolutions. In both duplexes, the bicyclic-C bases form pairs with the counter bases through hydrogen bonds, and stabilize the duplex formation in part by stacking interactions between X and the subsequent thymine base of the same strand.
• Self-Avoiding molecular Recognition Systems (SAMRS)
  - Nucleic Acids Symp Ser (Oxf) 52(1):129-130 (2008)
  Reported here is a "Self-Avoiding Molecular Recognition Systems" (SAMRS), a species of DNA that can bind via simple rules to natural DNA but cannot bind to other members of the same SAMRS species. A system having these properties has been achieved with 2-aminopurine-2'-deoxyriboside (A*), 2'-deoxy-2-thiothymidine (T*), 2'-deoxyinosine (G*) and N4-ethyl-2'-deoxycytidine. These were designed to form more stable base pairs with natural complements than with SAMRS complements, based on the number of hydrogen bonds. Thermal melting studies were performed using duplexes containing SAMRS components. All SAMRS species, A*, T*, G* and C*, formed more stable base pairs with natural complements, T, A, C and G than with SAMRS complements, T*, A*, C* and G* respectively. This property of SAMRS would be useful for avoiding to be produced undesired products derived from intra- and intermolecular interaction between primers in multiplexed polymerase chain reactions.
• Finding at-DNA - Kinetic Recognition of Long Adenine-Thymine Stretches by Metal-Ligand Complexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):131-132 (2008)
  High selectivity for long AT sequences can be attained by kinetically controlled DNA threading intercalation by binuclear ruthenium(II) complexes. The rate of intercalation is strongly correlated to the number of consecutive AT basepairs, being up to 2500 times faster with an AT polymer compared to mixed-sequence DNA.
• Unlocked nucleic acid (UNA) and UNA derivatives: Thermal denaturation studies
  - Nucleic Acids Symp Ser (Oxf) 52(1):133-134 (2008)
  A study on the thermal stability of duplexes formed between unlocked nucleic acid (UNA) modified DNA and RNA oligonucleotides and complementary DNA and RNA is presented. The acyclic UNA monomers are shown to induce a decrease in duplex thermal stability.
• Synthesis of 3'- and 6'-functionalised Locked Nucleic Acid (LNA) analogues
  - Nucleic Acids Symp Ser (Oxf) 52(1):135-136 (2008)
  Two locked nucleic acid (LNA) analogues, i.e. a 3'-C-hydroxymethyl derivative and a 6'(R)-hydroxymethyl derivative of the LNA thymidine monomer, have been synthesized efficiently using convergent strategies. The hydroxymethyl group at the 3'-position or the 6'-position provides a versatile starting point for various modifications of LNA.
• Formation of a stable triplex incorporating a CG interrupting site by a new WNA derivative containing 3-aminopyrazole as a nucleobase
  - Nucleic Acids Symp Ser (Oxf) 52(1):137-138 (2008)
  Triplex-forming oligonucleotides (TFOs) bind within the major groove of duplex DNA in a sequence-specific manner, and have attracted much interest as genomic tools. However, as the triplex DNA is formed by the interaction between the TFOs and homopurine/homopyrimidine sequences of the target duplex DNA, the stable triplex formation is prevented by one pyrimidine base in the homopurine strand. Previously, we developed the nucleoside analogues (WNA: W-shaped nucleoside analogues) that furnish an aromatic ring as a stacking part and a nucleobase as a recognition part onto the bicyclic skeleton. Selective recognition of a TA and a CG interrupting site has been achieved by WNA-{beta}T and WNA-{beta}C, respectively. In the subsequent study, it was found that the triplex formation by the WNA analogues depend on its neighbouring bases within the TFO. In this paper, we describe the synthesis and the evaluation of the triplex forming ability of WNA-{beta}3AP, having 3-aminopyraz! ole (3AP) as a nucleobase. It is remarkable that the TFO containing the WNA-{beta}3AP recognizes the CG interrupting site with high selectivity in the TFO sequence of 3'-GZG-5', in which the previous WNA-{beta}C did not show the stabilizing effect.
• Strand invasion properties and serum stability of {alpha}-tricyclo-DNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):139-140 (2008)
  {beta}-tricyclo DNA (tcDNA) is a third generation antisense oligonucleotide developed and studied extensively in our laboratory. We recently became interested in the pairing properties of its {alpha}-anomeric form and observed parallel duplex formation with natural DNA and RNA. These duplexes were of the same level of thermal stability as their natural counterparts. In addition {alpha}-tcDNA exhibited poor thermal stability of antiparallel duplexes within its own backbone series which makes it a canditate for double strand invasion applications. Here we report on the strand invasion properties of {alpha}-tc-DNA and its stability in human serum.
• Single-strand binding protein enhances invasion of a PNA strand to double-stranded DNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):141-142 (2008)
  We show that, in the presence of single-strand binding protein (SSB), one strand of peptide nucleic acid (PNA) efficiently invades double-stranded DNA (dsDNA) in sequence-specific manner, as shown in Figure 1. In the absence of SSB, this kind of strand invasion never occurs. This significant enhancement of invasion can be applied to in vivo applications.
• Introduction of linkers into PNA for versatile applications
  - Nucleic Acids Symp Ser (Oxf) 52(1):143-144 (2008)
  PNA (peptide nucleic acid) is widely used as a DNA recognition tool. In this study, we introduced linkers in the middle of PNAs to expand their applicability. By means of Tm measurement and gel-shift assay, it was confirmed that these PNA can recognize double stranded DNA through efficient invasion.
• Iso-Thioacetamido nucleic acids (isoTANA): Synthesis and DNA binding studies of Lyxo/Ribo oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):145-146 (2008)
  In the previous study, we developed a method to synthesize 5-atom thioacetamido linked TANA dimer blocks. Incorporation of the dimers in chimeric TANA-phosphodiester linked oligomers and the binding studies towards DNA/RNA preferences were reported. The selectivity towards RNA was found to be significantly high compared to DNA. The lyxo isomer of 3'-amino substitution in thymine was found to have higher 3'-endo conformation in monomer units. In this paper we present studies on synthesis of 3'-lyxo-3'-amino isomer of TANA, its tst and cst dimer units, and incorporation in oligomers. Preliminary results on DNA/RNA binding of these new backbone modified oligomers will be presented.
• Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy
  - Nucleic Acids Symp Ser (Oxf) 52(1):147-148 (2008)
  A new approach is described for the insertion of nitroxide spin-labels at specific positions within DNA oligomers. The latter bioconjugaison strategy is based on a click chemistry 1,3-dipolar cycloaddition between a spin-labeling reagent, namely the 4-azido-TEMPO, and alkyne modified uridine-containing oligonucleotides. This highly efficient labeling method was applied for site-specific incorporation of two TEMPO units within a set of double-stranded DNA constructs. Then the determination of the inter-nitroxide distances was achived by using a four-pulses DEER technique that successfully validates the new site-directed spin labeling strategy.
• A click chemistry approach towards nucleic acid major groove functionalization
  - Nucleic Acids Symp Ser (Oxf) 52(1):149-150 (2008)
  A synthetic strategy towards new aromatic nucleoside derivatives introducing additional aromatic functionality placed in the major groove of a modified DNA duplex is precented. The functionalities are introduced using Click Chemistry conditions and found to increase the overall duplex stability.
• Dynamics of 2-N-tert-Butylaminoxyladenosine Incorporated into Oligodeoxynucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):151-152 (2008)
  We synthesized oligodeoxynucleotide (ODN, 3) containing 2-N-tert-butylaminoxyladenosine (1) and studied EPR spectra of 1 and its duplexes with varied sequences. Duplex formation resulted in a broadening of spectrum and a significant decrease in peak-to-peak height and peak height ratio values. The h+/ho values in EPR spectra well correlated with stability of duplex which indicated ODN containing 1 has the potential to monitor DNA structure.
• Nitroxide Spin labeled RNA for long range distance measurements by EPR-PELDOR
  - Nucleic Acids Symp Ser (Oxf) 52(1):153-154 (2008)
  Long range distance measurement on RNA allow the determination of RNA folds. Here we report the site specific incorporation of nitroxide spin labels at U,C and A by "on column synthesis". PELDOR (Pulsed Electron Double Resonance) measurements of several RNAs in the range of 2-6nm were successful.
• New triarylamine-based far-red DNA stainers with high two-photon absorption properties
  - Nucleic Acids Symp Ser (Oxf) 52(1):155-156 (2008)
  A series of red emitting vinyl-triphenyamines (TP) have been synthesized and evaluated for their two photon absorption (2PA) properties. These compounds are virtually non fluorescent in the free state but exhibit a bright red fluorescence upon binding to doublestranded DNA, both in one- and two-photon absorption. This feature allows one- and two-photon confocal imaging in cells of nuclear DNA with an excellent contrast. Derivatizable analogues for covalent bioconjugation to oligonucleotides are described and variation on the structure is discussed.
• Role of Molecular Crowding in perturbing Quadruplex-Watson Crick Duplex Equilibrium.
  - Nucleic Acids Symp Ser (Oxf) 52(1):157-158 (2008)
  Herein, we address the sensitivity of the competitive equilibria between hoogsteen bonded quadruplex structure and hydrogen bonded Watson-Crick duplex structure. We used osmolytes as molecular crowding agents to mimick intracellular milieu and analysed their effect on Quadruplex-Duplex transition. We used telomeric quadruplex 5'Fluorescein-d[(G3 TTA)3 G3] as a model system and performed extensive Fluorescence Resonance Eenergy Transfer analysis for duplex formation in absence and presence of different concentrations of osmolytes (Glycerol and Ethylene Glycol). Overall the data shows that these molecular crowding agents stabilize quadruplex structure, delays duplex formation and thereby shifts the equilibrium towards quadruplex formation.
• Protein hnRNPA1 binds to a critical G-rich element of KRAS and unwinds G-quadruplex structures: implications in transcription
  - Nucleic Acids Symp Ser (Oxf) 52(1):159-160 (2008)
  The promoter of the KRAS proto-oncogene contains a critical nuclease hypersensitive element (NHE) forming G-quadruplex structures that are recognized by nuclear proteins: PARP-1, Ku70 and hnRNPA1. Here we have studied the interaction between hnRNPA1 (and its derivative UP1) and the G-quadruplexes of KRAS by EMSA, FRET and CD experiments. FRET and CD showed that hnRNPA1/UP1 is able to unfold the G-quadruplexes of KRAS and facilitate the quadruplex to duplex transformation. This finding strengthens our previous hypothesis that the transcription regulation of KRAS is mediated by G-quadruplex structures. Against this background we designed G4-decoy oligonucleotides specific for KRAS that exhibit a strong antiproliferative effect in pancreatic cancer cells.
• Structural stability analysis of the intermediates in the folding pathway of human telomeric hybrid-1 G-quadruplex based on fragment molecular orbital method
  - Nucleic Acids Symp Ser (Oxf) 52(1):161-162 (2008)
  The human telomeric DNA sequence d[AGGG(TTAGGG)3] has been found to form different type of G-quadruplex structure based on NMR1, X-ray crystallography2 and circular dichroism (CD). Recently human telomeric hybrid-1 G-quadruplex structure in K+ solution has been revealed by CD and NMR3,4,5. However, folding pathway of G-quadruplex structures is not clear to date. It is important to elucidate the intermediate structure of human telomeric hybrid-1 G-quadruplex for drug discovery in addition to having essential knowledge of telomere. In this study, we designed two types of triplex intermediate model from hybrid-1 NMR structure and evaluated their stabilities with ab initio Fragment Molecular Orbital (FMO) method6,7,8. The folding pathways of human telomeric hybrid-1 G-quadruplex structure are discussed.
• New platinum(II) complexes targeting the loops of the human telomeric G-quadruplex.
  - Nucleic Acids Symp Ser (Oxf) 52(1):163-164 (2008)
  Two novel series of platinum(II) complexes have been designed and shown to interact with the human telomeric G-quadruplex-DNA via different binding modes: i- terpyridine-platinum (Pt-tpy) complexes covalently interact with quadruplex-DNA via selective platination of adenine residues of the loops, their interaction being driven by the aromatic surface of the ligand; ii- platinum-quinacridine hybrid (Pt-MPQ) interacts with quadruplex-DNA via a dual noncovalent/covalent binding mode, targeting preferentially guanines constitutive of external G-quartets. Therefore, platinum complexes presented herein constitute potential agents for irreversible trapping of G-quadruplex. DNA.
• Ligand binding to tetra-end-linked (TGGGGT)4 G-quadruplexes: an electrospray mass spectroscopy study
  - Nucleic Acids Symp Ser (Oxf) 52(1):165-166 (2008)
  The binding properties of a series of known G-quadruplex ligands have been studied by ESI-MS experiments. The tetramolecular (TG4T)4 quadruplex and its analogues I and II blocked, respectively, at the 3' or 5'-end by a tetra-end-linker (TEL) unit were chosen as the ligands targets. The stoichiometries of the obtained complexes as well as the ligand affinity and selectivity to the different quadruplexes were determined to deduce the ligand binding site. The TEL derivatives I and II allowed the probing of the grooves contribution to the binding of ligands to G-quadruplexes, demonstrating that the 3' and 5' quartets are not equivalent binding sites for ligand end-stacking.
• A short C-rich PNA fragment capable to form novel G-quadruplex-PNA complexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):167-168 (2008)
  In this work we investigated the interaction between the short ac4a C-rich peptide nucleic acid (PNA) probe and two intramolecular G-quadruplex targets having the same G-tetrad core, but different folding topologies. The T(G4T)3G4T and the recently reported tetra-end-linked-(TG4T)4 G-rich oligonucleotides (GROs) were chosen and synthesized for this study. UV, CD, and MS experiments revealed the formation of novel 1:1 G-quadruplex-PNA complexes besides the expected DNA-PNA heteroduplexes.
• Human telomere RNA and DNA form an intermolecular G-quadruplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):169-170 (2008)
  For a long time, telomeres have been considered to be transcriptionally silent. Recently, Azzalin et al. demonstrated that telomeres are transcribed into telomeric repeat-containing RNA (TERRA) in mammalian cells. The telomere RNA was found to localize at the telomere DNA. These findings raise a possibility of that telomere RNA may be involved in an intermolecular G-quadruplex with the telomere DNA. In the current studies, we found that human telomere RNA and telomere DNA sequence can form hybrid-type parallel G-quadruplex structure. These results provide valuable information to allow understanding of the roles of human telomeric RNA in chromosome ends regulation and protection.
• Orientation of ends of G-quadruplex structure investigated with end-extended oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):171-172 (2008)
  The human telomere terminus can adopt the structure of a G-quadruplex. This structure has become an attractive target for anticancer drugs, because it effectively inhibits telomerase activity. In this study, we investigated the orientation of both 5' and 3' ends of the stable G-quadruplex structure. To verify the orientation, we designed end-extended G-quadruplex forming oligonucleotides. We carried out gel electrophoresis and the NMR analysis and found that the ends of the stable G-quadruplex structure are located on opposite faces of each of the quadruplexes.
• Molecular docking study of binding of TMPyP4 to a bimolecular human telomeric G-quadruplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):173-174 (2008)
  We carried out molecular docking simulation of binding of cationic porphyrin TMPyP4 to a bimolecular human telomeric G-quadruplex using DOCK6.1 to examine whether or not to reproduce the loop binding mode of TMPyP4 in the crystal structure. The simulation gave the two highest-ranked docking poses of TMPyP4, and the binding modes were external stacking on the terminal guanine-tetrad and groove binding.
• Human Telomeric RNA in G-quadruplex Structure
  - Nucleic Acids Symp Ser (Oxf) 52(1):175-176 (2008)
  Very recently, a breaking finding from two groups demonstrated that telomere DNA is transcribed into telomeric repeat-containing RNA in mammalian cells. Telomeric RNA, a newly appeared player in telomere biology, may be a key component of telomere machinery. However, structure and function of the telomeric RNA in chromosome ends have not yet been elucidated. In the current studies, we found that the human telomeric RNA sequence can form parallel G-quadruplex structure in the presence of K+ or Na+ ions. These results provide valuable information to allow understanding of the roles of human telomeric RNA in chromosome ends regulation and protection.
• Studies on the influence of inversion of polarity sites on the dG residues glycosidic conformation in quadruplex structures
  - Nucleic Acids Symp Ser (Oxf) 52(1):177-178 (2008)
  Insights into the influence of inversion of polarity sites on the dG residues glycosidic conformation in quadruplexes is presented. The NMR studies concern modified oligodeoxynucleotides based on the quadruplex forming sequence TGGGT.
• Structural analysis of r(GGA)4 found in RNA aptamer for bovine prion protein
  - Nucleic Acids Symp Ser (Oxf) 52(1):179-180 (2008)
  RNA aptamers for bovine prion protein (bPrP) were obtained by in vitro selection. It was found that the r(GGA) triplet repeat was frequently present in these aptamers. We already reported that both DNA and RNA containing the GGA repeat form unique quadruplex structures. The unique structures may be utilized for the recognition of bPrP by these aptamers. One of these aptamers contains four continuous repeat of r(GGA), r(GGA)4. Here, we analyzed the structure of r(GGA)4 under physiological ionic conditions by NMR. It was revealed on the basis of characteristic NOEs that an r(GGA)4 strand forms the quadruplex composed of one G:G:G:G tetrad plane and one G(:A):G:G(:A):G hexad plane. Furthermore, two monomers form a dimeric structure in a tail-to-tail manner. The dimeric quadruplex structure of r(GGA)4 is similar to that of d(GGA)4, but the difference between two structures was also noticed.
• NMR structural study of DNA oligomers containing alkylene crosslinked cyclic 2'-deoxyuridylate dimers
  - Nucleic Acids Symp Ser (Oxf) 52(1):181-182 (2008)
  A 2'-deoxyuridylate dimer cyclized via cross-linkage by ethylene (UetpU) or propylene (UprpU) linker was incorporated in DNA oligomer. Fluorescence resonance energy transfer (FRET) experiment showed that they bent at a sharp angle of approximately 90 degree. HMGB1 A-box protein, which selectively binds to bent DNA, binds to the UetpU DNA oligomer with high affinity, but not to the UprpU. In order to explain this difference, we have studied the solution structures of the UetpU and UprpU DNA oligomers using NMR. Most 1H signals except for 4', 5' and 5'' were assigned by 1H-1H two-dimensional NMR spectra and natural abundance 1H-13C HSQC spectra. Cross-peak patterns of 1H-1H NOESY spectra indicate that both oligomers have right-handed B-form DNA like structures and the cyclization in 2'-deoxyuridylates by alkylene crosslinking does not break Watson-Crick base pairs. Chemical shift differences between these two DNA oligomers are localized to the region of 2'-deoxyuridylate! dimer and its 3' side. These chemical shift differences and some characteristic NOE crosspeaks suggest the presence of the local structural differences in these regions between the UetpU and UprpU DNA oligomers.
• NMR assignments and the identification of the secondary structure of the anti-retroviral cytidine deaminase
  - Nucleic Acids Symp Ser (Oxf) 52(1):183-184 (2008)
  APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) is known to have a role in intrinsic cellular immunity against human immunodeficiency virus type1 (HIV-1). The antiretroviral activity of APOBEC3G (APO3G) is associated with the hypermutation of viral DNA through cytidine deamination. APO3G contains two cytidine deaminase domains that are characterised by highly conserved zinc-coordinating motif. It is known that only the C-terminal domain of APO3G (c-APO3G) has the catalytic activity. To shed light on the molecular mechanism of action by which APO3G inactivates HIV-1, we have undertaken the structural and binding studies by NMR. Here, we show the achievement of backbone assignments of c-APO3G and the identification of the secondary structure deduced from chemical shift index (CSI) and NOE data.
• Inhibition of human papillomavirus replication by using artificial zinc-finger nucleases
  - Nucleic Acids Symp Ser (Oxf) 52(1):185-186 (2008)
  Recently, we have designed artificial zinc-finger proteins (AZPs) that prevent a viral replication protein, E2, of human papillomavirus type 18 (HPV-18) from binding to its replication origin and demonstrated that the gene-delivered AZPs inhibited HPV-18 DNA replication in mammalian cells. In the present study, we examined a new approach to inhibition of DNA virus replication by using an AZP-nuclease fusion. In transient replication assays for HPV-18, the genedelivered AZP-nuclease fusion reduced the viral DNA replication rate significantly. Moreover, it was demonstrated by ligation-mediated PCR that viral DNA regions close to the AZP-binding site were cleaved in the cells by the AZP-nuclease. Thus, our results demonstrate that AZP-nucleases have potentials to inhibit replication of any DNA viruses whose replication mechanisms remain unsolved.
• Modulation of endogenous VEGF-A expression under hypoxia by using artificial transcription factors
  - Nucleic Acids Symp Ser (Oxf) 52(1):187-188 (2008)
  The vascular endothelial growth factor A (VEGF-A) gene is an attractive therapeutic target because both activation and repression of the gene are useful for treatment or cure of many diseases related to abnormal angiogenesis. To modulate the endogenous gene expression artificially, we previously designed a six-finger AZP to recognize a 19-bp DNA in the VEGF-A gene, and fused the AZP with a nuclear localization signal and a repressor domain to generate an artificial transcription factor (ATF). Using the ATF, we demonstrated efficient modulation of the VEGF-A expression. In the present study, we evaluate the ability of the ATF to modulate the gene expression under hypoxic conditions. Enzyme-linked immunosorbent assays (ELISA) for VEGF-A protein in the culture medium revealed that the gene-delivered ATF also repressed the expression of the endogenous VEGF-A gene under hypoxia.
• Generation of plants resistant to tomato yellow leaf curl virus by using artificial zinc-finger proteins
  - Nucleic Acids Symp Ser (Oxf) 52(1):189-190 (2008)
  Previously, we designed an artificial zinc-finger protein (AZP) for blocking a replication protein (Rep) of beet severe curly top virus (BSCTV) from binding to its replication origin and demonstrated that transgenic Arabidopsis plants expressing the AZP are completely resistant to the virus infection. Here we applied the AZP technology to tomato yellow leaf curl virus (TYLCV) infective to an important agricultural crop, tomato. We designed and constructed an AZP binding to the direct repeat to block the TYLCV Rep binding. In gel shift assays, we confirmed that the designed AZP has a higher affinity to the replication origin than that of Rep and that the AZP effectively inhibited the Rep binding to its replication origin in vitro. The AZP gene was then introduced into a plant genome with the help of Agrobacterium tumefaciens to generate the transgenic plants. We will discuss properties of the AZP-transgenic plants against TYLCV infection.
• Synthesis of locked S-type and locked N-type uridine monomer units for incorporation in 2'-5' RNA:3'-5'RNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):191-192 (2008)
  To delineate the structural requirements of 2'-5' RNA:3'-5' RNA duplexes, we report the synthesis of N-type locked, S-type locked and 3'-fluoro-2'-phosphoramidite building blocks. The consequent incorporation of these three novel monomers into 2'-5' linked oligomers and their biophysical implications on the stability of the said duplexes will have explicit importance towards development into therapeutic oligomers. The intrinsic stability of 2'-5' phosphodiester linkage as opposed to 3'-5' linked oligomers will be an added advantage
• Structural analysis of Musashi-RNA complex on the basis of long-range structural information
  - Nucleic Acids Symp Ser (Oxf) 52(1):193-194 (2008)
  Musashi protein is supposed to be involved in the regulation of differentiation of neural stem cells. Musashi binds to 3' untranslated region of target mRNA and represses the translation of mRNA. Musashi has two tandem RNA-binding domains (RBDs), RBD1 and RBD2. Both RBDs cooperatively bind to the target mRNA. Here, we determined the structure of RBD1-RBD2 in complex with target RNA. First, the structures of two RBDs in the complex were determined on the basis of short-range distance restrains derived from NOEs. However, the relative position of two RBDs was not determined due to the lack of long-range distance restraints across two RBDs. In order to overcome the situation, we introduced the paramagnetic center into Musashi by attaching MTSL carrying the NO radical. The long-range distance restraints (ca. 20-40 A) between two RBDs were derived from paramagnetic relaxation enhancement (PRE) caused by the paramagnetic center. The relative position of two RBDs was successf! ully determined on the basis of these longrange distance restraints. The change in the relative position of two RBDs on binding to the target RNA was also detected by PRE. The determined structure of RBD1-RBD2 in the complex has suggested how Musashi recognizes its target mRNA.
• Construction of a stable functional ribonucleopeptide complex by the covalent linking method
  - Nucleic Acids Symp Ser (Oxf) 52(1):195-196 (2008)
  We describe here a novel strategy to create a stable functional ribonucleopeptide (RNP) complex by the covalent linking method. Adenosine-5'-triphosphate (ATP)-binding RNP receptors were selected from the RNP library by in vitro selection. The RNA subunit of RNP is utilized to construct a ligand-binding cavity, while the peptide subunit can be functionalized independently. By introducing a fluorophore at the N-terminus of the Rev peptide subunit, the ATP-binding RNP receptor is successfully converted to a noncovalent complex of ATP-responsive fluorescent RNP sensor. Such a noncovalent RNP sensor could be covalently linked by the tethering the RNA to the fluorophorelabeled peptide subunit to form a stable RNP sensor without losing the original function.
• The specific interaction between metal cation and mismatch base pair in duplex RNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):197-198 (2008)
  We have already found that mercury (II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4{degrees}C. We have also found that silver (I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4{degrees}C. In the present study, to examine whether the specific interaction between metal cation and mismatch base pair can be also formed in duplex RNA, we investigated the effect of the metal cation on the thermal stability of homoduplex and heteroduplex RNA. Addition of mercury (II) cation increased the melting temperature of heteroduplex RNA containing U:U mismatch base pair by about 6{degrees}C. The thermal stability of homoduplex RNA containing U:A or A:U perfectly matched base pair and heteroduplex RNA containing A:A mismatch bas! e pair was not significantly changed by the addition of mercury (II) cation. On the other hand, addition of silver (I) cation increased the melting temperature of heteroduplex RNA containing C:C mismatch base pair by about 4{degrees}C. The thermal stability of homoduplex RNA containing C:G or G:C perfectly matched base pair and heteroduplex RNA containing G:G mismatch base pair was not significantly changed by the addition of silver (I) cation. We conclude that the specific interaction between metal cation and mismatch base pair can be formed in duplex RNA as in the case of duplex DNA.
• Selective recognition of a tetra-amino-acid motif containing phosphorylated tyrosine residue by ribonucleopeptide
  - Nucleic Acids Symp Ser (Oxf) 52(1):199-200 (2008)
  We describe here a ribonucleopeptide (RNP) receptor targeting a tetra-amino-acid motif containing phosphotyrosine, GpYSR. GpYSR-binding RNP receptors were obtained from an RNA-based RNP library by in vitro selection. These receptors have a higher affinity than those of previously obtained pY-binding RNP receptors. One of these RNP receptors exhibited unique specificity to the target GpYSR peptide over other tetra-amino-acid peptides derived from the tyrosine-phosphorylation sites of native proteins. The GpYSR-binding RNP receptor discriminated not only the phosphorylated tyrosine residue, but also its surrounding three amino acid residues. Thus, RNP receptors could target a defined pY-containing amino-acid sequence by expanding the recognition surface within the ligand-binding pocket of RNP.
• Development of ribonucleopeptide-based fluorescent sensors for biologically active amines based on the stepwise molding strategy
  - Nucleic Acids Symp Ser (Oxf) 52(1):201-202 (2008)
  General strategy for the development of fluorescent biosensors as a tracer of key' molecule in the cellular system would provide important breakthroughs for ubiquitous applications in the field of diagnosis and pharmacology in addition to our understanding of cellular events. The sophisticated design of fluorescent biosensors based on the organic synthesis is one of the promising approaches, but this type of biosensors frequently fail to maintain their performance in the cellular environment despite of laborious protocols. Another procedure for simultaneous preparation of a wide variety of fluorescent biosensors for the optical monitoring of a target molecule represents an especially attractive alternative. In our continuous efforts, we have recently developed a conceptually new strategy for coincidental production of fluorescent biosensors with diverse functions based on a framework of ribonucleopeptide (RNP). RNP-based fluorescent sensors were fabricated with a comb! ination of in vitro selection method and a successive modification of the peptide of RNP with a fluorophore. Each RNP composed of a ligand-binding RNA subunit and a fluorophore-tagged peptide motif facilitated the fluorometric detection of biologically active amines with a unique binding affinity and an inherent fluorescent signal.
• Significance of the N-terminal Histidine-rich Region for the Function of the Human Toll-like Receptor 3 Ectodomain
  - Nucleic Acids Symp Ser (Oxf) 52(1):203-204 (2008)
  Toll-like receptors (TLRs) are an essential component of the innate immune response to microbial pathogens. TLR3 is localized in intracellular compartments such as endosomes and signals in response to virus-derived double-stranded RNA (dsRNA). TLR3 localization within endosomes is required for ligand recognition, suggesting that acidic pH is the driving force for TLR3 ligand binding. To clarify the pH-dependent binding mechanism of TLR3 at the structural level, we focused on 3 highly conserved histidine residues clustered at the N-terminal region of the TLR3 ectodomain (ECD): H39, H60 and H108. Mutagenesis of these residues showed that H39, H60, and H108 were essential for ligand-dependent TLR3 activation in a cell-based assay. Furthermore, dsRNA binding to the recombinant TLR3 ECD depended strongly on pH and dsRNA length, and was reduced by mutations of H39, H60, and H108, demonstrating that TLR3 signaling is initiated from the endosome through a pH-dependent binding ! mechanism, and that a second dsRNA binding site exists in the N-terminal region of the TLR3 ECD. We propose a novel model for the formation of TLR3 ECD dimers complexed with dsRNA that incorporates this second binding site.
• Isolation of RNA aptamers specific for the 3' X tail of HCV
  - Nucleic Acids Symp Ser (Oxf) 52(1):205-206 (2008)
  The 3' end of the HCV genome, designated as the 3' X tail, comprises an almost invariant 98-nucleotide sequence containing three highly conserved stem-loop structures (3' SL1, 3' SL2, and 3' SL3). Since these sequences are all critical for the initiation of negative-strand synthesis and essential for viral replication, they are attractive targets for novel anti-HCV drugs. To obtain effective RNA aptamers specific for the 3' X tail, and with the aim of developing novel inhibitors of HCV replication, we performed in vitro selection of aptamers with specificity for the 3' X tail. In vitro selection, namely SELEX (systematic evolution of ligands by exponential enrichment) is a useful strategy for isolating nucleic acid sequences from a randomized oligonucleotide pool that have a high affinity for a target molecule. After four selection cycles, a pool of the 3' X tail-specific RNA aptamers were obtained. This RNA pool included 39 clones that could be divided into three main! classes (cSL1, cSL2, and cSL3) which harbor complementary sequences to the apical loops of 3' SL1, 3' SL2, and 3' SL3, respectively. Biochemical analyses are in progress to evaluate whether these RNA aptamers have the potential to block HCV replication.
• Subsites for substrate recognition by bacterial ribonuclease P
  - Nucleic Acids Symp Ser (Oxf) 52(1):207-208 (2008)
  We have prepared series of shape variant RNAs of a tRNA precursor and analyzed the substrate shape recognition by bacterial ribonuclease P ribozyme and holoenzyme. The results showed the evidence for the presence of subsites for the recognition of the trna shape. We will discuss a new model for substrate recognition and the role of the protein component.
• Analysis of the interaction between selected RNA-binding peptides and a target RNA containing a bulge and a GNRA-type tetraloop
  - Nucleic Acids Symp Ser (Oxf) 52(1):209-210 (2008)
  We have characterized the interaction between selected novel RNA-binding peptides and their target RNA. The RNA is comprised of two elements, a GCAA tetraloop, a member of the thermodynamically stable GNRA-type (where N is A or G, U, C; R is G or A) tetraloops, and a tri-purine bulge found in the frameshift stimutating structure on the human immunodeficiency virus type 1 (HIV-1) gag-pol mRNA. Peptides that bind specifically to the target RNA were selected from a combinatorial library based on arginine-rich motif (ARM) by a bacterial reporter system. We performed mutational studies using the reporter system and gel shift assays and found that the binding affinity and specificity of the RNA were mainly dependent on the GNRA-type tetraloop, and a modest contribution was also attributed to the bulge structure. Our finding reveals a novel mode of interaction by an RNA-peptide complex and expands our knowledge on the diversity of molecular recognition.
• Fluorescent Ligand as a molecular probe for the RNA structure
  - Nucleic Acids Symp Ser (Oxf) 52(1):211-212 (2008)
  In this report, we show the recognition of RNA secondary structure by exploiting a fluorescent 2,7-disubstituted 9H-xanthen-9-one derivative (2). The xanthone derivertive (2) especially bound to RNA and became non-fluorescent. Upon mixing with RRE model RNA, U-bulge RNA, or hairpin RNA, the fluorescence intensity of 2 was decreased in different intensity. The distinct fluorescent increase of 2 to each RNA seems to a good probe for The RNA structures.
• Identification of RNA Binding Specificity for the TET-family Proteins
  - Nucleic Acids Symp Ser (Oxf) 52(1):213-214 (2008)
  The TET-family proteins (TAF15, EWS and TLS) are the RNA binding proteins involved in multiple levels of cellular functions. The RNA binding domain of those proteins is known as the important region for cellular functions. But little is known about the RNA binding specificity of TET-family proteins. In order to investigate the RNA binding properties of the TET-family proteins, we performed electrophoretic mobility shift assay using recombinant Flag-tagged TLS and guanine-rich and RNAs. It was found that TLS binds to human telomeric RNA in the presence of KCl, but not in the presence of LiCl.
• Isoenergetic microarray mapping reveals differences in structure between tRNAiMet and tRNAmMet from Lupinus luteus
  - Nucleic Acids Symp Ser (Oxf) 52(1):215-216 (2008)
  Isoenergetic microarray mapping is shown to be an ideal method for probing subtle structural differences between initiator tRNAiMet and elongator tRNAmMet from Lupinus luteus. The differences in structure of both tRNAs cause significant dissimilarities in binding to microarrays probes.
• Interactions between antitumor drugs and vault RNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):217-218 (2008)
  It is supposed that ribonucleoprotein particle vault is involved in detoxification processes and thus is related to multidrug resistance. The vault is composed of three proteins and three vault RNAs, hvg-1, -2 and -3. The direct interactions between vault components and drugs were not reported. Recently, we revealed the interactions between vault RNAs and mitoxantrone. Here, we examined the interactions between hvg-2 and six antitumor drugs by a chemical shift perturbation method of NMR. It was found that in addition to mitoxantrone, hvg-2 can interact with two drugs basically in the same way using the same site. The difference in the affinity was also noticed among three drugs. Hvg-2 did not bind to the other three drugs. It is suggested that the common or closely related chemical structure of the positive three drugs is recognized by vault RNA.
• Isoenergetic microarray mapping - the advantages of this method in studying the structure of Saccharomyces cerevisiae tRNAPhe
  - Nucleic Acids Symp Ser (Oxf) 52(1):219-220 (2008)
  Isoenergetic microarrays were applied to study the structure of processed S. cerevisiae tRNAPhe and its unmodified transcript. Results of hybridization experiments demonstrate significant differences in binding of both RNAs. The microarray mapping approach provides similar structural information as traditional chemical or enzymatic mapping, but is superior in several ways.
• Crystal structures of RNA 3'-terminal phosphate cyclase and its complexes with Mg2++ATP, ATP or Mn2+
  - Nucleic Acids Symp Ser (Oxf) 52(1):221-222 (2008)
  RNA 3'-terminal phosphate cyclase (Rtc) is an enzyme related to RNA splicing, in which the 3'-terminal hydroxyl group of a truncated RNA is converted to the 2',3'-cyclic phosphate that is required prior to RNA ligation. This reaction may occur in the following two steps: (i) Rtc + ATP [->] Rtc-AMP + Ppi and (ii) RNA-N3' + Rtc-AMP [->] RNA-N>p + Rtc + AMP. In order to establish the reaction mechanism, Rtc of Sulfolobus tokodaii, overexpressed in E. coli, was crystallized in the following states, Rtc, Rtc-AMP, Rtc:AMP, Rtc:ATP and Rtc:Mn, and their crystal structures have been determined at 2.25, 2.25, 2.9, 2.4 and 3.2 A resolutions, respectively. Based on these structures, a possible reaction mechanism has been proposed.
• Lipophilic DNA-conjugates: DNA controlled assembly of liposomes
  - Nucleic Acids Symp Ser (Oxf) 52(1):223-224 (2008)
  DNA detection systems based on encoded solid particles have been reported but require often tedious and not generally applicable surface chemistry. In the present study a system comprised of a lipid-modified DNA probe sequence and unmodified DNA target sequences is used to non-covalently assemble liposomes. The process results in large liposome aggregates with dramatically different optical properties compared to individual liposomes in solution. The presented method enables fast and easy detection of target polynucleotides. Furthermore, the system displays remarkably sharp thermal transitions which enable detection of single nucleotide polymorphisms with greatly enhanced resolution compared to flourescence based methods.
• DNA Controlled Assembly of Soft Nanoparticles
  - Nucleic Acids Symp Ser (Oxf) 52(1):225-226 (2008)
  DNA-encoding of solid nanoparticles requires surface-chemistry, which is often tedious and not generally applicable. In the present study non-covalently attached DNA are used to assemble soft nanoparticles (liposomes) in solution. This process displays remarkably sharp thermal transitions from assembled to disassembled state for which reason this method allows easy and fast detection of polynucleotides (e.g. DNA or RNA), including single nucleotide polymorphisms as well as insertions and deletions.
• Poly (L-lysine)-graft-dextran acts as a nucleic acid chaperone for tetramolecular quadruplex formation
  - Nucleic Acids Symp Ser (Oxf) 52(1):227-228 (2008)
  DNA sequence 5'-TGGGGT-3'; TG4T takes an intermolecular quadruplex structure which composed of four TG4T strands associated together by four layers of G-quartets with parallel strand orientation. Interestingly, its association rate is considerably slow, hampering its application of DNA nanobiotechnology. We have reported that the cationic comb-type copolymer seemingly promoted the nucleation step of DNA hybridization to accelerate duplex and triplex formation. Then, we presumed that the copolymer also influenced kinetics of the quadruplex formation. In this study, the effect of the copolymer on the intermolecular quadruplex folding was investigated. The copolymer was found to accelerate both the association and dissociation of tetramolecular quadruplex. We concluded that PLL-g-Dex acts as a nucleic acid chaperone for tetramolecular quadruplex formation.
• New eximer-based tandem systems for SNP detection
  - Nucleic Acids Symp Ser (Oxf) 52(1):229-230 (2008)
  We report here the design and synthesis of new mono- and bis-pyrene-labeled oligo(2'-Omethylribonucleotide) tandems as perspective probes for SNP detection. The detection strategy is based on the eximer formation when two or more pyrene groups are brought into close proximity upon hybridization of the tandem components with DNA. The potential of SNP detection with tandems of pyrene-labeled oligo(2'-O-methylribonucleotides) by duplex melting curve analysis based on excimer fluorescence registration was demonstrated.
• Design of a fluorescent probe for DNA/RNA imaging
  - Nucleic Acids Symp Ser (Oxf) 52(1):231-232 (2008)
  We report new on-off fluorescent DNA probes. We used the fluorescence quenching by the exciton coupling effect of thiazole orange dyes to achieve turning off of fluorescence. Aggregation of dyes tethered to a single nucleotide in DNA dramatically changes the photophysical properties of the dyes, as demonstrated by the large spectral shifts relative to the absorption of the monomeric dyes binding to DNA. The photophysically designed DNA showed strong emission when it hybridized with the target strand, whereas the emission was suppressed strongly in the single-stranded state. The present approach using an excitonic interaction exhibits a quenching ability that is high enough to work as an on-off probe, and also includes many advantages quite different from conventional assays.
• Studies of oligodeoxyfluorosides (ODFs) as FRET probes for DNA hybridization
  - Nucleic Acids Symp Ser (Oxf) 52(1):233-234 (2008)
  Oligodeoxyfluorosides (ODFs) are a novel system of stacked, electronically interacting fluorophores built on the DNA scaffold. Here we describe early studies of these ODFs as potential universal FRET donors and as reporters of DNA hybridization.
• Sequence-selective fluorophore formation by furan-conjugated oligodeoxyribonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):235-236 (2008)
  We developed a mutation diagnosis system using a 1,N'-ethenoadenosine forming reaction. Furanderivatized oligodeoxyribonucleotides were synthesized and fluorescence properties were studied in the presence of complimentary strand under oxidative conditions. Strong emission at 430 nm was observed in the presence of A allele ODN.
• Application of partially double-stranded DNA probes to high-throughput SNPs genotyping
  - Nucleic Acids Symp Ser (Oxf) 52(1):237-238 (2008)
  We have focused on DNA strand exchange reaction (SER) with partially double-stranded (PDS) probes as a novel platform of SNPs genotyping. In this report, we elaborated the method for reliable and high-throughput SNPs typing. Competitive reactions using a couple of PDS probes designed for wild and mutant sequences significantly increase resolution efficiency to SNP types that is hardly discriminated by a non-competitive reaction with a single probe. Integration of the PDS probes with DNA microarray technology enable us to increase throughput efficacy. Simple and quick typing of fifteen SNPs with reliability as high as a ligase-based assay was demonstrated.
• Synthesis of a naphthalene diimide derivative having four ferrocene moieties as an electrochemical DNA hybridization indicator
  - Nucleic Acids Symp Ser (Oxf) 52(1):239-240 (2008)
  A new naphthalene diimide derivative carrying four ferrocene moieties, F4ND, was synthesized by the reaction of trimethylammoniummethylferrocene with the terminal amino moieties of two imide substituents of naphthalene diimide. Spectrophotometric binding studies of F4ND with calf thymus DNA showed its high affinity of 2 x 105 M-1 in 10 mM MES buffer and 1 mM EDTA (pH 6.25) containing 0.10 M NaCl. The Osteryoung square wave voltammetry (SWV) of a DNA probe-immobilized electrode before and after hybridization with target DNA gave a larger peak current in an electrolyte containing F4ND than that containing FND carrying two ferrocene moieties. The current peak difference between mismatched and fully matched DNA was also greater with F4ND than that with FND.
• Synthesis of a perylene diimide derivative having two ferrocene moieties as an electrochemical indicator for human telomeric DNA tetraplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):241-242 (2008)
  A water-soluble perylene diimide derivative carrying two ferrocene moieties, FPD, was synthesized in two steps as an electrochemical indicator for human telomeric DNA tetraplex. Spectrophotometric binding studies of FPD with AG3(T2AG3)3 revealed its specific binding to tetraplex DNA in 10 mM MES and 1 mM EDTA (pH 6.25) containing 0.10 M KCl. Cyclic voltammograms of AAT CCG TCG AGC AGA G(T2AG3)4 in an electrolyte containing 20 mM FPN showed single oxidation and reduction peaks based on the ferrocene moieties. The peak current for the oxidation process increased linearly with the scan rate, suggesting the effective concentration of FPN on this electrode.
• Synthetic Biology for Improved Personalized Medicine
  - Nucleic Acids Symp Ser (Oxf) 52(1):243-244 (2008)
  Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than $10,000 (as opposed to $100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and th! e Westheimer Institute have developed several of these, which will be the topic of this talk..
• Stability and Mismatch Discrimination of DNA duplexes containing 2,6-Diaminopurine and 2-Thiothymidine Locked Nucleic Acid bases
  - Nucleic Acids Symp Ser (Oxf) 52(1):245-246 (2008)
  Hybridization thermodynamics measured by differential scanning calorimetry (DSC) and UV spectroscopy (UVM) are reported for 8- and 14-mer oligonucleotides containing two new LNA bases: 2,6 diaminopurine (D) and 2-thiothymidine (2sT). Oligonucleotides containing D or 2sT bases are shown to have enhanced stability and improved discrimination for several of the possible mismatched base pairs.
• Sensitive DNA probe for photochemical ligation prepared in click chemistry
  - Nucleic Acids Symp Ser (Oxf) 52(1):247-248 (2008)
  We report a new photosensitive probe, that takes advantage of the electronic structural changes associated with the triazole ring formed in the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition. We succeeded in performing photoligation on the second time scale. Huisgen cycloaddition are employed to various labeling. As an example, we report a procedure for preparing the DNA probe for quick SNP typing.
• SPR-imaging based assays on an oligonucleotide-array to analyze DNA lesions recognition and excision by repair proteins
  - Nucleic Acids Symp Ser (Oxf) 52(1):249-250 (2008)
  An original oligonucleotide-array, coupled with SPR-imaging detection, has been developed to study biological interactions between DNA base lesions and DNA repair enzymes. This bioanalytical tool constitutes an efficient screening platform to quantify DNA repair activities and to search for new DNA repair inhibitors.
• Ion-Exchange Capture of Labeled Pyrrolidinyl Peptide Nucleic Acids for DNA Sequence Determination
  - Nucleic Acids Symp Ser (Oxf) 52(1):251-252 (2008)
  Peptide nucleic acid (PNA) is a new class of DNA mimic, which is potentially useful for diagnostic and therapeutic applications due to its high affinity and specificity of binding to target nucleic acids. The goal of this work is to develop a simple and sensitive fluorimetric-based detection of DNA sequences using new fluorescent labeled PNA probes previously developed in our laboratory. The method relies on the preferential absorption of PNA{middle dot}DNA hybrid (negatively charged) over unhybridized PNA (neutral) on an anion exchange solid support. This enables detection of the PNA-nucleic acid interactions, which will take place only when the PNA and DNA sequences are fully complementary directly on the solid support by fluorescence microscopy. With the high specificity of this new PNA-based technology, single mismatches in 9-13 bases target DNA sequences were readily distinguished. Based on this principle, a prototype for detection of single nucleotide polymorphis! ms (SNP) without the need for labeling the DNA substrates has been developed.
• New substituted aryldiazomethyl labels for high density DNA Chipbased nucleic acid testing
  - Nucleic Acids Symp Ser (Oxf) 52(1):253-254 (2008)
  DNA and RNA labeling and detection are key steps in nucleic acid testing, particularly for molecular diagnostics applications. Here, we report a new class of aryldiazomethyl labels that include in their structure a biotin moiety as a detectable unit and a nitro substituted phenyl diazomethyl as a reactive group. The greatest reactivity towards phosphates of nucleic acids, the water solubility and the stability of these new molecules were demonstrated. These very important properties, which are the main requirements for nucleic acid labeling in aqueous conditions using automated protocols within integrated diagnostic devices, make them the perfect labeling tools for hybridization-based analysis, especially for high-density DNA chips.
• Synthesis and Properties of Oligonucleotides Containing Silylated Pyrene Derivatives
  - Nucleic Acids Symp Ser (Oxf) 52(1):255-256 (2008)
  Preparation and properties of the novel fluorescently labelled oligonucleotides, containing the silylated pyrene derivatives are reported. The silylated pyrene derivatives were introduced into both 5'-terminus and C-5 position of the deoxyuridine derivatives. The fluorescent spectra indicated the possibility of the discrimination of the duplex formation by the simple denaturation.
• Enhanced amplification of polymerase chain reaction by addition of cosolutes derived from a cellular compatible solute
  - Nucleic Acids Symp Ser (Oxf) 52(1):257-258 (2008)
  We designed and synthesized artificial compatible solutes which can not only decrease the melting temperature of DNA duplexes dependent of their chemical structures but also improve the amplification of highly stable genome DNA sequence.
• Heat Activatable 3'-modified dNTPs: Synthesis and Application for Hot Start PCR
  - Nucleic Acids Symp Ser (Oxf) 52(1):259-260 (2008)
  Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95{degrees}C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.
• Synthesis of novel fluorophores for labelling of oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):261-262 (2008)
  Some novel fluorophores, viz. 2-(4-Carboxymethyl-naphthalene-1-carbonyl)-benzoic acid (1), 7, 12 -Dioxo-7, 12 dihydrobenzo[a] anthracene 5-yl]-acetic acid (2), [7,12-Dibenzylidine-7,12-Dihydro-benzo[a] anthracene-5-yl]-acetic acid (3), 2-[1-(4-carboxy methyl naphthalene-1-yl)-2-phenylvinyl]-benzoic acid (4) and (12-benzylidine-7-oxo-7, 12-dihydro-benzo[a] anthracene-5-yl)-acetic acid (5) have been designed and synthesized. These fluorophores have shown very good fluorescence signals in UV-visible region.
• Thymidine analogs as potential antiviral agents
  - Nucleic Acids Symp Ser (Oxf) 52(1):263-264 (2008)
  A dideoxythymidine analog, viz. 3'-N, N-dimethyl-2'-3'-dideoxythymidine (3) and its 5'-O-carboxyl ester prodrug derivatives (4-6) have been synthesized as probable antiviral agents. All these compounds have shown low to moderate level of antiviral activities against HIV, HSV 1 & 2, reovirus and many others.
• Design and synthesis of novel oxindoles as potential non-nucleosidic reverse transcriptase inhibitors against HIV
  - Nucleic Acids Symp Ser (Oxf) 52(1):265-266 (2008)
  We describe here the synthesis of novel oxindoles by cycloproponation of its diazo derivatives. The synthesis involves the use of allyl methyl sulfide and allyl alcohol as olefins and rhodium acetate as catalyst. These oxindoles have been designed to act as non-nucleosidic reverse transcriptase inhibitors (NNRTIs) against HIV.
• Synthesis and structural analysis of [4.3.0]-bicylcothymidine
  - Nucleic Acids Symp Ser (Oxf) 52(1):267-268 (2008)
  In the last decade our laboratory has synthesized and characterized the analogue tricyclo-DNA (tc-DNA) which shows promising properties as antisense agent. As part of ongoing work to establish a structure/affinity relationship of the bicyclo-DNA scaffold the synthesis of [4.3.0]-bicyclo-DNA was undertaken. Here we present an expedient synthesis and an X-ray structure of the corresponding thymidyl monomer.
• Nucleosides with 1,4-dioxane as sugar moiety
  - Nucleic Acids Symp Ser (Oxf) 52(1):269-270 (2008)
  A synthetic route towards novel nucleosides with 1,4- dioxane as the sugar moiety has been developed. The dioxane moiety features a second anomeric center, which has been phosphitylated giving a diastereomeric mixture of the corresponding phosphoramidites.
• New conformationally restricted DNA mimics
  - Nucleic Acids Symp Ser (Oxf) 52(1):271-272 (2008)
  Two novel bicyclic nucleotide monomers have been developed for use as DNA mimics. Melting temperature studies showed that these modifications decrease binding affinity towards complementary DNA and RNA.
• Greener Biocatalytic Approach to the Synthesis of Nucleosides and their Precursors
  - Nucleic Acids Symp Ser (Oxf) 52(1):273-274 (2008)
  Novel, efficient and selective biocatalytic acylation / deacylation strategies have been used for the greener synthesis of precursors of different bicyclonucleosides. Biocatalytic methodology has also been developed for the separation of pyrano- and furanonucleosides, which is otherwise almost impossible to achieve by usual chemical approach.
• Synthesis of double-headed nucleosides with the additional nucleobase in the 5'-C-position
  - Nucleic Acids Symp Ser (Oxf) 52(1):275-276 (2008)
  Three analogues of double-headed nucleosides with the additional nucleobase in the 5'(S)-C-position have been synthesised. A thymine has been attached through a methylene or an ethylene linker and an adenine through a methylene linker. Thermal hybridisation studies indicate that this 5' (S)-C-position is ideal for placing the additional base in the minor groove and obtain specific interstrand stacking effects in a (-3)-zipper arrangement. However, this specific interaction is decreasing when elongating the linker from a methylene to an ethylene linker. The successful syntheses of the two nucleoside analogues with thymine in the 5'-C-position as well as different approaches towards a corresponding adenine analogue are reported.
• Efficient Preparation of xanthine-containing oligodeoxynucleotide from oxanine-containing oligodeoxynucleotide, catalyzed by N{alpha}-acetyl-L-histidine
  - Nucleic Acids Symp Ser (Oxf) 52(1):277-278 (2008)
  Xanthine (Xan) and oxanine (Oxa) are major damage products from guanine (Gua) by NO- or HNO2-induced nitrosative deamination. Xan- and Oxa-containing oligodeoxynucleotides are essential substrates for the biochemical studies to reveal genotoxicity or mutagenesis raised by nitrosative oxidation. In previous study, we have developed chemical synthesis method for obtaining Oxa-ODN. Here, we proposed an efficient preparation method of Xan-ODN by incubating of Oxa-ODN in N{alpha}-acetyl-L-histidine (Ac-His) for 3 days at mild condition.
• Development of a novel selective detection method for 5-formyl-2'-deoxyuridine by fluorescent derivatization
  - Nucleic Acids Symp Ser (Oxf) 52(1):279-280 (2008)
  5-Formyl-2'-deoxyuridine (fdUrd) is derived from the oxidation of thymidine and is known to induce mutation (A:T to G:C) in DNA. We planned to develop a novel selective detection method for fdUrd in oligodeoxynucleotide (ODN) based on specific fluorescent derivatization of fdUrd using 2-aminothiophenol derivatives (ATs) as fluorogenic reagents. To achieve this goal, we first synthesized 5- (benzothiazol-2-yl)-2'-deoxyuridine derivatives (btdUrds) via the reaction between fdUrd and 5- substituted ATs. We report herein the synthesis and UV absorbance and fluorescence properties of btdUrds in different pH conditions.
• Synthesis of 3'-deoxyapionucleoside triphosphates and their incorporation into DNA by DNA polymerase
  - Nucleic Acids Symp Ser (Oxf) 52(1):281-282 (2008)
  Here, we report the synthesis of 3'-deoxyapionucleoside 3''-triphosphates (apioNTPs) and the analysis of their property as substrate of enzymatic polymerization. We established the large scale synthetic route of 3-deoxy-D-apiose from D-galactose, and regio- and stereo-selective glycosylation procedures. Resulting 3'-deoxyapionucleosides were then converted into their 3''-triphosphates. We carried out primer-extension reactions and found that Therminator polymerase, a variant of 9 {degrees}N DNA polymerase, was an efficient enzyme for incorporation of apioNTPs to synthesize DNA-dependent 3'-deoxy-D-apiose nucleic acids (apioNAs).
• Use of DNA and Click chemistries to synthesize combinatorial libraries of galactosyl-phosphodiester clusters
  - Nucleic Acids Symp Ser (Oxf) 52(1):283-284 (2008)
  Phosphotriester polyalkyne scaffolds bearing two to four alkynes were synthesized by DNA phosphoramidite chemistry. Then two galactosyl azide derivatives exhibiting different linkers were conjugated thanks to a Copper catalyzed Alkyne Azide 1,3-dipola cycloaddition (CuAAC) "click reaction". This reaction was performed on solid support assisted by microwaves. Thus, small libraries of 4, 8 and 16 galactosyl-clusters were obtained.
• Synthesis of nucleoside phosphorothio-, phosphorodithio- and phosphoroselenoate diesters via oxidative esterification of the corresponding H-phosphonate analogues
  - Nucleic Acids Symp Ser (Oxf) 52(1):285-286 (2008)
  It was found that nucleoside H-phosphonothioate, H-phosphonodithioate, and H-phosphonoselenoate monoetsers in the presence of iodine underwent rapid oxidative esterification with various hydroxylic components to produce in "one pot" reactions the corresponding nucleoside phosphorothioate, nucleoside phosphorodithioate and nucleoside phosphoroselenoate diesters in good yields. The transformation represents a novel alternative method for the synthesis of internucleotide phosphate bridge, bearing sulfur or selenium atoms. Mechanistic studies have revealed metaphosphate analogues as the probable intermediates, involved in the reactions.
• Synthesis and biochemical studies of tetraphosphate 5' mRNA cap analogs bearing bisphosphonate modification
  - Nucleic Acids Symp Ser (Oxf) 52(1):287-288 (2008)
  Dinucleotide cap analogs containing tetraphosphate bridge modified by replacing one of the bridging oxygen atoms with methylene group were synthesized. The analogs have been examined for their stability towards enzymatic hydrolysis by human DcpS and binding affinity for eIF4E. Their inhibitory properties in rabbit reticulocyte lysate (RRL) translational system were studied.
• The first examples of mRNA cap analogs bearing boranophosphate modification
  - Nucleic Acids Symp Ser (Oxf) 52(1):289-290 (2008)
  The syntheses of mRNA cap analogs modified with boranophosphate moiety at either the {alpha} or {beta}-position of the 5', 5'-triphosphate bridge (m7GpppBH3G, m7GppBH3pG and m7GppBH3pm7G) are described. The preliminary biological characterization of these compounds revealed that they have high affinity for translational factor eIF4E and high potency to inhibit cap-dependent translation in cell free system. The analogs modified at the {beta}-position were also found to be resistant to DcpS decapping pyrophosphatase.
• m7GTP{alpha}S is a strong and stable inhibitor of cap-dependent translation
  - Nucleic Acids Symp Ser (Oxf) 52(1):291-292 (2008)
  Two diastereomers of 7-methylguanosine 5'-O-(1-thiotriphosphate) have been synthesized and resolved by RP HPLC. Preliminary studies revealed that these new analogs of mRNA cap are characterized by high affinity for eIF4E, resistance towards DcpS pyrophosphatase and high potency to inhibit cap-dependent translation.
• Synthesis of a Photoresponsive {alpha}-Dideoxyuridine Triphosphate Derivative
  - Nucleic Acids Symp Ser (Oxf) 52(1):293-294 (2008)
  The synthesis of a photoresponsive {alpha}-5-cyanovinyl- 2',3'-dideoxyuridine-5'-triphosphate ({alpha}ddCUTP) is described. A one-pot enzymatic synthesis of photoresponsive {alpha}-5-cyanovinyl-2',3'-dideoxyuridine ({alpha}ddCU) labeled ODNs with terminal deoxynucleotideyl transferase (TdT) would be a powerful tool for the development of modified long-chain DNA and branched DNA unable to be prepared by DNA synthesizer or enzymatic ligation.
• Bisphosphonate mRNA cap analog attached to Sepharose for affinity chromatography of decapping enzymes
  - Nucleic Acids Symp Ser (Oxf) 52(1):295-296 (2008)
  m7GTP-Sepharose is routinely used for cap binding protein isolation. Here we present the synthesis of a new affinity resin containing a mononucleotide cap analog resistant to hydrolysis by DcpS. The resin has been designed in order to identify and purify Arabidopsis thaliana DcpS and other pyrophosphatases. The binding efficiency of the new resin to eIF4E protein was compared with standard m7GTP-Sepharose. The utility of non-hydrolysable resin was demonstrated on yeast extract.
• Chemical Models and Their Mechanistic Implications for the Transformation of 6-Cyanouridine 5'-Monophosphate Catalyzed by Orotidine 5'-Monophosphate Decarboxylase
  - Nucleic Acids Symp Ser (Oxf) 52(1):297-298 (2008)
  Orotidine 5'-monophosphate decarboxylase (ODCase) catalyzes an unprecedented transformation of 6- cyanouridine 5'-monophosphate (6-CN-UMP) into barbiturate nucleoside 5'-monophosphate (6-hydroxyuridine 5'-monophosphate, BMP). The reactions of 6- cyano-1,3-dimethyluracil toward various nucleophilic conditions have been studied as chemical models in order to understand the possible mechanism for the ODCase-catalyzed transformation of 6-CN-UMP.
• A versatile synthetic approach for the development of libraries of 5', 3'-bis-conjugated oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):299-300 (2008)
  A versatile approach to develop libraries of diverse 5',3'-bis-conjugated oligonucleotides (ODNs) is here described. The usage of ad hoc derivatized solid supports, to which the first nucleoside unit is attached through a phosphate linkage, opens easy synthetic access to a large variety of hybrid bis-conjugated oligomers. The G-quadruplex forming d(5'TGGGAG3') sequence, as a potential anti-HIV agent, has been here used as a model system.
• Synthesis and properties of oligonucleotides having a chemically stable 2-(trimethylsilyl)benzoyl group
  - Nucleic Acids Symp Ser (Oxf) 52(1):301-302 (2008)
  Acyl groups such as benzoyl and phenoxyacetyl have been used as a fundamenmtal tool for protection of reactive hydroxyl and amino groups in chemical synthesis of DNA and RNA oligomers. It is well known that such protecting groups can be easily cleaved under basic conditions. Here we report 2-(trimethylsilyl)benzoyl (TMSBz), i.e., a chemically stablized benzoyl group that is substituted with a trimethylsilyl group at its 2-position. To the best of our knowledge, the TMSBz group is the most resistant to basic conditions as an acyl group in nucleic acid chemistry.
• Synthesis of a new internucleosidic linkage: the Borononucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):303-304 (2008)
  The synthesis of a borononucleotide analogue of thymidine and its association with various nucleosides, nucleotides and saccharides is described.
• Design and Chemical Synthesis of a 2',4'-BNA Probe for 7,8-Dihydro-8-oxoguanine-containing Double Stranded DNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):305-306 (2008)
  7,8-Dihydro-8-oxoguanine (8-OxoG), caused by DNA oxidation, is considered to be a marker of oxidative stress, and also to be a cause of genomic diversity in organism because of its mutagenic potential. Here, we designed and synthesized a 2',4'-BNA probe for 8-OxoG-containing double stranded DNA (dsDNA). The designed probe showed an 8-OxoG:C base pair selective triplex-forming ability.
• Synthesis of C8-N-Acetylarylamine 2'-dG-adducts and their sitespecifically incorporation into oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):307-308 (2008)
  Beside the predominately found C8-NH-arylaminedG damages also C8-N-acetylarylamine-lesions of 2'-dG seems to play an important role in the induction of the chemical carcinogenesis. A new synthetic pathway leading to these DNA-adducts using different aromatic amines have been developed. The C8-dG-adducts were converted into the corresponding phosphoramidites and incorporated site-specifically into oligonucleotides giving damaged DNA-strands. With damaged DNA hybrids studies with respect to their thermal stability (UV melting temperature analysis) and circular dichroism were performed.
• Synthesis and properties of covalently linked antiparallel and parallel DNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):309-310 (2008)
  We synthesized covalently linked antiparallel stranded (aps) and parallel stranded (ps) DNA duplexes. These modified duplexes contained an ethylene-linked T-T base pair at the duplex end. Thermal denaturation analysis of these modified duplexes indicated that the aps and ps DNA duplexes have similar thermal stabilities.
• Some Observations on Detritylation in Solid-Phase Oligonucleotide Synthesis
  - Nucleic Acids Symp Ser (Oxf) 52(1):311-312 (2008)
  The quality of oligodeoxyribonucleotides prepared by solid phase synthesis under different acid treatment time was compared. Much shorter acid delivery time did not lead to significant decrease of the full length products.
• Foldamers derived from nucleoside beta-amino acids: a new twist on the DNA helix
  - Nucleic Acids Symp Ser (Oxf) 52(1):313-314 (2008)
  Peptides derived from a thymidine beta-amino acid have been prepared by solid-phase synthesis and their conformation investigated by NMR. Interestingly, NMR and modelling studies indicate that the tetramer and octamer form an unusual 8-helical conformation. Studies are currently underway to investigate the synthesis of peptides derived from the other deoxyribonucleosides with the intention of examining the association between helices capable of nucleobasepairing.
• 2'-Methylseleno modified oligoribonucleotides synthesized for X-ray crystallography
  - Nucleic Acids Symp Ser (Oxf) 52(1):315-316 (2008)
  Site-specifically modified 2'-methylseleno RNA represents a valuable derivative for phasing of X-ray crystallographic data. Several successful applications in three-dimensional structure determination of nucleic acids, such as the Diels-Alder ribozyme,1 have relied on these modifications.2 Currently, preparation of 2'-methylseleno modified oligoribonucleotides is restricted to the 2'-O-[(triisopropylsilyl)oxy]methyl (TOM)3,4,5 and 2'-O-tert.-butyldimethylsilyl (TBDMS) RNA synthesis methods. Recently, we have introduced synthetic routes to 2'-methylseleno phosphoramidite building blocks of all four standard nucleosides, adenosine, uridine, guanosine, cytidine, that are tailored for the 2'-O-bis(acetoxyethoxy)methyl (ACE) RNA solid-phase synthesis method.6
• Chiral Phosphonate Internucleotide Linkage: A Promising Modification for Chimeric Oligonucleotides?
  - Nucleic Acids Symp Ser (Oxf) 52(1):317-318 (2008)
  We have synthesized two different groups of oligonucleotides containing chiral isopolar nonisosteric phosphonate internucleotide linkages, and studied their properties in combination with natural phosphodiester ones. The improved synthetic procedures for the monomers preparation are also reported.
• Incorporation of 3'-S-phosphorothiolates into RNA: potential applications in RNAi
  - Nucleic Acids Symp Ser (Oxf) 52(1):319-320 (2008)
  Using solid-phase synthesis, oligoribonucleotides containing multiple 3'-S-phosphorothiolate modifications have been successfully synthesized, purified and characterized, utilizing a 2'-deoxyuridine phosphorothioamidite monomer.
• Optimized Fluoride Ions Treatment for Release of Base-Sensitive 2'-O-Modified Oligoribonucleotides from Various Solid Supports
  - Nucleic Acids Symp Ser (Oxf) 52(1):321-322 (2008)
  To synthesize oligoribonucleotides containing basesensitive 2'-O-modifications, we used the Q-linker or a silyl linker anchored to CPG or polystyrene resin. A fluoride ions treatment was optimized to release oligoribonucleotides from the solid-support and to remove TBDMS groups without affecting 2'-O-acyloxymethyl groups.
• Chemical synthesis of RNA including 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine
  - Nucleic Acids Symp Ser (Oxf) 52(1):323-324 (2008)
  Recently, novel base-modified uridine derivatives, 5-taurinomethyluridine ({tau}m5U) and 5-taurinomethyl-2-thiouridine ({tau}m5s2U) have been discovered from mammalian mitochondrial tRNAs, and these modified ribonucleosides were found to exist at the first position of the anticodon. We herein report efficient reactions for the synthesis of RNA oligomers including these basemodified ribonucleosides.
• Development of new methods for the synthesis of RNA oligonucleotides having baselabile functional groups
  - Nucleic Acids Symp Ser (Oxf) 52(1):325-326 (2008)
  In the previous study, we developed a method for O-selective phosphorylation, i.e., "the activated phosphite method" for DNA synthesis without base protection. However, the O-selectivity was low in RNA synthesis when the activated phosphite method was used. In this paper, we developed two new methods for synthesis of RNA oligomers having base-labile functional groups on polymer supports. One is the N-unprotected phosphoramidite method involving P(III)-N bond cleavage. The selectivity of the phosphorylation in RNA synthesis increased to more than 99% by posttreatment of the undesired P(III)-N bonds with 6-nitoro-HOBt and was independent of the kind of protecting groups at the 2' position. Another method is the RNA synthesis involving deprotection of protecting groups of the nucleobases under acidic conditions. It was found that an N,N-dialkylaminomethlene (DAF) group could be easily removed by heat-induced deprotection using HOBt as an acidic promoter.
• Synthesis and properties of 2'-modified-4'-thioRNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):327-328 (2008)
  As a part of our ongoing research projects, we envisioned the synthesis of 2'-modified-4'-thioRNA, such as 2'-fluoro-4'-thioRNA (2'-F-4'-thioRNA) and 2'-O-methyl-4'-thioRNA (2'-OMe-4'-thioRNA) to enhance the potential of 4'-thioRNA. Appropriately protected 2'-deoxy-2'-fluoro-4'-thiouridine (6), -cytidine (10), and - adenosine (18), substrates for the synthesis of novel modified RNAs were successfully synthesized. The fullymodified RNA consisting of 2'-deoxy-2'-fluoro-4'-thionucleosides was synthesised, and we examined its abilities of hybridization and stability against nucleases. It was found that 2'-F-4'-thioRNA shows high hybridization ability to complementary RNA. Furthermore, 2'-F-4'-thioRNA has strong resistance toward degradation in 50% human plasma.
• Synthesis and properties of oligonucleotides containing 4'-selenoribonucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):329-330 (2008)
  We report herein the synthesis and properties of oligonucleotides (ONs) containing 4'-selenoribo nucleosides. 4'-Selenouridine derivative 1, prepared via the stereoselective seleno-Pummerer reaction, was converted into 4'-selenouridine 9 and -cytidine 10 phosphoramidites. The resulting 4'-selenouridine unit was stable under a standard phosphoramidite protocol including oxidation step and was able to be introduced in ONs (5'-UUUUUU-3', and 5'-UUUUUU-3'). This is the first example of ONs synthesis containing 4'-selenoribonucleosides.
• A convenient approach towards 2'-O-modified RNA-oligonucleotides on solid support using universal nucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):331-332 (2008)
  Here we report a novel synthetic pathway for an efficient modification of RNA-oligonucleotides on solid -support using universal nucleosides bearing a 2'-O-aminopropyl-tether.
• Stereocontrolled synthesis of backbone-modified oligonucleotides via diastereopure H-phosphonate intermediates
  - Nucleic Acids Symp Ser (Oxf) 52(1):333-334 (2008)
  Synthesis of stereoregulated backbone-modified oligodeoxyribonucleotides via the corresponding diastereopure H-phosphonate intermediates is described. The diastereopure H-phosphonate intermediates were synthesized by using the nucleoside 3'-oxazaphospholidine monomers, and were stereospecifically converted into a variety of P-stereoregulated backbone-modified oligonucleotides, such as phosphorothioates, boranophosphates, and phosphoramidates, on a solid-support.
• Stereocontrolled synthesis of oligonucleoside phosphorothioates and PO/PS-chimeric oligonucleotides by using oxazaphospholidine derivatives
  - Nucleic Acids Symp Ser (Oxf) 52(1):335-336 (2008)
  Solid-phase synthesis of oligonucleotides having a fully-modified stereoregular phosphorothioate backbone or a stereoregular phosphate/phosphorothioate chimeric backbone was achieved by using diastereopure nucleoside 3'-bicyclic oxazaphospholidine derivatives and {beta}-cyanoethyl phosphoramidites as monomer units and N-(cyanomethyl)pyrrolidinium trifluoromethanesulfonate (CMPT) as an acidic activator. The trans-isomers of the oxazaphospholidines were obtained exclusively by the reaction of the 3'-OH of appropriately protected nucleosides and the corresponding 2-chloro-1,3,2-oxazaphospholidine derivative. The trans-isomers were configurationally stable and did not epimerize almost at all even in the presence of CMPT. As a result, the formation of phosphorothioate internucleotide linkages using the oxazaphospholidine derivatives and CMPT proceeded without any loss of diastereopurity. In addition to the synthesis of stereoregular phosphorothioate linkages, the synthesis ! of phosphate internucleotide linkages through the same method was studied. As a result of the study, stereoregular phosphate/phosphorothioate chimeric oligonucleotides as well as stereoregular oligonucleoside phosphorothioates were efficiently synthesized by using the same method.
• Towards Fluorous-Phase Synthesis of Nucleoside {beta}-Peptides
  - Nucleic Acids Symp Ser (Oxf) 52(1):337-338 (2008)
  We have recently shown that peptides derived from nucleoside {beta}-amino acids adopt an unusual 8-helical conformation in solution1. These {beta}-peptide homooligomers were constructed using Fmoc solid-phase peptide synthesis protocols (SPPS); however, we found these procedures to be somewhat limiting in terms of scale and lacking a quantitative method of monitoring reactions. Preliminary investigations into the use of fluorous dendrons as an alternative to SPPS have been conducted and show that coupling reactions with modified nucleoside (1) and fluorous dendrimer (2) are possible in the presence of a glycine linker. Significantly, success of the coupling reactions could be monitored by NMR without loss of materials or termination of the synthetic sequence and this promises much for the future of both oligonucleotide and polypeptide synthesis.
• Translesion synthesis across the (6-4) photoproduct and its Dewar valence isomer by the Y-family and engineered DNA polymerases
  - Nucleic Acids Symp Ser (Oxf) 52(1):339-340 (2008)
  We analyzed the translesion synthesis across the UV-induced lesions, the (6-4) photoproduct and its Dewar valence isomer, by using human DNA polymerases {eta} and {iota} in vitro. The primer extension experiments revealed that pol {eta} tended to incorporate dG opposite the 3' component of both lesions, but the incorporation efficiency for the Dewar isomer was higher than that for the (6-4) photoproduct. On the other hand, pol {iota} was likely to incorporate dA opposite the 3' components of the (6-4) photoproduct and its Dewar isomer with a similar efficiency. Elongation after the incorporation opposite the UV lesions was not observed for these Y-family polymerases. We further analyzed the bypass ability of an engineered polymerase developed from Thermus DNA polymerase for the amplification of ancient DNA. This polymerase could bypass the Dewar isomer more efficiently than the (6-4) photoproduct.
• Thiophosphoramidites scale up and their use in the synthesis of phosphorodithioate linkages
  - Nucleic Acids Symp Ser (Oxf) 52(1):341 (2008)
  The replacement of two non-bridging oxygen atoms with sulfur atoms in the phosphodiester linkage of DNA creates a phosphorodithioate (PS2) linkage (1). Like natural DNA, it is achiral at phosphorus. Additionally, recent research has demonstrated that this analog is completely resistant to nuclease degradation (2). Moreover, PS2-ODNs bind proteins with a higher affinity than their phosphodiester analogues (3,4) suggesting that PS2-ODNs may have additional utility in the form of sulfur-modified phosphate ester aptamers (thioaptamers) (4-7) in therapeutic and diagnostic applications. The biological interest and promise of the PS2-ODN has spawned a variety of strategies for synthesizing, isolating, characterizing and purifying these compounds over the past two decades. However, the building blocks of the PS2-ODN, thiophosphoramidites, are only now commercially available after recent successful developments at AM Biotechnologies (www.thioaptamer.com). This presentation, wil! l address (a) thiophosphoramidite production scale-up, (b) screening of suitable activators for effectively catalyzing the thiophosphoramidites, (c) screening of sulfurizing reagents for effective synthesis of PS2-ODN linkage(s), and (d) procedures for deprotecting PS2-ODNs. Finally, a novel bead-based thioaptamer selection procedure will be summarized that enables efficient selection of oligonucleotide affinity agents that have a limited number of PS2 linkages.
• Novel Advanced Universal Solid Supports for Oligonucleotide Synthesis
  - Nucleic Acids Symp Ser (Oxf) 52(1):343 (2008)
  A novel series of advanced universal solid supports for oligonucleotide synthesis that require no or very minor modifications to the standard protocols were developed. This permitted a more straightforward preparation of synthetic 2'-deoxy- and 2'-O-substituted oligonucleotides and their phosphorothioate analogs.
• Synthesis of four colors fluorescently labelled 3'-O-blocked nucleotides with fluoride cleavable blocking group and linker for array based Sequencing-by-Synthesis applications
  - Nucleic Acids Symp Ser (Oxf) 52(1):345-346 (2008)
  Reversible terminators having a fluoride cleavable 3'-O-blocking group are presented. Each nucleotide triphosphate is labelled by a fluorescent dye cleavable by the same reagent. We present here their synthesis, cleavage experiments and polymerase incorporation tests for a possible use in a process of Sequencing-by-Synthesis.
• DNA Glue: 1-, 2- and 4-Ethynylpyrenes in the Structure of Twisted Intercalating Nucleic Acids (TINAs), DNA Duplexes/Triplexes and Interstrand Excimer Formation
  - Nucleic Acids Symp Ser (Oxf) 52(1):347-348 (2008)
  Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo-pair in the middle of the duplex resulted in formation of an excimer band at 505 nm for both 1- and 4-ethynylpyrene analogues.
• BODIPY-modified uridines as potential fluorescent probes for nucleic acids that are recognized by DNA-polymerases
  - Nucleic Acids Symp Ser (Oxf) 52(1):349-350 (2008)
  A new type of BODIPY-modified uridines that contain the fluorophore attached to the 5-position of uridine via a short phenylene bridge have been prepared and characterized by methods of the optical spectroscopy and electrochemistry. The spectroscopic and redox properties can be manipulated by substituent and ligand exchange. After synthetic incorporation into DNA via phosphoramidite chemistry the canonical base pairing is maintained due to the rigid phenylene bridge. Moreover, primer extension experiments show that BODIPY-modified uridines are still recognized as thymidine derivatives by DNA-polymerases and adenine is inserted as the correct counter base.
• Cholesterol-Linked Pyrene Excimer Molecular Beacon with Enhanced Cell Permeability
  - Nucleic Acids Symp Ser (Oxf) 52(1):351-352 (2008)
  Covelently labeled pyrene excimer molecular beacon (MB) with cholesterol moiety has been developed for enhanced the cellular delivery of MB.1 Pyrene units were covalently attached into adenosine and incorporated to oligonucleotides at the complementary locations in opposite strands in the middle positions of hairpin stems. The system behaves as an effective MB that changes color from green to blue upon duplex formation. A cholesterol unit was also attached into a free terminus of one of these hairpins. The cholesterol-linked MBs enhanced the cellular delivery of the MBs and showed similar cell permeability to conventional transfection methods. These structurally simple cholesterol-based MB systems, which can be synthesized very efficiently, have good potential for opening up new and exciting opportunities in the field of in vivo biosensors.
• Fluorogenic probe triggered by reduction for nucleic acids sensing
  - Nucleic Acids Symp Ser (Oxf) 52(1):353-354 (2008)
  A reduction-triggered fluorescence probe with a new fluorogenic compound derivatized from rhodamine 110 was developed for sensing oligonucleotides. The chemistry to activate the compound involves the reaction between the azide group of rhodamine derivatives and reducing reagents, with the fluorescence signal appearing after reduction of the azide group. The reaction proceeds under biological conditions to produce fluorescence signal within 10-20 min in the presence of target DNA or RNA. The probes were successfully applied to the detection of oligonucleotides in solution and endogenous RNA in bacterial cells.
• Intracellular mRNA imaging with a hybridization sensitive fluorescent nucleotide
  - Nucleic Acids Symp Ser (Oxf) 52(1):355-356 (2008)
  We reported a technique for detecting intracellular RNA with a newly developed functional probe. To detect mRNAs, a polyA tail targeting probe was synthesized. This probe showed quite large enhancement in the presence of its complementary RNA. In living cultured cells, the probe also showed strong fluorescence. This technique was effective for intracellular mRNA detection.
• Fluorometric sensing of conformational switching of DNA; The use of fluorescence labeled C8-alkylamino substituted 2'-deoxyguanosine
  - Nucleic Acids Symp Ser (Oxf) 52(1):357-358 (2008)
  C8-alkylamino substituted 2'-deoxyguanosine was incorporated into a DNA sequence and then labeled with pyrene. The conformational change from B- to Z-form was monitored using CD and fluorescence spectroscopy for both labeled and unlabeled ODNs.
• Design of an efficient self-quenched molecular beacon for SNPs genotyping
  - Nucleic Acids Symp Ser (Oxf) 52(1):359-360 (2008)
  Quencher free molecular beacon with an excellent signal to noise ratio was designed and used for SNPs genotyping.
• Design of an ultimate quencher free molecular beacon containing pyrrolocytidine-guanine base pair
  - Nucleic Acids Symp Ser (Oxf) 52(1):361-362 (2008)
  A novel quencher free molecular beacon was designed in which fluorophore-labelled pyrrolocytidine was placed away from the stem terminal. This new type of MB was used for the detection of a target DNA with an excellent efficiency.
• Targeting specific gene by alkylating pyrrole-imidazole polyamides
  - Nucleic Acids Symp Ser (Oxf) 52(1):363-364 (2008)
  Targeting specific genes or gene products by small molecules is novel approach of cancer chemotherapy. We have developed sequence-specific DNA alkylating agents, conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. These compounds efficiently alkylate DNA at a targeted sequence and inhibit gene expression caused by alkylation at template strand of coding region. Recently, histone H4c gene could be targeted by polyamide-chlorambucil (Chl) conjugates. Thus, we designed and synthesized polyamide seco-CBI conjugates 1-5 targeting histone H4c coding sequence. High resolution denaturing polyacrylamide gel electrophoresis (PAGE) showed polyamide seco-CBI conjugates alkylated at the histone H4c coding sequence.
• Sequence-specific alkylation of DNA by pyrrole-imiclazole polyamides through cooperative interaction
  - Nucleic Acids Symp Ser (Oxf) 52(1):365-366 (2008)
  We designed and synthesized an alkylating N-methylpyrrole (Py) -N-methylimidazole (Im) polyamideand l-(chloromethyl)-5-hydroxy-l,2-dihydro-3H benz[e]indole (seco-CBI) conjugate 1. DNA alkylatingactivities of conjugate 1 were evaluated by high-resolution denaturing polyacrylamide gelelectrophoresis with DNA fragment containing humantelomere repeat sequence. These results revealed thatsimultaneous treatment of the DNA fragment withconjugate 1 and Distamycin A (Dist) alkylate at the 5'-GGTTAGGGTTA-3' sequence effectively due tocooperative interaction. Moreover, this combination hasachieved 11-base-pair (bp) recognition. Thus, it issuggested that the utilization of one alkylating agentpossessing long Py-Im polyamide moiety and short Py-Im polyamide partner can be an expedient way to attainthe extension and precision of the recognition of DNAsequence.
• Site-specific modification by functionality-transfer oligonucleotide with the photo-inducible reactivity
  - Nucleic Acids Symp Ser (Oxf) 52(1):367-368 (2008)
  Previously, we reported that S-vinyl thioguanosine analogs exhibited the efficient functionality-transfer reaction with selectivity toward cytidine. This technique was applied to the inhibition and the labelling of DNA or RNA. In this study, we aimed at developing functionality-transfer oligonucleotide with the photo-inducible reactivity. This photo-induced transfer reaction proceeded only to dmC but not to dC and dT at the target site of the complementary ODN.
• Photochromic Nucleobase: Reversible Photoisomerization, Photochemical Properties and Photoregulation of hybridization
  - Nucleic Acids Symp Ser (Oxf) 52(1):369-370 (2008)
  We have developed a set of photochromic nucleobases (PCNs), which reversibly change the photochemical and physical properties such as fluorescence intensity upon cis-trans photoisomerization by the external light stimuli. PCNs showed very rapid and efficient reversible photoisomerization by illumination at specific wavelength. In addition, photoisomerization can be iteratively performed by alternate illumination with 250[~]350 nm and 350[~]500 nm light without any side reactions. We demonstrated a new type of the photoregulation of hybridization using this switching property.
• Synthesis and incorporation of fluorescent C-nucleoside analogue into oligodeoxyribonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):371-372 (2008)
  Fluorescent nucleoside analogues were synthesized and derived to protected nucleoside phosphoramidite. The nucleoside analogue bearing 3-aminobenzonitrile was sensitive to the surrounding environment. 3-Aminobenzonitrile is a similar size with pyrimidine base. Therefore, it is expected that the nucleoside little perturbs the formation of duplex. This nucleoside could be incorporated into DNA.
• Enzymatic Synthesis of multi Spin-labeled DNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):373-374 (2008)
  Electron paramagnetic resonance (EPR) spectroscopy was applied extensively in studies of nucleic acid structures and dynamics. Two modified 2'-deoxyuridine triphosphates were synthesized bearing a spin label linked to the base by a rigid linker to ensure a tight coupling of spin label dynamics. The incorporation of both spin-labeled nucleotides could be shown in primer extension reactions in presence of DNA polymerases from eukaryotic, prokaryotic, and archaic origin. In further experiments we were able to introduce multiple spin labels during primer extension reactions.
• Synthesis and Relaxivities of Oligonucleotides with TEMPO-labeled Sugar Moiety
  - Nucleic Acids Symp Ser (Oxf) 52(1):375-376 (2008)
  5-Uridin derivative, UUT, having TEMPO radical at the 2'-position of sugar moiety, was prepared and characterized by X-ray crystallography. The single strands, ssUUT, incorporating UUT and the double strands, dsUUT, with complimentary strand were also obtained. Their EPR spectra and relaxivities (25 MHz and 0.59 T) in water were measured. The values of {tau}R obtained from the simulation of EPR spectra and the water-proton relaxivities, r1 and r2, increased in order of UUT, ssUUT, and dsUUT.
• Structure-activity relationship of an antisense oligonucleotide-two Cu(II) complex conjugate as an artificial ribonuclease
  - Nucleic Acids Symp Ser (Oxf) 52(1):377-378 (2008)
  We previously demonstrated that an antisense 2'-O-methyloligonucleotide, with two terpyridine*Cu(II) complexes at contiguous internal sites, was highly active as a sequence-specific artificial ribonuclease. Two kinds of terpyridine-linked uridine derivatives (Ut and tUL) were used for its construction, and the residue on the 5'-side (Ut) was the derivative with terpyridine attached to the 2'-oxygen via a short linker arm. To examine the structure-activity relationship of this type of RNA cleaver, we synthesized an analogous RNA cleaver with the inosine counterpart (It) on the 5'-side, since inosine can base-pair with A, U or C. Using the RNA cleavers and the RNA oligomer substrates, we examined the effect of base-pair formation at the Ut (or It) and tUL sites on the activities of the cleavers. The cleavage reactions revealed that, for this type of RNA cleaver, a base-pair at the 5'-side and no base-pair at the 3'-side were required for high activity. In addition, for t! he 5'-side-It residue, a normal base-pair (I-C pair) was needed.
• Hydrolysis of Single Linkage in Long DNA using Oligonucleotide-Multiphosphonate Conjugates and CeIV/EDTA
  - Nucleic Acids Symp Ser (Oxf) 52(1):379-380 (2008)
  Only one phosphodiester linkage in long DNA was hydrolyzed simply by using two components: oligonucleotide-multiphosphonate conjugates ("gapmers") and CeIV/EDTA (Fig. 1).
• New Methodology to Search for DNA Binding Ligands Based on Duplex DNA-Templated Click Chemistry
  - Nucleic Acids Symp Ser (Oxf) 52(1):381-382 (2008)
  We have developed a new methodology for searching new molecules that bind to dsDNA using DNA-templated click chemistry. The click reactions between the minor groove binding peptide and acridine intercalators were accelerated by addition of dsDNA. Furthermore, resulting peptide-acridine conjugate showed strong binding to dsDNA. These results indicate that DNA-templated click chemistry is applicable to search new DNA binding peptide-acridine conjugates from random peptide library.
• DNA Conjugation by Staudinger Ligation
  - Nucleic Acids Symp Ser (Oxf) 52(1):383-384 (2008)
  Two 5 modified 2'-deoxyuridin triphosphates and a 7 modified 2'-deoxy-7-deazaadenosine were synthesized carrying a terminal azide linked to the base. For probing the sterical influence on incorporation and Staudinger ligation different sized flexible linkers were chosen. All three nucleotides can completely replace their natural counterparts in primer extension as well as polymerase chain reactions (PCR) using Pwo DNA polymerase. For azide labeled primer extension products subsequent conjugation of suitably functionalized phosphines via Staudinger ligation was achieved, e.g. for the conjugation of biotin as an affinity tag.
• A new linker group for functionalization of nucleic acids
  - Nucleic Acids Symp Ser (Oxf) 52(1):385-386 (2008)
  A new linker bearing two orthogonal precursor groups - an amino group for the reaction with electrophilic agents, and also a terminal triple bond for the reaction with an aliphatic azido group (1,3-dipolar cycloaddition reaction) for the introduction of the different residues into nucleosides is designed and synthesized. 5'-Triphosphates of various nucleosides bearing proposed linker group have been obtained.
• Synthesis of oligonucleotide conjugated pyrrolepolyamide- and pyrrole-imidazole polyamide-2'-deoxyguanosine hybrids as novel gene expression control compounds
  - Nucleic Acids Symp Ser (Oxf) 52(1):387-388 (2008)
  DNA oligonucleotide conjugated pyrrolepolyamide and pyrrole-imidazole polyamide-2'-deoxyguanosine hybrids were efficiently synthesized by a post-synthetic modification method through condensation of the 2-fluoro-2'-deoxyinosine moiety of oligonucleotide 9 and FmocNH-PyXPyPy (X = Py or Im) derivatives.
• Circular DNA Formation on the Bimolecular Triplex of Anthracene-modified Oligonucleotide Conjugate
  - Nucleic Acids Symp Ser (Oxf) 52(1):389-390 (2008)
  DNA is usually found in nature as a double stranded helix but there are many other structural forms that it can adopt. Triple helical DNA structures are one of these arrangements that DNA can form through some folding interactions. These structures have attracted great interest in the field of DNA recognition due to their higher affinity and selectivity. Here we introduced a bimolecular triplex structure formed by a anthracene-DNA conjugate through a well characterized photodimerization reaction in the presence of complementary target and evaluated the system for the target discrimination by using a mismatch target. Results indicated that the system could provide moderate discrimination.
• Synthesis of the Peptide Nucleic Acid (PNA) Incorporating 2-Amino-6-vinylpurine Derivative and Evaluation of the Reactivity
  - Nucleic Acids Symp Ser (Oxf) 52(1):391-392 (2008)
  Previously, we established a new strategy of synchronous cross linking reaction activated by hybridization to target genes. In this strategy, the highly reactive cross-linking agent, 2-amino-6-vinylpurine nucleoside analog, is generated from its stable precursors by a hybridization-promoted activation process with selectivity to cytosine. In this paper, we wish to report the synthesis of the peptide nucleic acids (PNAs) incorporating 2-amino-6-vinylpurine derivatives and the evaluation of their reactivity to target DNA.
• Construction of an aminooxy derivative for RNA and DNA labeling
  - Nucleic Acids Symp Ser (Oxf) 52(1):393-394 (2008)
  We previously reported that the reactivity of a primary amine is increased by connecting the amine to an aromatic residue with an alkyl linker. This strategy was applied to the construction of a reagent that could form a covalent bond with an aldehyde group. We synthesized a new reagent that consisted of aminooxy and aromatic groups and biotin. The new reagent showed a higher reaction rate to apurinic/apyrimidinic (AP) sites in DNA than a conventional aldehydereactive probe (ARP). This reagent also efficiently reacted with 2', 3'-dialdehyde groups, which were generated by the oxidation of the 2', 3'-terminal vicinal hydroxy groups of RNA. The reaction efficiency of the reagent closely related to the structure of the target molecules. We describe the synthesis and chemical properties of the new reagent.
• Effective Synthesis of Photosensitive Oligodeoxynucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):395-396 (2008)
  In this paper, the synthesis of various vinyl substituted photosensitive pyrimidine nucleosides and nucleotides is described; starting from 5-Iodo-2'- deoxyuridine (IdU) or oligodeoxynucleotides (ODN) containing IdU, which has been attached using an automated batch stop-flow microwave apparatus. The utility of the Pd(0) cross-coupling to photosensitive pyrimidine is expanded herein to include the reaction of glass-supported ODN containing IdU under Heck and Suzuki conditions.
• RNA containing pyrrolocytidine base analogs: increased binding affinity and fluorescence that responds to hybridization
  - Nucleic Acids Symp Ser (Oxf) 52(1):397-398 (2008)
  6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. These analogs produce increased binding affinity to both RNA and DNA targets when incorporated into oligoribonucleotides. The fluorescence observed for the single-stranded oligomers is quenched upon duplex formation with either RNA or DNA targets. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside.
• RNA containing pyrrolocytidine base analogs: good binding affinity and fluorescence that responds to hybridization
  - Nucleic Acids Symp Ser (Oxf) 52(1):399-400 (2008)
  6-Phenylpyrrolocytidine and 6-methoxymethylene-pyrrolocytidine are base-modified nucleosides with remarkable fluorescence properties. When incorporated into RNA, these analogs enhance binding affinity towards RNA and DNA targets with a concomitant change in their fluorescence upon duplex formation. The fluorescence response depends on the nature of the 6-substituent and the sequence position of the modified nucleoside. The fluorescence response of these structurally conservative, well-tolerated fluorescent nucleosides may be exploited as probes in the study of nucleic acid processing enzymes.
• Exceptional Fluorescence and Hybridization Properties of a Phenylpyrrolocytosine in Peptide Nucleic Acid
  - Nucleic Acids Symp Ser (Oxf) 52(1):401-402 (2008)
  A phenylpyrrolocytosine derivative possessing bis-(ortho-aminoethoxy) substitution has been designed, synthesized and incorporated into PNA resulting in oligomers that demonstrate increased binding affinity for DNA targets. The phenylpyrrolocytosine is an exceptionally fluorescent modified nucleobase possessing a high quantum yield while the fluorescence intensity displays useful environmental sensitivity.
• Synthesis of RNA Dimer and Trimer Blocks and Their Uses
  - Nucleic Acids Symp Ser (Oxf) 52(1):403 (2008)
  Since RNA interference was first observed in plants, worms, and then mammalian cells, siRNA becomes a potent therapeutic approach to cure diseases. However, the synthesis of RNA oligonucleotide usually gives lower purities and yields than DNA oligonucleotides due to 2'- hydroxyl group. To be an affordable medicine, many approaches for higher purities and yields have been attempted in RNA oligoncucleotide synthesis. New RNA phosphoramidites and other types of monomer, solution phase synthesis, hybrid of solid-solution phase synthesis, and novel supports are among them. We have developed an efficient method to synthesize siRNA using RNA dinucleotide blocks in solid phase synthesis. Various RNA dimer phosphoramidites were synthesized and applied for siRNA synthesis giving the highly pure RNA oligonucleotides comparable to DNA oligonucleotides. The RNA trimer blocks were also synthesized and its application will be discussed as well.
• Easy method for the synthesis of labeled oligonucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):405-406 (2008)
  We developed a new strategy for labeling oligonucleotides. Labels bearing an acceptor substituted azide moiety, e.g. a sulfonyl azide substituent are used during oligonucleotide synthesis instead of conventional dye phosphoramidites. Azides are well known to react with trivalent phosphor compounds to phosphor amidates and therefore they could be used instead of an oxidizer during oligonucleotide synthesis. Because N-Alkyl or N-Aryl phosphor amidates are hydrolyzed especially under acidic conditions, we used acceptor substituted azides as reactants, which results in remarkable stabilization of the corresponding amidates. This method is suitable to introduce labels at any internucleosidic linkage of an oligonucleotide and could be used for synthesis of any kind of labeled or polylabeled detection probes. Probes synthesized with these new labeling reagents are evaluated in Real Time PCR. They show the same performance like probes synthesized by conventional means. Since! the labeling reagents could be easily synthesized and since excess reagent could be recycled and used for further labeling reactions, this method represents a very cost effective way for the synthesis of labeled oligonucleotides.
• Synthesis of oligonucleotides terminally modified by bulky and negatively charged substituents and their hybridization with RNA targets
  - Nucleic Acids Symp Ser (Oxf) 52(1):407-408 (2008)
  2'- O-methyl RNAs having phosphorylated cyclohexane groups at the 5' and 3'-terminal bases were synthesized and their hybridization properties toward RNA targets were studied. It was proved that the duplex of the modified 2'- O-methyl RNAs and the target RNA longer than the 2'- O-methyl RNA was less stable than that with the short target. In addition, it was also clarified that the selectivity toward the short target was improved by the introduction of the phosphate group in comparison with that without the phosphate group.
• Folding pathways of hybrid-1 and hybrid-2 G-quadruplex structures
  - Nucleic Acids Symp Ser (Oxf) 52(1):409-410 (2008)
  The G-rich sequence in the human telomeric DNA can form the G-quadruplex structure. The G-quadruplex structure has become an attractive target for the anticancer drugs, because it effectively inhibits telomerase activity. Recently, the human telomere G-quadruplex in K+ solution has been determined as a hybrid structure. This structure is called hybrid-1. More recently, the hybrid-2 G-quadruplex has been determined by NMR. Hybrid-2 G-quadruplex differs from hybrid-1 in its loop arrangement and strand orientation. Here, we propose the folding pathways of hybrid-1 and hybrid-2 G-quadruplex structures. In hybrid-1, we proposed two pathways. In one pathway, the random coil forms triplex-1; in another pathway, the random coil forms chair-1. Similarly, we proposed two pathways in hybrid-2. In one pathway, the random coil forms triplex-2; in another pathway, the random coil forms chair-2. The folding pathways of human telomeric hybrid-1 G-quadruplex and hybrid-2 G-quadruplex s! tructures were proposed using MO calculation and molecular modelling.
• Mechanism of DNA elongation during de novo DNA synthesis
  - Nucleic Acids Symp Ser (Oxf) 52(1):411-412 (2008)
  Under isothermal conditions, short oligodeoxynucleotides (ODNs) were elongated to long DNA by Vent(exo-), a thermophilic DNA polymerase, in the presence of dNTPs. Short ODNs (14-28 nt) were designed to form hairpin structures based on the sequence we obtained from de novo DNA synthesis in the presence of restriction enzyme Tsp509I. As short as 14-nt-long DNA could be elongated to longer than 20000 nucleotides by Vent(exo-) at 65{degrees}C in 1 h. The high efficiency of elongation at very low concentration (<1 nM) supported the THF-SPE (terminal hairpin formation and self-priming extension) mechanism we purposed for DNA elongation during de novo DNA synthesis. The hairpin structure forms at a DNA duplex end as a self-priming complex, followed by strand displacement extension to longer DNA. The highly efficient elongation attributes to the successive repetition of the process of THF-SPE.
• Thermodynamics of DNA structures under molecular crowding conditions with neutral and positive charged cosolutes
  - Nucleic Acids Symp Ser (Oxf) 52(1):413-414 (2008)
  The condition in a living cell is molecularly crowded with various biomolecules. Especially, in cell nucleus, there are high concentrations of histone proteins around DNA. Here, we analyzed quantitatively the effects of cosolutes inducing molecular crowding and histone-mimicking peptides on the thermodynamics of DNA structural formation via Watson Crick or Hoogsteen base pairs. The free energy changes for DNA structure formations with Hoogsteen and Watson-Crick base pairs decreased and increased when the concentration of poly(ethylene glycol) 200 was increased from 0 to 40 wt%, respectively. Moreover, it was observed that a histone-mimicking peptide, which is a part of the binding site of histone H3, stabilized DNA structures only with Hoogsteen base pairs. These results show that a cell nucleus mimicking condition such as a molecular crowding condition in the presence of histone proteins, stabilize DNA structures containing Hoogsteen base pairs and destabilize those w! ith Watson-Crick base pairs, leading to structural polymorphism of various DNA sequences under molecular crowding conditions that mimic those found in cell nucleus.
• Mechanism of chiral-selective tRNA aminoacylation and the origin of amino acid homochirality
  - Nucleic Acids Symp Ser (Oxf) 52(1):415-416 (2008)
  The aminoacylation of tRNA, a process in which amino acids first encounter RNA, may be closely related to the origin of L-amino acid homochirality in biological systems. A clear preference for L-amino acids as opposed to D-amino acids was noted in the efficient nonenzymatic aminoacylation of an RNA minihelix (progenitor of the modern tRNA) by an aminoacyl phosphate oligonucleotide. The steric clash between the side chains of D-amino acid and the terminal adenosine of the minihelix is the stereochemical mechanism underlying the chiral selectivity of the aminoacylation. Subtle structural changes dependent on sugar puckering could affect the positioning of the amino acids and hence the chiral selectivity. These results clearly suggest that tRNA aminoacylation may have been a critical step in determining L-amino acid homochirality.
• Effect of Locked Nucleic Acid (LNA) modification on Hybridization Kinetics of DNA duplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):417-418 (2008)
  The effect of locked nucleic acid (LNA) modification on hybridization kinetics of DNA duplex formation has been studied at varying salt concentration (Na+ and Mg2+) using surface-plasmon resonance. The study suggested that the increased stability of LNA containing duplexes mainly originates from the slower dissociation rates constants of the duplexes. An increase in salt concentration increased the binding affinity of the individual duplexes by raising their association rate constants. Monitoring the change in binding affinity with respect to salt concentration revealed that of LNA-associated enhancement in helical stability mainly results from the changes in the non-electrostatic interactions upon duplex formation.
• Promotion of Triplex Formation by 2'-O,4'-C-Aminomethylene Bridged Nucleic Acid (2',4'-BNANC) Modification
  - Nucleic Acids Symp Ser (Oxf) 52(1):419-420 (2008)
  We examined the effect of 2'-O,4'-C-aminomethylene bridged nucleic acid (2',4'-BNANC) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The melting temperature of the pyrimidine motif triplex at pH 6.8 with 2',4'-BNANC modified TFO was significantly higher than that observed with unmodified TFO. The 2',4'-BNANC modification of TFO increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the 2',4'-BNANC backbone modification of TFO could be a key chemical modification and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.
• Photoinduced excess electron injection into DNA duplexes containing mismatched base pairs
  - Nucleic Acids Symp Ser (Oxf) 52(1):421-422 (2008)
  A series of DNA containing photoinduced electron donors and mismatched DNA base pairs have been prepared and applied for the chemical investigation of excess electron transfer (EET) in the duplex DNA. As the electron donors, phenothiazine (PTZ) with a flexible linker was tethered to the 5'-end or in the middle of the sequences, or diaminostilbene (DAS) was covalently linked to form a hairpin structure. The presence of mismatched base pair lowered EET efficiency in the DAS-capped DNA hairpins, on the other hand, efficient EET beyond the mismatch site was observed in the PTZ-conjugated DNA.
• Development of template-directed reversible DNA photocrosslinking
  - Nucleic Acids Symp Ser (Oxf) 52(1):423-424 (2008)
  We describe a novel reversible DNA interstrand photocrosslinking reaction via the artificial nucleoside (X). Oligodeoxynucleotide (ODN) containing X can be photocrosslinked by irradiation at 366 nm for 1 s, and the photocrosslinked ODN can be split by irradiation at 312 nm for 60 s.
• Thermodynamic, Counterion and Hydration Effects for the Incorporation of Locked Nucleic Acid (LNA) Nucleotides in Duplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):425-426 (2008)
  A Locked Nucleic Acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O, 4'-C-methylene bridge added to the ribose ring that is known to exhibit enhanced hybridization affinity towards complementary DNA and RNA, however the underlying thermodynamic basis for this observation are poorly understood. We have evaluated the influence of LNA residues on hybridization thermodynamics, counterions and hydration of DNA*DNA and DNA*RNA heteroduplex using spectroscopic and calorimetric techniques. Thermodynamic analysis for duplex formation using UV and differential scanning calorimetry suggested that LNA-induced stabilization results from a large, favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. The heat capacity change, {Delta}Cp, accompanying the duplex formation, obtained through DSC, has also been reported and has been used to furnish thermodynamic parameters at 37 {degrees}C. Furtherm! ore, it was observed that relative to the formation of unmodified duplex, the formation of LNA-modified duplexes was accompanied by a higher uptake of counterions and a lower uptake of water molecules.
• On the Stereochemistry of C3'-O-P-CH2-O-C4'' Phosphonate Internucleotide Bond, a Phosphate Isostere
  - Nucleic Acids Symp Ser (Oxf) 52(1):427-428 (2008)
  The work deals with structural evaluation of the internucleotide phosphonate C3'-O-P-CH2-O-C4'' linkage which is an isosteric alternative to natural phosphodiester bond. A thorough description of its stereochemical features was made possible now by matching the results from additional NMR data obtained from the synthesised 13C-labeled dimeric model compounds and the new findings provided by the extended MDS and ab initio studies. It completed the earlier assumptions. The obtained overall picture in terms of specifying the linkage conformational preferences shows explicitly why the respective phosphonate oligonucleotides differ in properties from those with related phospodiester chain.
• Temperature-dependent changes of RNA secondary structures monitored by 19F-NMR spectroscopy
  - Nucleic Acids Symp Ser (Oxf) 52(1):429 (2008)
  19F-NMR spectroscopy is shown to be a useful tool for monitoring hybridization of nucleic acids.
• Effect of Linker Length on DNA Duplexes Containing a Mismatched O6-2'-Deoxyguanosine-Alkyl Interstrand Cross-Link
  - Nucleic Acids Symp Ser (Oxf) 52(1):431-432 (2008)
  DNA duplexes containing a directly opposed O6- alkyl-2'-deoxyguanosine interstrand cross-link were synthesized to serve as structural mimics of lesions formed by the bifunctional chemotherapeutic alkylating agents busulfan and hepsulfam. One of the key steps to prepare the necessary bis-phosphoramidites involved the Mitsunobu reaction between a diol linking two protected 2'-deoxyguanosine nucleosides at the O6 position. These bis-phosphoramidites were incorporated into 11-bp DNA duplexes by solid phase synthesis to produce cross-linked DNA probes in high yields. UV thermal denaturation studies revealed that these interstrand cross-linked containing oligonucleotides were stabilized compared to a DNA duplex containing a central 2'-deoxyguanosine mismatch. The duplex containing the four carbon cross-link was stabilized by 10{degrees}C relative to the seven carbon linker. Molecular models of these duplexes that were geometry optimized by the AMBER force field suggest that ! the seven carbon cross-link was less efficiently accommodated in the major groove of the duplex relative to the four carbon linker, accounting for the observed destabilization.
• Reactivity of Thymine Doublet in Single Strand DNA with Osmium Reagent
  - Nucleic Acids Symp Ser (Oxf) 52(1):433-434 (2008)
  Osmium oxidation has been frequently utilized for thymine-targeting DNA sequencing because of its high selectivity. In this study, we investigated the reactivity of various DNAs containing multiple thymine bases. A thymine doublet was more easily oxidized than a single thymine in single strand DNA.
• The reaction of cytosine with bisulfite by base flipping from the duplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):435-436 (2008)
  Methylation of cytosine at the C5-position in DNA plays a major role in epigenetic gene control. The detection of 5-methylcytosine was designed with the base flipping from the duplex using NC and CGG/CGG triad. Using bisulfite and hydroxylamine, the reaction of cytosine in 11-mer duplex produced the adduct of bisulfite and hydroxylamine, which was isolated by HPLC and identified by MALDI-TOF MS. The duplex containing 5mC in 5mCGG/5mCGG did not react with bisulfite and hydroxylamine under the same conditions. Further this method enables cytosine adjacent to mismatch guanine to react with bisulfite and hydroxylamine selectively.
• Stereoselective formation of a cyclobutane pyrimidine dimer by using N4-acetyl protection of the cytosine base
  - Nucleic Acids Symp Ser (Oxf) 52(1):437-438 (2008)
  The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. As a part of our study on the synthesis of CPD-containing oligonucleotides, we have prepared properly-protected thymidylyl-(3' 5')-N4-acetyl-2'-deoxycytidine, and the solution of this compound was UV-irradiated using acetophenone as a sensitizer. In this reaction, hydrolysis of the acetylamino group occurred, and a trans-syn cyclobutane thymine-uracil dimer with the syn-anti conformation around the glycosidic bonds was formed stereoselectively.
• Oligonucleotides damaged with Carcinogenic Aromatic Amines at the C8- and N6-Position of 2'-Deoxyadenosine
  - Nucleic Acids Symp Ser (Oxf) 52(1):439-440 (2008)
  The synthesis of C8- and N6-adducts of 2'- deoxyadenosine with carcinogenic aromatic amines is described. Furthermore, the adducts were converted into their corresponding phosphoramidites. These were applied to solid phase DNA synthesis to obtain the sitespecifically modified oligonucleotides that were characterized and investigated regarding melting temperature, circular dichroism spectra and in some cases by enzymatic degradation assays.
• Reactivity of oxanine: efficient fabrication of DNA microarray by using oxanine-containing DNA oligomer as probe molecule
  - Nucleic Acids Symp Ser (Oxf) 52(1):441-442 (2008)
  Oxanine (Oxa), a mutagenic lesion generated from guanine by nitrosative oxidation, can make a covalent bonding with -NH2 or -SH group since Oxa possesses O-acylisourea conformation in the base-ring structure. We employed such unique reactive functionality of Oxa for fabrication of DNA-immobilized system. When DNA oligomer with Oxa at 5'-end (Oxa-end DNA) is spotted on amine-functionalized surface, the DNA probe can be immobilized without any involvement of activation step or any treatment of chemical linker. We further optimized the fabrication conditions for efficient immobilization of Oxa-end DNA.
• Analysis for complexity of clustered DNA damage generated by heavy ion beams
  - Nucleic Acids Symp Ser (Oxf) 52(1):443-444 (2008)
  Among numerous DNA damaging factors, ionizing radiation produces the damage showing very unique structure. Since ionizing radiation passes through a target DNA as a beam, the respective induced lesions locate close together around the track. Such damage aggregation on target DNA called "clustered DNA damage" is thought to be a major cause of the specific and serious effect of ionizing radiation. However, we have less knowledge about the structure of clustered DNA damage, which seems very important in its biological impact. Therefore, we evaluated procedure to analyze the structure of clustered DNA damage induced by ionizing radiation. In the present study, we used polyacrylamide gel electrophoresis to separate oligodeoxyribonucleotide (ODN) substrates containing clustered DNA damage with post-modification with aldehyde reactive probe. The designed procedure well counted the number of abasic site in the model substrate of clustered DNA damage.
• Base recognition abilities of 2'-deoxynucleoside N-oxide derivatives and their substrate specificity in enzyme reaction
  - Nucleic Acids Symp Ser (Oxf) 52(1):445-446 (2008)
  The main products obtained by oxidation of cytosine and adenine bases with hydrogen peroxide are cytosine and adenine N-oxide derivatives. There is a possibility that these N-oxide derivatives are mutagenic in genomic DNA, such as 8-oxoguanine or thymine glycol. Although the chemical synthesis and properties of 2'-deoxynucleoside N-oxide derivatives have been well established, little has been reported about the chemical and biochemical behavior of DNA oligomers containing these modified 2'-deoxynucleoside. In this study, we examined their base recognition ability by Tm experiments and computer modeling, and their substrate specificity in enzyme reaction. It was found that the Tm values of in DNA-DNA, DNA-RNA duplexes incorporating 2'-deoxynucleoside N-oxide derivatives were significant low, while the one-point incorporation of these modified derivatives into the 3'-terminal site of a DNA oligomer by DNA polymerase occurred accurately selecting the complementary G or T ! base on a template DNA oligomer.
• pH effect on the one-electron photooxidation of 5-methylcytosine with naphthoquinone sensitizer
  Yamada H Tanabe K Ito T Nishimoto S - Nucleic Acids Symp Ser (Oxf) 52(1):447-448 (2008)
  The pH effect on the one-electron photooxidation of 5-methyl-2'-deoxycytidine (dmC) by sensitization with 1,4-naphthoquinone (NQ) was investigated. Upon photoirradiation of dmC in the presence of NQ under slightly acidic conditions such as pH 5.0, 5-formyl-2'- deoxycytidine (dfC) was formed efficiently, whereas similar NQ-photosensitized oxidation of dmC proceeded to lesser extent under neutral or basic conditions. Under slightly acidic conditions, dmC radical cation favors to undergo irreversible deprotonation at the C(5)-methyl group to form a methyl-centered radical, leading to a higher yield of the alkali-labile oxidation products including dfC. In contrast, the dmC radical cation competitively undergoes deprotonation at the exocyclic N(4)-amino group under neutral or basic conditions, resulting in a decreased yield of NQ-photosensitized oxidation products.
• Synthesis, Biophysical and Repair Studies of O6-2'-Deoxyguanosine Adducts by Escherichia coli OGT
  - Nucleic Acids Symp Ser (Oxf) 52(1):449-450 (2008)
  Oligonucleotides containing modified 2'-deoxyguanosines bearing a seven carbon linker at the O6- atom with either a terminal hydroxyl or 2'- deoxyguanosine group have been synthesized as potential intermediates formed during repair of interstrand cross-linked DNA. Repair of these substrates with Escherichia coli OGT was investigated with an assay involving cleavage of the unmodified duplex with the restriction endonuclease PvuII followed by analysis of the products by denaturing polyacrylamide gel electrophoresis. Duplexes containing these modifications were repaired by OGT suggesting that direct repair may play a role, in combination with other repair pathways, in reversing interstrand crosslink DNA damage.
• X-Ray Crystallographic Identification of Bisulfite-Uracil Adduct as Sodium 5,6-Dihydrouracil 6-Sulfonate
  - Nucleic Acids Symp Ser (Oxf) 52(1):451-452 (2008)
  DNA methylation at position 5 of cytosine residues plays an important role in the gene function control. The analytical method for determining the sites of 5- methylcytosine residues utilizes bisulfite treatment of genomes. Cytosines in DNA are converted into uracils by this treatment, while 5-methylcytosines remain unaltered. The bisulfite treatment followed by amplification by polymerase chain reaction and by sequencing the resulting DNA allows determination of the 5-methylcytosine sites in the original. In this chemical modification, key intermediates are those formed by addition of bisulfite across the 5,6-double bond of pyrimidine ring. Their structures were proposed in 1970 as 5,6-dihydropyrimidine 6-sulfonates, but not its 6-sulfurous acid ester, on the basis of spectral data. X-ray analysis has now been performed for a single crystal of sodium bisulfite-uracil adduct and the results showed its structure as sodium 5,6-dihydrouracil 6-sulfonate monohydrate, thus ! providing definite evidence for the C(6)-sulfonate structure.
• Effect of backbone-modification of oligodeoxyribonucleic acid on primer extension reactions
  - Nucleic Acids Symp Ser (Oxf) 52(1):453-454 (2008)
  We synthesized two types of modified DNA templates in which the phosphodiester linkage was replaced with an amide-type linker [-CH2C=ONH-] or an amine-type linker [-CH2CH2NH-]. Primer extension reactions were performed using these modified DNA templates to investigate the effect of backbone modification on polymerase reactions. Our results indicated that the polymerase reaction was affected much more by the insertion of the cationic flexible amine-type linker than by insertion of the neutral rigid amide-type linker.
• Towards the Replication of xDNA, a Size-expanded Unnatural Genetic System
  - Nucleic Acids Symp Ser (Oxf) 52(1):455-456 (2008)
  Here we study the viability of an unnatural genetic system with size-expanded geometry (xDNA). xDNA contains base pairs 2.4 A larger than those of natural DNA. The expanded geometry is expected to be problematic for the natural high-fidelity replication machinery required to process genetic information. However, initial studies with a variety of DNA polymerases are promising in demonstrating replication of these unnatural bases. The results suggest the future possible viability of fully functional unnatural genetic systems, and give insight into the steric limits of some natural DNA polymerases.
• Sequences around the unnatural base pair in DNA templates for efficient replication
  - Nucleic Acids Symp Ser (Oxf) 52(1):457-458 (2008)
  Unnatural base pairs, compatible with PCR amplification, could potentially increase the versatility of nucleic acids. We recently reported an unnatural base pair, between 7-(2-thienyl)-imidazo[4,5-b]pyridine (denoted by Ds) and 2-nitropyrrole (denoted by Pn), which specifically and efficiently functions in PCR. Toward the efficient incorporation of extra, functional components into DNA fragments, we examined the influence of the sequences around the unnatural base pair and the dependence of the substrate concentrations on the selectivity and efficiency of replication by DNA polymerase.
• Radiolytic Ligation of Oligodeoxynucleotides Possessing Disulfide Bond
  - Nucleic Acids Symp Ser (Oxf) 52(1):459-460 (2008)
  Hypoxic X-radiolysis of diluted aqueous solutions was performed to generate hydrated electrons that induced one-electron reduction of oligodeoxynucleotides (ODNs) possessing a disulfide bond. Upon hypoxic irradiation, reductive ligation reaction between two types of ODNs occurred in the presence of a template ODN strand, resulting in the formation of a prescribed ODN in substantial amounts.
• Regulation of Protein-protein Interaction via Assembly of Coiled-coil Domain
  - Nucleic Acids Symp Ser (Oxf) 52(1):461 (2008)
  The protein-protein interaction presides the various biological events in life. Toward the understanding of their functions and networks, various techniques to regulate the specific protein functions are developed and applied so far. Here we examined the novel method to regulate the protein-protein interactions via coiled-coil assembly.
• Comparison of the chemical properties of a novel amino-linker with various amino modifications
  - Nucleic Acids Symp Ser (Oxf) 52(1):463-464 (2008)
  We previously reported a series of new amino-linkers, consisting of an aminoethyl carbamate structure (Komatsu, 2008). We have now examined the chemical properties of oligonucleotides modified with an ssH-linker, which is the simplest and most cost-effective derivative of the series. Although it was previously shown that monomethoxytrityl protection on a primary amine of the ssH-linker was cleaved under weakly acidic conditions (1% acetic acid), we found that the deprotection also proceeded in aqueous buffer solutions (pH 6.0, 7.0). The MMT group was removed much faster than other commercially available amino-linkers, and this property enabled the ssH-modified oligonucleotides to be conveniently purified with a cartridge column. Furthermore, the ssH-modified oligonucleotides were utilized in on-support labeling reactions. As compared with other amino-linkers, the ssH-linker was superior in terms of its purification and reaction efficiencies.
• DNA Strand Exchange on Liposome Surfaces
  - Nucleic Acids Symp Ser (Oxf) 52(1):465 (2008)
  We demonstrate that both the rate and yield of DNA strand exchange is significantly enhanced on the surface of positively charged liposomes compared to in bulk solution, using a FRET setup.
• Direct Preparation of Sticky-Ended Duplexes within PCR by Using Caged Primers
  - Nucleic Acids Symp Ser (Oxf) 52(1):467-468 (2008)
  Control of the terminal structures of PCR products is crucially important to facilitate molecular biology and biotechnology. Here, we report a new method to prepare PCR products having desired sticky ends directly after thermal cycles. When a pair of caged primers is used, polymerase reaction is site-selectively terminated in front of the caged nucleotide, and the 5'- portion of the primer remains single-stranded throughout the reaction. Successive removal of the photo-cleavable protecting group gives restriction-enzyme free sticky ends on the product. We succeeded in applying this technique to make a recombinant vector bearing GFP gene.
• Efficient PCR amplification by an unnatural base pair system
  - Nucleic Acids Symp Ser (Oxf) 52(1):469-470 (2008)
  Expansion of the genetic alphabet by an unnatural base pair system enables the site-specific incorporation of extra functional components into nucleic acids and proteins. In this system, PCR amplification of DNA templates containing unnatural base pairs is essential for modern biotechnology. We present a new unnatural base pair system, in which DNA duplexes containing the unnatural base pairs can be efficiently amplified by PCR. The system also provides a method for the site-specific incorporation of functional components into amplified DNA fragments by PCR, using unnatural base substrates linked with functional groups of interest.
• Azide-alkyne "click" reaction performed on oligonucleotides with the universal nucleoside 7-octadiynyl-7-deaza-2'-deoxyinosine
  - Nucleic Acids Symp Ser (Oxf) 52(1):471-472 (2008)
  Oligonucleotides containing 7-substituted 7-deaza-2'- deoxyinosine derivatives bearing alkynyl groups were prepared. The octa-1,7-diynyl derivative was functionalized with the non-fluorescent 3- azidocoumarin by the Huisgen-Sharpless-Meldal cycloaddition to afford a highly fluorescent oligonucleotide conjugate. The ambiguous base pairing character and the clickable side chain allows the incorporation of almost any reporter molecule to DNA.
• Temperature-gradient dependant detection of target DNA oligomers using DNA-immobilized open tubular capillary column
  - Nucleic Acids Symp Ser (Oxf) 52(1):473-474 (2008)
  A DNA-immobilized open tubular capillary column (DNA-immobilized OTC) system was developed for analysis and separation of target DNA oligomers. By combining the DNA-immobilized OTC column with a nano/micro-flow pump and a high-sensitivity UV detector, we succeeded in small-scale selective detection of target DNA oligomers (> 0.02 pmol). We designed a temperature-gradient strategy for the efficient separation of target DNA.
• Toward the discovery of new antifungal agents: The design and validation of a novel 2'P-RNA probe and high throughput screening assay against 2'-phosphotransferase Tpt1p
  - Nucleic Acids Symp Ser (Oxf) 52(1):475-476 (2008)
  We report the solid-phase synthesis of novel 2'P-RNA probes for use in fluorescence polarization (FP) ligand binding assays that screens for inhibitors of the yeast 2'- phosphotransferase Tpt1p. The probe was synthesized by utilizing silyl phosphoramidite chemistry and a phosphoramidite synthon containing an orthogonal (DMT) protecting group at its 2'-position. Regioselective removal of the 2'-DMT group and phosphitylation of the unmasked 2'-hydroxyl group afforded the desired 2'P-RNA sequence
• New Strategies for DNA Polymerase Library Screening
  - Nucleic Acids Symp Ser (Oxf) 52(1):477-478 (2008)
  Engineered enzymes are of increasing importance for a plethora of biotechnical applications. Especially DNA polymerases are workhorses in biochemical technologies in particular the polymerase chain reaction (PCR), cDNA cloning procedures, genome sequencing and in diagnostic applications. DNA polymerase mutant libraries can be used for the screening of non-standard reaction conditions or substrates e.g. the efficient amplification of difficult templates like ancient DNA. We are convinced that these fascinating enzymes can be optimized and costum-made for a specific application to result in more robust and reliable systems. To our knowledge, all known screening methods for DNA polymerase mutants are focused and thus limited to the screening of a single reaction or one new function. We developed improved strategies for multiplexed DNA polymerase screening that will be presented.
• Codon based mutagenesis using trimer phosphoramidites
  - Nucleic Acids Symp Ser (Oxf) 52(1):479 (2008)
  A set of Trimer phosphoramidites was synthesized covering all 20 amino acid codons. These trimer phosphoramidites can be added during synthesis using standard DNA chemistry. A Reaction Factor (RF) was determined empirically for each trimer to compensate for differences in their relative rate of reaction during coupling. It is therefore possible to introduce an equimolar mix of all 20 amino acid codons, or subsets thereof, at any location within a sequence. By mutating a gene at the codon level rather than at individual bases, it is possible to avoid codon bias, frame-shift mutations and the introduction of stop codons, making Trimer phosphoramidites a highly efficient tool for the exploration of sequence space in proteins.
• Intracellular small RNA-agarose: Preparation and application for the analysis of proteins interacted with small RNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):481-482 (2008)
  Recent study suggests that newly found small RNAs, such as microRNA, aptamer RNA and so on, have the important roles on the eukaryotic cell function, and several intracellular small RNAs are identified. However, the exact role of small RNA including the protein factors interacted with them has not been clarified. To search the novel interaction between the intracellular small RNA and specific proteins, we prepared the intracellular small RNA-agarose derived from the egg extract of African claw frog, Xenopus laevis. Using this agarose, several Xenopus proteins those might interact with small RNA were obtained. Among them, four proteins strongly interacted with small RNA were analyzed by mass spectroscopy. Result indicated that they could be heat shock protein 90, heat shock protein 70, and ATP synthase {alpha} and {beta} subunits, respectively. In the present paper, the biological utility of intracellular small RNA-agarose is discussed.
• Site-specific gene manipulation of fluorescent proteins using artificial restriction DNA cutter
  - Nucleic Acids Symp Ser (Oxf) 52(1):483-484 (2008)
  Two of three amino acid residues, which compose the chromophore of the enhanced green fluorescent protein (EGFP), were converted to others by using artificial restriction DNA cutter (ARCUT). The vector prepared by ARCUT was easily connected with the insert by using oligonucleotide additive and resultant fluorescent protein such as blue fluorescent protein (BFP) was successfully expressed in cells.
• OLIGONUCLEOTIDES ARE POTENT ANTIOXIDANTS ACTING MAINLY AS METAL-ION CHELATORS
  - Nucleic Acids Symp Ser (Oxf) 52(1):485-486 (2008)
  We explored by ESR the potential of 2'-deoxy-oligonucleotides as biocompatible inhibitors of the Fe(II)/Cu(I)/(II)-induced *OH formation from H2O2. d(A)5, (2'-OMe-A)5, d(A)7, d(A)20, and d(T)20, proved highly potent antioxidants (IC50: 5-17 or 48-85 {micro}M in inhibiting Fe(II)/Cu(I)- or Cu(II)-induced H2O2- decomposition), representing 40 to 215 - fold increase of potency as compared to Trolox. The antioxidant activity does not depend on the oligonucleotides' length or composition. The primary inhibition mechanism by oligonucleotides is metal-ion chelation and the secondary is radical scavenging. 1H-, 31P-NMR and ESR data suggest that Cu coordination involves adenine bases and 1-2 phosphates. We propose the use of short, metabolically stable oligonucleotides as highly potent and long-lived (t1/2 ca. 20 h) antioxidants that may prevent oxidative damage.
• RNA aptamers specifically interact with the Fc region of mouse immunoglobulin G
  - Nucleic Acids Symp Ser (Oxf) 52(1):487-488 (2008)
  We have designed the in vitro selection method to obtain some aptamers such as a general antibody-probing agent, which might bind to the constant regions of mouse immunoglobulin G (IgG) subclasses. As a consequence, one of the selected aptamers found to recognize mouse IgG1, 2a, and 3 subclasses. According to the binding assay, it is suggested that this aptamer recognizes the constant regions of mouse IgG subclass. In addition, this aptamer could recognize the only native form of mouse IgGs but the denatured IgGs. These features show the advantage of the aptamer as an antibody-probing agent rather than the usual secondary antibodies.
• A multisubstrate deoxyribonucleoside kinase from plants
  - Nucleic Acids Symp Ser (Oxf) 52(1):489-490 (2008)
  Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy.
• Influence of 3'-azido-2',3'-dideoxyguanosine treatment on amounts of TERT and POT1 in human HL60 cells
  - Nucleic Acids Symp Ser (Oxf) 52(1):491-492 (2008)
  In human cells, TERT (telomerase reverse transcriptase) is involved in the synthesis of telomere DNA, and POT1 (protection of telomeres 1) is believed to be a regulator of telomere length. We have reported that long-term treatment of human HL60 cells with 50 {micro}M 3'-azido-2',3'-dideoxyguanosine (AZddG) caused telomeres to shorten significantly during early passages (up to 40-50 days), but that telomere length was then stabilized at [~]2 kbp during later passages. Additionally, cell growth rates showed no obvious change during culture in the presence of 50 {micro}M AZddG. Western blot analysis of these cells showed that the amounts of TERT and POT1 expressed were increased significantly and slightly, respectively. Furthermore, telomeric 3' G-overhangs (G-tails) of AZddG-treated cells were lengthened. These findings suggest that HL60 cells may develop resistance to telomere erosion induced by AZddG.
• Isolation and characterization of RNA aptamers specific for the HCV minus-IRES domain I
  - Nucleic Acids Symp Ser (Oxf) 52(1):493-494 (2008)
  The minus-IRES ((-)IRES), corresponding to the 3'-terminal end of the negative strand of hepatitis C virus (HCV) RNA, is well conserved among HCV subtypes. The higher order structure of (-)IRES is essential for HCV replication, because the viral RNA dependent RNA polymerase, NS5B, recognizes it as the initiation site for plus-strand synthesis of the HCV genome. To inhibit the "de novo" synthesis of plus-strand RNA molecules, we performed an in vitro selection procedure for RNA aptamers that are specific for (-)IRES domain I. Among the selected aptamers, one RNA aptamer had two binding sites for the (-)IRES domain I. We found that this aptamer inhibited plus-strand synthesis by about 50%, suggesting that both binding sites are important for binding to its target within the (-)IRES domain I.
• Expanding substrate specificity of nucleoside 2'-deoxyribosyltransferase
  - Nucleic Acids Symp Ser (Oxf) 52(1):495-496 (2008)
  Nucleoside 2'-deoxyribosyltransferase (NDT) is usedto synthesize unnatural 2'-deoxyribonucleosides, modified mostly on the heterocyclic base. Here we describe a strategy for improving 2,3-dideoxyribosyl(ddR) transfer activity of NDT by combining mutagenesis and in vivo selection in E. coli.
• Purification of eukaryotic translation factors from wheat germ for reconstitution of protein synthesis
  - Nucleic Acids Symp Ser (Oxf) 52(1):497-498 (2008)
  The wheat germ cell-free protein synthesis is a powerful and versatile method for preparation of proteins based on the accumulated DNA sequence information. As the cell extract used for it contains many factors that are unknown or do not directly involve in protein synthesis, details of the translation reaction is yet to be understood. Therefore, we have decided to try reconstitution ofprotein synthesis, which would be useful for better understanding of the mechanisms supporting eukaryotic protein synthesis and translational regulation and probably applicable to synthetic biology. In the present study, we fractionated an extract from crude wheat germ according to published protocols to obtain the fractions containing the eukaryoticelongation factors (eEFs) 1A, 1B, and 2. The eEF1A and eEF2 fractions supported polyphenylalanine synthesis.
• Suppression of bcr-abl mRNA by Chemically Modified siRNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):499-500 (2008)
  Synthesis of 21nt siRNAs bearing chemically modified dangling ends with a novel nucleobase wasachieved. Evaluation of gene silencing of bcr-ablchimeric gene derived from Philadelphia chromosome by thus obtained chemically modified siRNAs was performed using human leukaemia cell line K-562 and resulted in efficient suppression of the targeted gene. siRNAs whose sense strands were modified with a novel base were found to be more effective than a native siRNA and that siRNAs whose antisense strands were modified with a novel base showed largely decreased silencing effects on the contrary.
• Tc-DNA modified siRNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):501-502 (2008)
  We investigated the biological activity of siRNAscarrying tc-DNA modifications at various positions in the sense strand. The siRNAs were directed to thecoding region of the enhanced green fluorescent protein (EGFP) mRNA. HeLa cells were transfected with the EGFP plasmid and variable concentrations of siRNA duplexes. The antisense effect was quantified on the protein level by fluorescence activated cell sorting (FACS). We found that 3'-end modification as well as modifications at 3-4 further positions either in the 3'- orthe 5'-region of the sequence were well tolerated, some of them leading to higher repression of gene expression than wild type RNA.
• Synthesis and silencing properties of siRNAs possessing lipophilic groups at their 3'-termini
  - Nucleic Acids Symp Ser (Oxf) 52(1):503-504 (2008)
  Short interfering RNAs (siRNAs) conjugated with lipophilic groups at their 3'-termini were synthesized. The silencing activities of these siRNAs were examined by a dual-luciferase assay. It was found that the '-end of the passenger strand (sense strand) was better than the guide strand (antisense strand) for connecting bulky lipophilic molecules, particularly a rigid molecule such as cholesterol.
• Nanocircular RNAs for RNA interference
  - Nucleic Acids Symp Ser (Oxf) 52(1):505-506 (2008)
  We designed and synthesized dumbbell-shaped nanocircular RNAs for RNA interference applications, which consist of a stem and two loops1. RNA dumbbells are specifically recognized and cleaved by the human Dicer enzyme, and are thus transformed into double-strandedRNA in cells, although this RNA is resistant todegradation in serum. The structure was optimized tomaximize its RNAi activity. The most potent activity was achieved when the stem length was 23 base pairs. The RNAi activity is prolonged by the shape of the molecule, an endless structure, compared with that of normal siRNA.
• MicroRNAs as biomarkers in Tomato Leaf Curl Virus (ToLCV) disease
  - Nucleic Acids Symp Ser (Oxf) 52(1):507-508 (2008)
  MicroRNAs are [~]21- 25 nt long RNA species that are critical regulators of transcriptome across theeukaryotes. Growing number of evidences clearly supports their involvement in plant leaf development. ToLCV infection severely affects the morphology of mature tomato leaves. To investigate the mechanism underlying the virus- host interaction, we focussed our studies on expression of microRNAs and the irrespective targets under normal and ToLCV infection. We have cloned Myb33, ARF4 homolog, Argonaute1, Apetala2, SBP transcription factor and RBOH from tomato and checked their expression by RT-PCR. Our work suggests that miR159 is upregulated while miR164 and miR171 are downregulated under viral infection. Our studies shed light on the impact of ToLCV infection on host transcriptome.
• Micro RNA like inhibition of HIV-1 replication
  - Nucleic Acids Symp Ser (Oxf) 52(1):509-510 (2008)
  In this study, we investigated an RNA (R-{Psi}-sgRNA) that suppresses HIV-1 replication. This RNA is expressed by a plasmid vector (pR-{Psi}-sgRNA-ter) that was constructed accidentally. To examine if this effect is caused by RNA interference, R-{Psi}-sgRNA was synthesized in vitro and treated with the Dicer enzyme, an important RNase III enzyme for RNA interference. The RNA was cleaved into fragments of approximately 20 nucleotides. We then performed an HIV-1 p24 assay with the RNA fragments to evaluate their effect on HIV-1 replication. HIV-1 replication was suppressed.
• The HIV-2 TAR RNA domain as a potential source of viral-encoded miRNA. A reconnaissance study.
  - Nucleic Acids Symp Ser (Oxf) 52(1):511-512 (2008)
  Recently, it has been reported that HIV-1 TAR RNA element releases functionally competent miRNAs upon processing by Dicer enzyme. Here, we extend the analysis of miRNA viral-encoding potential to the TARRNA of the HIV-2. Using in vitro Dicer cleavages and computer-aided analysis we have found that the 124-mer TAR RNA domain, present at the 5' end of HIV-2mRNAs, putatively encodes pre-miRNAs. When deduced sequences of the viral-encoded miRNAs were matched against the database of human mRNA 3'-UTRs, it appeared that two miRNA candidates may target a large number of cellular transcripts.
• In vitro selection of RNA aptamer to hemin
  - Nucleic Acids Symp Ser (Oxf) 52(1):513-514 (2008)
  A new RNA aptamer-binding hemin was synthesized by the in vitro selection (SELEX) method. A pool of 103 bases single strand DNAs containing a randomized sequence of 59 bases was synthesized. The pool was incubated with hemin on hemin-immobilized affinity column. Bound RNAs were eluted off with hemin solution and amplified by PCR. After 3 rounds of selection process, the selected RNAs were cloned and sequenced. Some RNA aptamers, which have affinity to hemin, was selected.
• Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide
  - Nucleic Acids Symp Ser (Oxf) 52(1):515-516 (2008)
  Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Pseom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a! powerful antisense molecule to inhibit the expression of mRNA having a point mutation.
• Self-catalyzing functions of DNA
  - Nucleic Acids Symp Ser (Oxf) 52(1):517-518 (2008)
  We already reported that 281 bp DNA was degraded to 5'-dNMP by treatment at 70 {degrees}C and pH 7.5 for 1 h in the presence of 10 mM Mn ions, and the detailed results are published on Biosci. Biotechnol. Biochem., Vol. 71, 2670-2679 (2007). The degradation was accelerated by 100 mM NaCl. More than 80 bp DNA prepared by PCR using human ZNF 219 cDNA as the template were degraded into 5'- dNMP. Fifty bp DNA prepared by PCR us- ing the synthetic F and R primers of 22 mer was degraded into unknown material besides dNMP. Single-strand 281 b DNA prepared by Strandase (Novagen) was suggested not to be degraded into dNMP but to be degraded into the unknown material. Only double-strand DNA is presumed to be degraded into dNMP, therefore the double-strand structure is considered to be necessary for the degradation into dNMP. Furthermore, the unknown material was found in the ppt. fraction after centrifugation of the reaction mixture in the case of 34mer only G oligomer, while 5'! -dGMP were found not to be degraded into any mater- ial. The m/z of the unknown material prepar- ed from 34mer only G oligomer was determin- ed to be 266 by LC-TOFMS. The elucidation of the conversion mechanism is under investigation.
• Molecular crowding effect on metal ion binding properties of the hammerhead ribozyme
  - Nucleic Acids Symp Ser (Oxf) 52(1):519-520 (2008)
  Although metal ion is essential for the DNA and RNA folding, there are a limited number of reports describing the influence of molecular environments on the metal ion binding. Here, we investigated the binding properties of Mg2+ and Na+ toward the hammerhead ribozyme in the presence of PEG [poly(ethylene glycol)] as a cosolute that changes the solvent property of the reaction. PEG8000 (PEG with the average molecular weight of 8000) increased the reaction rate at lower Mg2+ concentrations but not at higher concentrations. We also found that PEG8000 unchanged the number of Mg2+ bound while the binding cooperativity of Na+ in the reaction without divalent metal ion was altered. The change of Na+ condensation by cosolute has also been reported for diffusely bound Na+ to Watson-Crick base pairs. It is thus supposed that diffusely bound Na+ stabilizes the ribozyme active form in the absence of divalent metal ion. This study provides insights into the RNA-metal ion interactio! n and the hammerhead ribozyme activity under the molecular crowding condition occurred in a cell and on biosensor devices.
• DNAzymes and ribozymes carrying 2'-C-methyl nucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):521-522 (2008)
  DNAzymes and Ribozymes find applications as inhibitors of gene expression and in detection systems such as biosensors, among others. In particular, we are interested in the properties of hammerhead ribozymes carrying 2'-C-methylnucleotides and 10-23 DNAzymes containing (2'R) or (2'S)- 2'-C-methyl 2'-deoxynucleotides. In this work a new synthesis of 2'-C-Methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies as well as the stability under oligonucleotide synthesis conditions is discussed.2'-C-methyl-2'-deoxynucleosides show differential preferred conformations depending on the configuration of the 2'-carbon. The influence of these modifications on the activity of 10-23 DNAzymes is also assessed.
• Mutation analysis of the base-pair connecting two functional modules in the DSL ribozyme
  - Nucleic Acids Symp Ser (Oxf) 52(1):523-524 (2008)
  The class DSL ribozyme is one of artificial RNA enzymes generated by module-based molecular design. In the structure of this ribozyme, two most important functional modules are connected by a U-A base-pair. We have examined the possible importance of this base-pair by site-directed mutation experiments using the DSL-1S ribozyme and its derivative possessing altered modular organization. The analysis indicated that the DSL-1S ribozyme preferred U-A pair at the positions whereas the derivative preferred A-U pair.
• Effective cleavage of structured RNAs by tandems of 10-23 DNAzymes with 3'-odified oligo(2'-O-methylribonucleotide)-effectors
  - Nucleic Acids Symp Ser (Oxf) 52(1):525-526 (2008)
  Catalytic activity of DNAzymes targeted to IGF-I and MDR1 mRNA can be regulated by the combination of 10-23 DNAzyme with 3'-modified oligo(2'-O-methylribonucleotides) that are favorable as effectors due to their high affinity to RNA and nuclease resistance. We have demonstrated that the DNAzyme constructions designed were able to bind and cleave long structured RNA transcripts effectively under simulated physiological conditions.
• Simple and Rapid Colorimetric Detection of Low-Weight Molecules Using Aptazymes in Combination with Noncrosslinking Gold Nanoparticle Aggregation
  - Nucleic Acids Symp Ser (Oxf) 52(1):527-528 (2008)
  We developed a method for simply and rapidly detecting cofactors of aptazymes with high sensitivity using a unique noncrosslinking gold nanoparticle aggregation. Applying this method to a theophylline dependent aptazyme, 100 {micro}M, 10 {micro}M, and 1 {micro}M theophylline were detected easily by the naked eye within 10 min, 20 min, and 65 min, respectively. This method is applicable to other cleavase-aptazymes without altering the probe-DNA sequence.
• Design and Synthesis of Peptide-Oligonucleotide Conjugates as Potential Artificial Ribonucleases
  - Nucleic Acids Symp Ser (Oxf) 52(1):529-530 (2008)
  A series of peptide-oligonucleotide conjugates was synthesized. The peptide fragment was attached to the middle part of DNA sequence. For this purpose the new amidophosphite dC with linker at C5-position was synthesized.
• Kinetics of dCTP incorporation opposite to 7,8-dihydro-8-oxoguanine with different 5' nearest neighbors by yeast polymerase {eta}
  - Nucleic Acids Symp Ser (Oxf) 52(1):531-532 (2008)
  Translesion synthesis (TLS), an important mechanism in cells refers to bypassing the DNA damage blockage on replication fork. Yeast TLS polymerase {eta} (pol{eta}) is able to bypass 7,8-dihydro-8-oxoguanine (8-oxoG) on DNA with high fidelity by incorporation of dCTP opposite 8-oxoG rather than dATP to avoid G to T transversion mutation. We have shown the 5' nearest base next to 8-oxoG affects the G to T mutation by yeast and human pol{eta}previously. In this study, the insertion efficiency of dCTP opposite 8-oxoG in various DNA sequences was kinetically investigated using yeast pol{eta}. Based on Km and Vmax, we demonstrated that the insertion efficiencies were also influenced by the 5' neighboring nucleotide next to 8-oxoG. The lowest Vmax/Km was observed when cytosine was 5' neighbouring base to 8-oxoG, in agreement with previous results in which dCTP incorporation to 8-oxoG was lowest when cytosine is on the 5'-side next to the lesion.
• 6-Aryl- and 6-Heteroarylpurines via Cyclotrimerization
  - Nucleic Acids Symp Ser (Oxf) 52(1):533-534 (2008)
  Transition metal complex catalysed cocyclotrimerization of 6-alkynylpurines 1 with various diynes 2 and 6-(diynyl)purines 4 with nitriles 5 enabled to synthesize series of substituted 6-arylpurines 3 and 6-heteroarylpurines 6 in good yields. The obtained 6-aryl- and 6-heteroarylpurines were tested for cytostatic activity.
• Synthesis and some transformation of acyclic nucleotide phosphonate analogues with triple bond.
  - Nucleic Acids Symp Ser (Oxf) 52(1):535-536 (2008)
  A series of novel group of unsaturated phosphonate analogues of purine and pyrimidine nucleotides with triple bond was synthesized using easily available synthon.
• Prolinol-Based Nucleoside Phosphonic Acids: Synthesis and Properties
  - Nucleic Acids Symp Ser (Oxf) 52(1):537-538 (2008)
  Commercially available trans-4-hydroxy-L-proline has been used as a starting material for the synthesis of prolinol-based nucleotide analogues with N-phosphonomethyl moiety attached to the nitrogen atom of prolinol ring. The synthetic methodology based on the inversion of configuration at both 1- and 4- positions led, in result, to all diastereoisomeric O-protected 4- -mesyloxyprolinol-N-methylphosphonates. Alkylation of nucleobases using the synthons afforded the nucleotide analogues corresponding to {alpha}- and {beta}-nucleotides in both L- and D-series. The NMR-based conformational study of {alpha}- and {beta}-nucleotides in aqueous solution performed at two different pH values securing either N-fully protonated or deprotonated forms revealed in both cases ocurrence of the same mostly populated conformer. All final prolinol-based nucleoside phosphonic acids were tested for cytotoxic and antiviral properties, but no significant activity was found.
• A step further in the SATE mononucleotide prodrug approach
  - Nucleic Acids Symp Ser (Oxf) 52(1):539-540 (2008)
  Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3'-azido-2',3'- dideoxythymidine) are reported herein. These mononucleotide prodrugs (pronucleotides) are characterized by the presence of polar (amino or hydroxyl) functions on the SATE biolabile phosphate protections. Whereas pronucleotides incorporating an amino residue in the vicinity of the thioester functionality display low chemical stability, the introduction of one or two hydroxyl groups on the SATE moiety confers high resistance of the resulting prodrugs towards esterase hydrolysis. Thus, one of these pronucleotides, derivative 2, was able to cross a Caco-2 cell monolayer mainly in intact form, probing that its further development is warranted as a possible HIV-pronucleotide candidate.
• Coupled biocatalysts applied to the synthesis of nucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):541-542 (2008)
  Biocatalytic procedures offer a good alternative to the chemical synthesis of nucleosides since biocatalyzed reactions are regio- and stereoselective and afford reduced by-products contents. Among them, enzymatic transglycosylation between a pyrimidine nucleoside and a purine base catalyzed by nucleoside phosphorylases or microorganisms that contain them, has attracted considerable attention. In addition, the combination to other enzymatic steps has been explored. In this work we investigate the coupled action of nucleoside phosphorylases with other enzymatic activities: deaminase and phosphopentomutase. Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Therefore, another strategy, based on microbial transglycosylation followed by deamination, is here explored. The direct use of furanose 1-phosphate, the intermediate in the transglycosylation reacti! on, is an attractive alternative when pyrimidine nucleosides are not available. Its preparation from the more stable furanose 5-phosphate and phosphopentomutase is here applied to different sugars and bases.
• What are the consequences of freezing the anomeric effect in nucleosides?
  - Nucleic Acids Symp Ser (Oxf) 52(1):543-544 (2008)
  The consequences of freezing the orientation of the oxygen's lone pair orbitals --which determines whether the anomeric effect is operative or not-- were studied theoretically and experimentally in two oxobicyclo-[3.1.0]hexane nucleosides (1 and 2). The results showed significant differences in the properties of these molecules, which correlated with the magnitude of the n2 [->] {sigma} * delocalization.
• First enantioselective synthesis of (-)-neplanocin B
  - Nucleic Acids Symp Ser (Oxf) 52(1):545-546 (2008)
  (-)-Neplanocin B, the natural isomer of a component of the neplanocin familly was enantioselectively synthesized.
• Synthesis of some derivatives of 3'-C-methyl pyrimidine nucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):547-548 (2008)
  Attempts for the synthesis of 2'-deoxy-2'-fluoro nucleoside derivative of 3'-C-methyluridine were reported. The corresponding parent nucleoside was chosen as the starting material. However, the 2'-deoxy-2'-fluoro nucleoside was not obtained. Nevertheless, the new and unexpected nucleoside derivatives obtained as well as some proposals of mechanism regarding their formation will be presented.
• A one step synthetic approach to L-pyrimidine nucleosides using Natural Phosphate coated with potassium iodide as catalyst
  - Nucleic Acids Symp Ser (Oxf) 52(1):549-550 (2008)
  The one-step synthesis of several {alpha}-L-arabino and {beta}-L- xylonucleosides was performed in good yields under mild conditions by N-glycosylation of 1-O-acetyl-L- arabino and -xylofuranose using Vorbruggen type reaction conditions with or without Microwave irradiation.
• Synthesis of functionalized {delta}-amino alcohol stereoisomers with a cyclopentane skeleton. Hydroxylated azidocarbanucleoside precursors
  - Nucleic Acids Symp Ser (Oxf) 52(1):551-552 (2008)
  Five stereoisomers of functionalized {delta}-amino alcohols with a cyclopentane skeleton were synthetized in enantiomerically pure form from racemic {gamma}-lactam 1.
• Design, Synthesis And Evaluation Of Constrained Methoxyethyl (cMOE) and Constrained Ethyl (cEt) Nucleoside Analogs
  - Nucleic Acids Symp Ser (Oxf) 52(1):553-554 (2008)
  Antisense drug discovery technology is a powerful method to modulate gene expression in animals and represents a novel therapeutic platform.1 We have previously demonstrated that replacing 2'Omethoxyethyl (MOE, 2) residues in second generation antisense oligonucleotides (ASOs) with LNA (3) nucleosides improves the potency of some ASOs in animals. However, this was accompanied with a significant increase in the risk for hepatotoxicity.2 We hypothesized that replacing LNA with novel nucleoside monomers that combine the structural elements of MOE and LNA might mitigate the toxicity of LNA while maintaining potency. To this end we designed and prepared novel nucleoside analogs 4 (S-constrained MOE, S-cMOE) and 5 (R-constrained MOE, R-cMOE) where the ethyl chain of the 2'O-MOE moiety is constrained back to the 4' position of the furanose ring. As part of the SAR series, we also prepared nucleoside analogs 7 (S-constrained ethyl, S-cEt) and 8 (Rconstrained Ethyl, R-cEt) wher! e the methoxymethyl group in the cMOE nucleosides was replaced with a methyl substituent. A highly efficient synthesis of the nucleoside phosphoramidites with minimal chromatography purifications was developed starting from cheap commercially available starting materials. Biophysical evaluation revealed that the cMOE and cEt modifications hybridize complementary nucleic acids with the same affinity as LNA while greatly increasing nuclease stability. Biological evaluation of oligonucleotides containing the cMOE and cEt modification in animals indicated that all of them possessed superior potency as compared to second generation MOE ASOs and a greatly improved toxicity profile as compared to LNA.
• Synthesis and Conformational Analysis of Novel 2', 3'-Didehydo-2', 3'-dideoxy-4'-selenonucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):555-556 (2008)
  The structure of 2', 3'-didehydro-2', 3'-dideoxynucleosides (d4Ns) was applied to design the novel bioisosteric 4'-seleno-d4Ns as potential inhibitors of human immunodeficiency virus reverse transcriptase (HIV RT). Conversion of 2', 3'-dihydroxyl groups of 4'-selenoribofuranosyl pyrimidines into the olefin was accomplished by treatment of cyclic 2', 3'-thiocarbonate with 1,3-dimethyl-2-phenyl-1,3,2-diazaphospholidine.
• Synthetic study of muraymycins using Ugi-four component reaction
  - Nucleic Acids Symp Ser (Oxf) 52(1):557-558 (2008)
  The synthetic study of muraymycins (MRYs), which have potent antibacterial activity, is described. The key elements of our approach include the synthesis of L-epicapreomycidine via a C-H amination reaction and a conversient assemblage to construct of the framework of muraymycins using Ugi-four component reaction. First, isonitrile 4 was prepared from uridine in 14 steps. The precursor of carboxylic acid component 15 was synthesised via the C-H amination reaction, formation of cyclic guanidine structure. Muraymycin D2 analogs were synthesized by a model Ugi-four component reaction.
• Total syntheses of a North methanocarba Puromycin analog and its dinucleotide derivative
  - Nucleic Acids Symp Ser (Oxf) 52(1):559-560 (2008)
  North methanocarba Puromycin analog 5 and its di-nucleotide derivative 6 were synthesized from D-ribosein respectively 18 and 19 steps, in order to be tested for peptidyl transfer efficiency in ribosomes.
• Regioselective glycosylation of 7-azapteridines and conversion of the 7-azapteridine nucleosides into 6-azapurine nucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):561-562 (2008)
  The regioselective glycosylation of reumycins (3) reacted with 1-O-acetyl-2,3,5-tri-O-benzoyl-{beta}-D-ribofuranose (4) and BSTFA in acetonitrile at 90 {degrees}C followed by reaction of SnCl4 in dioxane at room temperature afforded the 1-(2',3',5'-tri-O-benzoyl-{beta}-Dribofuranosyl)- 6-methylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-diones (5) (toxoflavin type nucleosides), while the similar alkylations with 1-bromo-2,3,5-tri-Obenzoyl- {beta}-D-ribofuranose (6) and KHCO3 in DMF at 100 {degrees}C gave predominantly the 8-(2',3',5'-tri-Obenzoyl- {beta}-D-ribofuranosyl)-6-methylpyrimido[5,4-e]- [1,2,4]triazine-5,7(6H,8H)-diones (7) (fervenulin type nucleosides). On the other hand, treatment of the 7- azapteridine nucleosides (5 and 7) in alkali solution at room temperature yielded the corresponding 1-({beta}-Dribofuranosyl)- 5-methyl-1H-imidazo[4,5-e][1,2,4]- triazin-6(5H)-ones (8) and 7-({beta}-D-ribofuranosyl)-5- methyl-5H-imidazo[4,5-e][1,2,4]triazin-6(7H)-ones (9) [! 6-azapurine nucleosides] by benzilic acid rearrangement. Some 7-azapteridine nucleosides (5 and 7-9) showed antitumor activities and anti-coccidiosis activities.
• Improved Synthesis of Nucleoside Diphosphate Glycopyranoses
  - Nucleic Acids Symp Ser (Oxf) 52(1):563-564 (2008)
  A new efficient method for the synthesis of nucleoside diphosphate glycopyranoses is based on cycloSalapproach. This new chemical synthesis uses cyclosaligenyl nucleoside phosphate triester as an active phosphate ester. In comparison with other known methods NDP sugars can be synthesised in short reaction times and convincing chemical yields. The method allows the preparation of anomerically pure NDP glycopyranoses.
• Exploring synthetic routes to nucleoside alkynylphosphonates
  - Nucleic Acids Symp Ser (Oxf) 52(1):565-566 (2008)
  Alkynylphosphonates belong to a very interesting family as they may be viewed as precursors to a wide range of functionalized derivatives. Considering our ongoing research on 5'-mononucleotides of biological interest, we embarked on the synthesis of such compounds. Despite a limited number of steps, the nucleosidic pathway appeared disappointing. Therefore, an osidic pathway was explored and proved to be interesting. The corresponding optimization study and the synthesis of a new series of nucleoside alkynylphosphonates are described herein.
• Reactive oxygen species generation through NADH oxidation by pterin derivatives
  - Nucleic Acids Symp Ser (Oxf) 52(1):567-568 (2008)
  Pterin is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities. In previous study, we have elucidated that 6FP derivatives in which the 3-position is modified possess reactive oxygen species (ROS), which are involved in the modulation of a variety of cell functions, generation activities through the oxidation of NADH to NAD+ in the dark at neutral pH. In the present study, we have demonstrated that the ROS generation activity by 6FP derivative is enhanced in the presence of 3-methyl-1-phenyl-2-pyrazolin-5-one. In this reaction, 3-methyl-1-phenyl-2-pyrazolin-5-one is reacted with the formyl group on the 6-position of 6FP derivative to give the activated product. The present results would be helpful for designing pharmaceutical ROS generation system in vivo.
• Synthesis of Nucleoside 5'-S-methylphosphonates and Related Compounds
  - Nucleic Acids Symp Ser (Oxf) 52(1):569-570 (2008)
  Phosphonate analogs of mono- and oligonucleotides with the P-C-O-C5' moiety have interesting biochemical and biophysical properties. A series of novel compounds, S-methylphosphonate-based nucleotides, with the P-C-S-C5' linkage was prepared as monomers for solid phase synthesis of modified oligonucleotides. Replacement of the 5'-oxygen atom with more nucleophilic, bulky, and lipophilic sulfur atom may influence the physicochemical and biological properties of nucleoside 5'-S-methylphosphonates and chimeric oligonucleotides as well.
• "Reverse Fleximers": Introduction of a series of 5-substituted carbocyclic uridine analogues
  - Nucleic Acids Symp Ser (Oxf) 52(1):571-572 (2008)
  Nucleosides are ubiquitous in biological systems and as such, have been a focus of medicinal chemistry research in the search for new and potent therapeutic compounds. There are a number of modified nucleosides on the market, however increasing reports of resistance by mutation of either the enzyme binding site or the pathway that they are designed to interrupt are surfacing. As shown in recent reports, a candidate that can change conformation and still maintain recognition by the target enzyme would be highly desirable, and it is for this reason that flexible substrates have recently been sought as potential therapeutics. With this goal in mind, we have begun investigation into novel flexible scaffolds capable of overcoming viral resistance mechanisms resulting from binding site mutations.
• Synthesis of N-1-alkyl analogues of cyclic inosine diphosphate ribose (cIDPR) by a new solid phase approach
  - Nucleic Acids Symp Ser (Oxf) 52(1):573-574 (2008)
  Herein we report an efficient solid-phase synthesis of some N-1-alkyl-substituted analogs of cyclic inosinediphosphate-ribose (cIDPR), a mimic of cyclic ADP-ribose (cADPR) which has been described as an agonist of the cADPR/Ca2+ signalling system. The proposed synthetic strategy uses a polystyrene support bearing inosine by a 2',3'-acetal linkage which is converted into several N-1-alkylinosine-bis-phosphate derivatives which in turn were cyclized by a solid-phase pyrophosphate bond formation.
• Total synthesis of a xylo-Puromycin analog
  - Nucleic Acids Symp Ser (Oxf) 52(1):575-576 (2008)
  N6-bis-demethylated xylo-Puromycin analog 2 was synthesized in 56% over 6 steps from adenosine 3, invol-ving a Mattocks bromo acetylation, a regio- and stereo-selective ribo-epoxide ring opening with sodium azideand an efficient Staudinger-Vilarrasa coupling reactionfor which the conditions have been optimized.
• Pyrrolidine analogues of nucleosides and nucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):577-578 (2008)
  Among the structurally diverse nucleoside phosphonic acids, several compounds possessing strong antiviral properties have been found. Our effort in this area was focused to the synthesis of novel compounds - pyrrolidine-based nucleoside phosphonic acids and their derivatives.
• New Efficient Synthesis of Nucleoside Diphosphate Glycopyranoses
  - Nucleic Acids Symp Ser (Oxf) 52(1):579-580 (2008)
  A new access for the synthesis of nucleoside diphosphate glycopyranoses has been developed based on the cycloSal-concept. Using this approach, excellent chemical yields were obtained within short reaction times. In comparison to other methods the cycloSalconcept allows a fast and efficient preparation of anomerically defined nucleoside diphosphate glycopyranoses.
• Highly {beta}-selective, N-iodosuccinimide-mediated nucleosidation to bicyclo- and tricyclo-nucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):581-582 (2008)
  The N-iodosuccinimide (NIS) induced nucleosidation of protected natural bases with bicyclo[3.3.0] sugar precursors was investigated. It was found that this method is particularly suited for the synthesis of N1- pyrimidine nucleosides and provides a selective entry into N7-purine nucleosides. This nucleosidation is {beta}- specific and is a valuable alternative to the Vorbruggen method.
• New and efficient Synthesis of Nucleoside Polyphosphates and Nucleoside Monophosphate Sugars
  - Nucleic Acids Symp Ser (Oxf) 52(1):583-584 (2008)
  A new and efficient method for the synthesis of nucleoside di- and triphosphates, dinucleoside tetraphosphates and nucleoside monophosphate sugars is described. This new route is based on cycloSalnucleosyl-phosphate triesters as active ester that underlie fast conversion to the corresponding products.
• Synthesis of 5-ethynyl-1-{beta}-D-ribofuranosyl-1H-[1,2,3]triazole-4-carboxylic acid amide (isosteric to EICAR) and its derivatives
  - Nucleic Acids Symp Ser (Oxf) 52(1):585-586 (2008)
  The synthesis of 5-ethynyl-1H-[1,2,3]triazole-4-carboxylic acid amide riboside 1 and its derivatives exploits Pd(0)-catalyzed cross-coupling reactions. The iodinated key intermediate 3a, when coupled with alkynes affords 5-alkynylated products 1b,c,e,f in diverse yields. Methanolysis of 1b and 1c provides the title compound 1 and the 5-propynyl derivative 1d, respectively. When coupled with methyl acrylate, 3a gives the E-isomer 4c, although in low yield, while the other 5-iodo precursor 3b undergoes reduction to 4b.
• Piperidine nucleosides and nucleotides
  - Nucleic Acids Symp Ser (Oxf) 52(1):587 (2008)
  A novel series of racemic piperidin-3-yl and piperidin-4-yl derivatives of nucleobases and their phosphonate derivatives were prepared.
• Novel nucleotide phosphonate analogues with 1,2-oxaphosphol-3-enering skeleton.
  - Nucleic Acids Symp Ser (Oxf) 52(1):589-590 (2008)
  A series of novel group of carbocyclic phosphonate analogues of nucleotides with 1,2-oxaphosphol-3-ene ring skeleton was synthesized using easily available 1-(diethoxyphosphonyl)buta-1,2-dienes.
• From Ribonucleoside 5'-Aldehydes to Ribonucleoside 5'-C-phosphonates as Building Blocks for Oligonucleotide Synthesis
  - Nucleic Acids Symp Ser (Oxf) 52(1):591-592 (2008)
  [IMG] /medium/nrn299i1.gif" ALT="Formula "> The synthetic approach towards selected monomers of ribonucleosidyl-5'-C-phosphonates I compatible with the phosphotriester condensation method is presented, together with the surprising and unexpected effect of TEA.3HF on the phosphonate moiety observed uponremoval of the O-silyl protecting groups.
• Design and Synthesis of 1-({beta}-D-Ribofuranosyl)imidazo[4,5-c]pyrazoles as 5:5 Bicyclic Analogs of Purine Nucleosides
  - Nucleic Acids Symp Ser (Oxf) 52(1):593-594 (2008)
  5-Alkylaminopyrazole nucleosides underwent nitro-sation to give the corresponding N1-ribosylated 5-alkyl-amino-4-nitrosopyrazoles. The intramolecular cyclo-dehydration reactions of these 5-alkylamino-4-nitroso-pyrazoles were carried out in pyridine at refluxtemperature to afford the ring-closure N-1 ribosylatedimidazo[4,5-c]pyrazoles in good yields.
• Synthesis and antiviral evaluation of 7-fluoro-7-deaza-2-aminopurine nucleoside derivatives
  - Nucleic Acids Symp Ser (Oxf) 52(1):595-596 (2008)
  Three 7-fluoro-7-deaza-2-aminopurine nucleoside derivatives were synthesized and evaluated as potential inhibitors of RNA virus replication, including hepatitis C virus (HCV).
• New depot forms of AZT and 3TC based on their phosphonate derivatives: anti-HIV activity and pharmacokinetic parameters
  - Nucleic Acids Symp Ser (Oxf) 52(1):597-598 (2008)
  New phosphonate depot forms of AZT and 3TC were synthesized and their anti-HIV properties in cell systems, cellular uptake, intracellular transformations and some pharmacokinetic and toxicological data were studied.
• Design and development of curcumin bioconjugates as antiviral agents
  - Nucleic Acids Symp Ser (Oxf) 52(1):599-600 (2008)
  A number of curcumin bioconjugates with fatty acid, dipeptide and folic acid, viz. di-O-decanoyl curcumin (2), di-O-tryptophanylphenylalanine curcumin (3), di-O-bis-({gamma}, {gamma})folyl curcumin (4), C4-ehyl- O-{gamma}-folyl curcumin (5) and 4-O-ethyl-O-{gamma}-folyl curcumin (6) have been synthesized. Conjugates 2-6 have shown good antiviral property with EC50 ranging between 0.019-0.105 {micro}M against a wide range of viruses, like HIV, HSV, VSV and many others.
• The Novel unsaturated acyclic nucleoside analogues: cytostatic and antiviral activity evaluations
  - Nucleic Acids Symp Ser (Oxf) 52(1):601-602 (2008)
  The novel pyrimidine (3-6) and purine (12-19) acyclic nucleoside analogues containing (Z) 4-amino or 4- aminohydrochloride-2-butenyl side chain (Fig.) were synthesized to evaluate their antiviral and cytostatic activity potency.
• Synthesis and study of 9-deazaguanosine derivatives as potential inhibitors of RNA virus replication
  - Nucleic Acids Symp Ser (Oxf) 52(1):603-604 (2008)
  9-Deazaguanosine and the {alpha} and {beta} anomers of its 2'-C-methyl counter part, have been synthesized and evaluated against a broad range of RNA viruses, including hepatitis C virus.
• Synthesis and antiviral activity of novel derivatives of 2'-{beta}-C-methylcytidine
  - Nucleic Acids Symp Ser (Oxf) 52(1):605-606 (2008)
  A series of novel derivatives of 2'-C-{beta}-methylcytidine, involving nucleosides modified in the "upper part" of the pyrimidine base (N4- and/or 5-position), has been synthesized and evaluated for their inhibitory effect on in vitro replication of the hepatitis C virus and the yellow fever virus (both Flaviviridae)
• Synthesis of Pyrimidine Analog of Fluoroneplanocin A as Potential Anti- HCV Agent
  - Nucleic Acids Symp Ser (Oxf) 52(1):607-608 (2008)
  N-Hydroxycytosine nucleoside 3 was synthesized as potential anti-HCV agent, starting from D-ribose using an iodine-fluorine exchange reaction by a help of BuLi, a RCM reaction, a stereoselective reduction and a Mitsunobu reaction as the key steps.
• Synthesis and antiviral evaluation of ({+/-})-4'-ethynyl-5'-difluorocarbocyclic-d4T analogue
  - Nucleic Acids Symp Ser (Oxf) 52(1):609-610 (2008)
  Synthesis of ({+/-})-4'-ethynyl-5'-difluorocarbocyclic-d4T analogue 8, in which the furanose ring oxygen of usual nucleosides is replaced with a geminal-difluoromethylidene group, was carried out. Electrophilic fluorination with Selectfluor(R) was applied to construct a gem-difluorocyclopentenone system to give 12. Regioselective introduction of thymine base was performed under the Mitsunobu conditions by employing the 4-methoxycarbonyl derivative 13. Antiviral evaluation of 8 was also examined.
• Synthesis and antiviral evaluation of a seven-membered sugar ring nucleoside analog, 9-(5-deoxy-{beta}-D-allo-septanosyl)-adenine
  - Nucleic Acids Symp Ser (Oxf) 52(1):611-612 (2008)
  The first example of a nucleoside analogue bearing a 5'-deoxy-{beta}-D-allo-septanose as the sugar moiety was synthesized and evaluated as a potential inhibitor of several virus replication.
• Synthesis of Novel Cyclopropyl Nucleoside Analogues as Potential Antiherpetic Agent
  - Nucleic Acids Symp Ser (Oxf) 52(1):613-614 (2008)
  Synthesis of novel cyclopropyl pyrimidine and purine nucleoside derivatives was successfully achieved using one pot reactions including an alkylation, an oxiranering opening reaction and a lactonization and a hydroboration-oxidation as the key steps in order to find new antiherpetic agent.
• Carbocyclic L-Nucleoside Analogs as Potential Antiviral Agents
  - Nucleic Acids Symp Ser (Oxf) 52(1):615-616 (2008)
  The syntheses of several carbocyclic L-nucleoside analogs starting from enantiomerically pure (1R,2S)-2-benzyloxymethylcyclopent-3-enol are described. The key step is the coupling of a protected nucleobase with different cyclopentane derivatives following a modified Mitsunobu protocol.
• Synthesis and antiviral evaluation of 4-fluoropyrazole-3-carboxamide nucleoside derivatives
  - Nucleic Acids Symp Ser (Oxf) 52(1):617-618 (2008)
  A series of novel 4-fluoro-1H-pyrazole-3-carboxamide nucleoside analogues were synthesized and evaluated as potential inhibitors of RNA virus replication, including hepatitis C virus (HCV).
• Base-Modified Ribonucleosides as Potential Anti-Hepatitis C virus Agents
  - Nucleic Acids Symp Ser (Oxf) 52(1):619-620 (2008)
  Four novel series of base modified ribonucleoside analogues were synthesized and evaluated as potential anti-HCV agents. For two compounds notable anti-HCV activity was obserwed The triphosphates of bicyclic pyrimidine ribonucleosides were studied as substrates/inhibitors of HCV RNA-dependent RNA polymerase (RdRp, NS5B protein) and RNA helicase/NTPase (NS3 protein).
• Synthesis and Antiviral Evaluation of Conformationally Fixed Bicyclohexanyl Nucleosides with Ethenyl Group at C3'-Position
  - Nucleic Acids Symp Ser (Oxf) 52(1):621-622 (2008)
  Conformationally fixed bicyclo[3.1.0]hexanyl nucleosides 1 and 2 with C3'-ethenyl group were successfully synthesized and evaluated against various viruses. Carbenoid intramolecular cycloaddition and diastereoselective Grignard reaction were employed as the key reaction steps.
• Synthesis of conformationally locked carbocyclic nucleoside phosphonates to probe the active site of HIV-1 RT
  - Nucleic Acids Symp Ser (Oxf) 52(1):623-624 (2008)
  The conformationally locked carbocyclic nucleoside phosphonates 2 and 2' and key intermediates for the synthesis of 3 and 3' were prepared from a chiral cyclopentene derivative and epicholorohydrine, respectively. The structure of the nucleoside precursor 6 was confirmed by X-ray crystallography. These carbocyclic nucleoside phosphonates were designed to probe their binding interactions at the active site of HIV-1-RT.
• Molecular mechanisms in two cell death-types, necrosis and apoptosis, induced 5-fluoro-2'-deoxyuridine
  - Nucleic Acids Symp Ser (Oxf) 52(1):627-628 (2008)
  We report that anticancer 5-fluoro-2'-deoxyuridine (FUdR) shows cytotoxicity against mouse cancer cell line FM3A cells, using a progeny clone F28-7 and its variant F28-7-A. In this process, the cell-death morphology is different between F28-7 and F28-7-A cells, that is, necrosis in F28-7 but apoptosis in F28-7-A cells. Recently we have investigated the gene and protein expression profiles of necrosis and apoptosis induced by FUdR using transcriptomic and proteomic analysis. In the proteomic analysis of these cells before their exposure to FUdR, the nuclear inner-membrane protein lamin B1 is up-regulated in F28-7 but not in F28-7-A, suggesting that lamin B1 may possess a function to regulate the morphology of cell-death. A knockdown of lamin B1 expression in F28-7 cells has now been performed by use of the small interfering RNA technique, resulting in a decrease of the lamin B1-expression level down to the level in F28-7-A. Remarkably, the FUdR-induced death morphology ! of this knocked-down F28-7 was apoptosis, definitely different from the necrosis that occurs in the FudRtreated original F28-7. This finding suggests a new role for lamin B1 as a regulator in the cell death.
• Thymidine phoshorylase as a target for antiangiogenesis treatment
  - Nucleic Acids Symp Ser (Oxf) 52(1):629 (2008)
  Thymidine phosphorylase (TP) has emerged as a promising target for antiangiogenesis treatment of cancer. Angiogenesis, the formation of blood vessels, is essential for tumors to grow in order to be supplied with nutrients and oxygen. The association of TP with angiogenesis was demonstrated in several clinical studies in various tissue types. It has been postulated that the angiogenic effect of TP is related to its enzymatic activity, which catalyzes the breakdown of thymidine to thymine and deoxyribose-1- phosphate (dR-1-P). The latter, in its parent form or in its sugar form, deoxyribose, may play a role in angiogenesis. It may interfere in cellular energy metabolism or be substrate in a chemical reaction generating reactive oxygen species. L-deoxyribose and a specific TP inhibitor, TPI, can reverse these effects, supporting the role of the enzymatic reaction and that of the sugar. Although TP is usually high in the tumor, we also observed a high expression in tumoras! sociated stromal cells and macrophages. In order to elucidate the mechanism of TP induced angiogenesis we have investigated the association of TP with angiogenesis, the effect of thymidine and its metabolites on angiogenic parameters (e.g. invasion), the modulation by TPI, the formation and retention of the sugar metabolites of thymidine, and the potential signalling pathways involved in the angiogenic process. We used cell lines without/low TP expression (Colo320 and RT112) and TP transfected variants (Colo320TP1 and RT112/TP). Intrinsic TP expression in cancer cells did not stimulate these cells to invade more. On the other hand, Colo320 and Colo320TP1 cells could attract endothelial cells to a high extent, but Colo320TP1 did not attract them to a higher extent. RT112/TP cells attracted more endothelial cells than RT112 (2 fold). The difference between the RT112's and Colo320's may be related to different formation of sugars. Exposure of tumor cells to thymidine resulted ! in a rapid formation of dR-1-P, which was rapidly degraded to ! deoxyribose and further metabolized to other sugar derivatives. Of the possible sugars that can be produced by the conversion of TdR, dR-5-P seems to accumulate the most. dR accumulated 3 fold higher extent in RT112/TP than in Colo320/TP1 cells. dR could be converted to advanced glycation endproducts (AGE), however this was to a lower extent than ribose. Thymidine also induced several signalling pathways in the cells, involved in migration and invasion, such as the Focal adhesion kinase (FAK), which subsequently stimulated p70/S6 phosphorylation. The latter is a downstream kinase of rapamycin and its phosphorylation is inhibited by rapamycin, an mTOR inhibitor. The association between rapamycin and TP was shown by the protection by thymidine of rapamycin induced cytotoxicity, while TPI inhibited the effect of thymidine addition. These studies clearly show a mechanistic link between TP, signalling pathways, and cell migration.
• A Proposal of the Structure of Modified Nucleosides Expected to be highly Anti-Viral Active and Lowly Toxic.
  - Nucleic Acids Symp Ser (Oxf) 52(1):631-632 (2008)
  The structure of modified nucleosides expected to be highly anti-viral active and lowly toxic is proposed.
• ppGpp analogues as antibacterial compounds
  - Nucleic Acids Symp Ser (Oxf) 52(1):633-634 (2008)
  Immediately after sensing the inception of amino acid starvation, bacteria respond pleiotropically with the stringent response via RelA, mainly resulting in the accumulation of the signal molecule (p)ppGpp. A series of analogues of ppGpp that inhibit RelA activity was prepared in order to control the ability of bacteria cells to react to the changes in their environment. Some of those compounds presented very clear inhibitory effect on both Gram positive and negative bacteria in vitro.
• Hetero-expanded Purine Nucleosides. Design, Synthesis and Preliminary Biological Activity
  - Nucleic Acids Symp Ser (Oxf) 52(1):635-636 (2008)
  Several thieno-expanded purine nucleoside analogues were synthesized for use as tools in ongoing investigations into nucleic acid structure and function in our laboratories. The inclusion of the thiophene ring system in the nucleoside endows the purine scaffold with advantages not previously available in other reported expanded purines. The synthesis and preliminary biological studies are reported herein.
• Biologically active 2-5A analogs containing 3'-O,4'-C-propylene adenosine as potent RNase L agonists
  - Nucleic Acids Symp Ser (Oxf) 52(1):637-638 (2008)
  Novel 2-5A analogs composed of 3'-O, 4'-C-alkylene adenosine were synthesized as potent RNase L activators. When we examined their properties, including their RNase L activating ability and their stability to exonuclease, we found that these 2-5A analogs showed high RNase L activation and high resistance to enzymatic degradation without the modification of an essential adenosine at the second position.
• Synthesis and Antitumor Activity of 1-{beta}-4-Selenoarabinofuranosyl cytosine (4'-Seleno-ara-C)
  - Nucleic Acids Symp Ser (Oxf) 52(1):639-640 (2008)
  4-Seleno analogue of 1-{beta}-arabinofuranosyl cytosine (ara-C) was synthesized via 4'-selenouridine as a key intermediate, which was easily prepared from D-ribose. The arabino configuration was achieved by chemoselective ring opening of the 2,2'-anhydro-4'-selenouridine. The synthesized 4'-seleno-ara-C showed potent antitumor activity (IC50 = 1.5 {micro}M) against stomach cancer cells (SNU638).
• Design and Synthesis of Truncated 4'-Thioadenosine Derivatives as Potent and Selective A3 Adenosine Receptor Antagonists
  - Nucleic Acids Symp Ser (Oxf) 52(1):641-642 (2008)
  We have established structure-activity relationships of novel truncated D-4'-thioadenosine derivatives from D-mannose as potent and selective A3 adenosine receptor (AR) antagonists. At the human A3 AR, most of N6-substituted analogues showed high potency and selectivity and acted as pure antagonists in a cyclic AMP functional assay. Among compounds tested, 2-chloro-N6-3-chlorobenzyl and N6-3-chlorobenzyl analogues displayed very high binding affinities (Ki = 1.66 nM and 1.5 nM, respectively) at the human A3 AR. Truncated 4'-thioadenosine derivatives studied here are regarded as an excellent template for the design of novel A3 AR antagonists to act at both human and murine species.
• Synthesis and Biological Activity of 7-Deaza-7-ethynyl-2'-deoxy-2'-fluoro-2'-C-methyladenosine and its 2'-C-Methyl-ribo Analogue
  - Nucleic Acids Symp Ser (Oxf) 52(1):643-644 (2008)
  In our search for improved therapeutic agents against HCV we synthesized 7-deaza-7-ethynyl-2'-C-methyladenosine (1) and its 2'-deoxy-2'-fluoro analogue 2. The corresponding nucleoside triphosphates were efficient chain terminators of the HCV NS5b polymerase with IC50's of 0.75 {micro}M and 0.4 {micro}M respectively. However, only the ribo-nucleoside 1 exhibited activity in a Huh7 cell based replicon assay with an EC50 of 0.09 {micro}M. In order to overcome the lack of activity of the fluoro analogue 2 we synthesised several phosphoroamidate prodrugs.
• Synthesis of 2-Chloro-N6-Substituted-4'-thioadenosine-5'-N, N-dialkyluronamides as Potent and Selective A3 Adenosine Receptor Antagonists
  - Nucleic Acids Symp Ser (Oxf) 52(1):645-646 (2008)
  The highly selective A3 receptor agonist, 4'-thio-Cl-IB-MECA was successfully converted into selective A3 receptor antagonists by appending a second N-alkyl group on the 5'-uronamide position. This result indicates that the hydrogen bonding ability of the 5'-uronamide is essential for the conformational change required for the receptor activation. Among compounds tested, a N6-(3-bromobenzyl) derivative with 5'-dimethyluronamide exhibited the highest binding affinity (Ki = 9.32 nM) at the human A3 AR with very low binding affinities to other AR subtypes.
• Effects of CpG- oligodeoxynucleotides on dendritic cell development
  - Nucleic Acids Symp Ser (Oxf) 52(1):647-648 (2008)
  Dendritic cell (DC) development begins in the bone marrow and immature progenitors reach their sites of residence in lymphoid organs. The mechanism of DC development in the bone marrow and in peripheral lymphoid organs is poorly understood. Here, we examined the effects of synthetic oligodeoxynucleotides containing a CpG motif (CpG-ODNs) on the development of DC from the bone marrow cells. Approximately 15% of bone marrow cells expressed CD11c surface antigen in the in vitro culture for 8 days in the presence of IL-3. However, the addition of a phosphorothioate-modified CpG-ODN (PTO-CpG-ODN), but not a phosphodiester-modified ODN (PO-CpG-ODN), suppressed the expression of CD11c surface antigen. Also, we examined the effects of CpG-ODNs on the maintenance of DCs resident in the gastrointestinal lymphoid tissues, where immature progenitors may be challenged by a variety of ODN derived from microflora. The population of CD11c+B220intGr-1low cells decreased by the addition! of PTO-CpG-ODN when the lymphocytes from mesenteric lymph nodes as well as spleen are cultured for 2 days in the presence of GM-CSF. In contrast, this population increased in the case of the lymphocytes from Peyer's patches. It thus follows that PTO-CpG-ODN plays a regulatory role in the differentiation of bone marrow cells into DCs and in the functional maintenance of DCs at peripheral lymphoid organs.
• Synthesis and Biological Evaluation of 5''-Iodoneplanocin A and Its Aanlogues
  - Nucleic Acids Symp Ser (Oxf) 52(1):653-654 (2008)
  5''-Iodoneplanocin A was enantiopurely synthesized as potential antiviral and antitumor agents starting from D-ribose using an addition-iodination-elimination reaction, a RCM reaction and an oxidative rearrangement.
• Boranophosphate siRNA-aptamer chimeras for tumor-specific downregulation of cancer receptors and modulators
  - Nucleic Acids Symp Ser (Oxf) 52(1):655-656 (2008)
  There is a need for novel, effective, and cell- and gene-specific therapeutics for cancer. Modified oligonucleotides can be used to modulate specifically and potently the expression of several genes that are upregulated in breast and prostate cancer and have been found to be causal to the tumor phenotype. Synergistic downregulation of these genes may be a potent therapeutic intervention. We are investigating the use of boranophosphate (BP) analogues of RNA as promising candidates for enhancing the potential of three relatively new, gene-specific, anticancer strategies: (1) Tumor-targeted borane siRNA against a combination of genes that control metabolism and transduction; (2) Tumor-specific modified aptamers against prostate specific membrane antigen (PSMA) and ERB2 in breast cancer as delivery agents; and (3) Cancer cell obliteration by cell-specific radiation therapy: Boron-Neutron-Capture-Therapy.
• Pyrimidine Acyclic Nucleoside Phosphonates and Phosphorylated Analogs (Part 2): Syntheses and Investigation of Their Inhibitory Effects Towards Human Thymidine Phosphorylase
  - Nucleic Acids Symp Ser (Oxf) 52(1):657-658 (2008)
  Various methods were used in the syntheses of a number of pyrimidine acyclic nucleoside phosphonates and their derivatives such as a nucleophilic fluorination, Suzuki-Miyaura coupling reactions and phosphorylation. These new compounds were further investigated for their potential biological activity. Based on results obtained the ability to inhibit human thymidine phosphorylase some of them was found.
• Synthesis of conformationally locked carbocyclic 1,3-diazepinone nucleosides as inhibitors of cytidine deaminase
  - Nucleic Acids Symp Ser (Oxf) 52(1):659-660 (2008)
  We synthesized a series of carbocyclic nucleoside inhibitors of cytidine deaminase (CDA) based on a seven-membered 1,3-diazepin-2-one moiety. In the key step, the seven-membered ring was formed by a ring-closing-metathesis reaction. Therefore, the bis-allylurea moiety had to be protected by benzoylation in order to obtain an orientation suitable for ring closure. To our surprise, the analogue built on a flexible sugar template (4) showed a 100-fold stronger inhibition of CDA than the derivative with the preferred south-conformation.
• 9-Deazaguanine derivatives: synthesis and inhibitory properties as multi-substrate analogue inhibitors of mammalian PNPs
  - Nucleic Acids Symp Ser (Oxf) 52(1):661-662 (2008)
  9-(5',5'-Difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) and its related analogues were designed as multi-substrate analogue inhibitors of purine nucleoside phosphorylase (PNP) on the basis of the X-ray crystallographic data obtained for the binary complex of 9- (5',5'-difluoro-5'-phosphonopentyl)guanine (DFPP-G) with calf spleen PNP. One of these analogues, homo- DFPP-DG was found to be a very potent PNP inhibitor at an intracellular ([~] 1 mM) phosphate concentration.
• Thermodynamic studies of interactions of calf spleen PNP with acyclic phosphonate inhibitors
  - Nucleic Acids Symp Ser (Oxf) 52(1):663-664 (2008)
  The Gibbs binding energy and entropy/enthalpy contributions to the interaction of calf spleen purine nucleoside phosphorylase (PNP) with the novel multisubstrate analogue DFPP-DG, as well as with DFPP-G and (S)-PMP-DAP were determined by fluorescence and calorimetric studies. Results were compared with findings for guanine - a natural reaction product and inhibitor.
• Nucleoside Phosphonic Acids in Thymidine Phosphorylase Inhibition: Structure - Activity Relationship
  - Nucleic Acids Symp Ser (Oxf) 52(1):665-666 (2008)
  A number of structurally diverse nucleoside phosphonic acids have been tested against human recombinant thymidine phosphorylase and human platelets supernatant using 2'-deoxy-5-nitrouridine as the substrate. We have selected several inhibitors working at micromolar level as lead structures for further evaluation.
• Crown ether ring-fused nucleosides: synthesis and conformational properties
  - Nucleic Acids Symp Ser (Oxf) 52(1):667-668 (2008)
  We here describe the synthesis of a series of novel bicyclic ribonucleoside derivatives, with 18-crown-6 ether moieties attached via their ribose 2- and 3- positions, as first examples of crown ether ring-fused nucleosides, to be evaluated as antiviral and/or antitumoral agents.
• Simple and universal method to determine dissociation constants for enzyme/ligand complexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):669-670 (2008)
  A simple and in principle universal method is proposed for measuring enzyme/ligand dissociation constants. The method is based on measuring enzyme activity remaining after heat treatment in the absence and in the presence of ligands. The method is especially suitable for enzymes interacting with nucleosides, nucleosides and oligonucleotides since for such enzymes convenient spectrophotometric assays are available.
• On the analysis of fluorimetric titration curves of purine nucleoside phosphorylase
  - Nucleic Acids Symp Ser (Oxf) 52(1):671-672 (2008)
  The steady-state fluorimetric titration curves for trimeric purine nucleoside phosphorylase (PNP) by two ligands, were analysed using the DynaFit program. Results of this analysis indicate that three binding sites of PNP molecule interact with each other and that the character of this interaction is different for both ligands. The DynaFit program is very useful in studies of oligomeric proteins, but for detection of non-interacting sites some independent tests are necessary.
• Effects of Arginine Residue Introduction upon Interaction and Complexation Behavior of Peptide Ribonucleic Acids (PRNAs) with RNA: Synthesis and Properties of {alpha}-Containing Arginine
  - Nucleic Acids Symp Ser (Oxf) 52(1):673-674 (2008)
  A novel nucleic acid model using {alpha}-peptide ribonucleic acid ({alpha}-PRNA) possessing alternative {alpha}- PRNA/arginine sequences, where the arginine residue would be expected to stabilize the complex not only by the conventional hydrogen-bonding interactions between the complementary nucleobase pairs but also through the electrostatic interactions between positively charged side-chain groups and RNA's phosphate anions on the backbone. These {alpha}-PRNA form stable sequencespecific complex with the complementary RNA.
• Photo-Switched DNA-Binding of a Photochromic Spiropyran
  - Nucleic Acids Symp Ser (Oxf) 52(1):675 (2008)
  Photochromic molecules, or photochromes, can be reversibly isomerized between two, more or less, thermally stable forms by exposure to light of different wavelengths. Spiropyrans is an important group of photochromes which in their colorless initial state consist of two ring systems connected at a spiro carbon in a closed orthogonal fashion (1c in Scheme 1). Upon irradiation by UV-light, the C-O bond in the pyran ring is broken resulting in the formation of an open planar molecule absorbing in the visible (1o in Scheme 1). Illumination by visible light switches the photochrome back to its initial closed state. Because planar molecules are known to bind to DNA through intercalation, the open form is much more likely to intercalate DNA than the closed isomer. This, together with the fact that interconversion between the two isomers can be controlled by photonic means, enables light controlled DNA-binding. This is a highly novel research area, as the approach in virtually! all previous studies aiming at controlling DNA-associated processes by photochromic means has been to covalently link the photochrome to short single-stranded oligonucleotides. Our approach surmounts the need of covalently modifying the targeted DNA molecules, making it much more suitable for any practical biological application. [IMG]/medium/nrn341i1.gif" ALT="Formula "> Scheme 1 Structures of the two isomeric forms of the spiropyran photochrome used in this study. Here we report the results of DNA-binding studies of spiropyran 1c/1o. Linear dichroism and absorption experiments show that the open form 1o intercalates DNA whereas the closed form 1c shows no or only weak interactions with DNA. Photocycling experiments, where the spiropyran repeatedly has been interconverted between the two isomers in presence of DNA, have shown that it is possible to reversibly control the DNA-binding process using light as external stimuli. We believe that the results from this study co! uld be valuable in the design of future light-activated prodru! gs for e.g. cancer treatment.
• Synthesis of Alkylated Poly(1-vinylimidazole) for a New pH-Sensitive DNA Carrier
  - Nucleic Acids Symp Ser (Oxf) 52(1):677-678 (2008)
  The poly(1-vinylimidazole) (PVIm) with sever al alkyl groups has been synthesized as a new pH-sensitive DNA car rier. The resulting alkylaed PVIm (PVIm-R) was water -soluble in spite of the hydrophobic alkyl groups and the deprotonation of the imidazole groups at physiological pH. Hemolysis assay showed the PVIm-R enhanced membr ane disruptive ability at endosomal pH, owing to the protonation of the imidazole groups with a pKa value around 6.0. Agarose gel r etardation assay proved that the quaternary alkylated imidazole groups worked as DNA binding groups. The resulting PVIm-R/DNA binary complex showed significant gene expr ession.
• Antisense Inhibition of Human Telomerase by Phosphorothioate Oligonucleotide-Peptide Conjugates
  - Nucleic Acids Symp Ser (Oxf) 52(1):679-680 (2008)
  Oligonucleotides can be covalently linked to peptides composed of any sequence of amino acids by SPFC. The peptides incorporated into the conjugates include nuclear localizing signals (NLS), nuclear export signals (NES), membrane fusion domain of some viral proteins and some designed peptides with amphipathic character. Evaluation of biological properties of DNA-peptide conjugates indicated that (a) the conjugates could bind to target RNA and dsDNA with increased affinity, (b) the conjugates were more resistant to cellular nuclease degradation, (c) the conjugates-RNA hybrids could activate RNase H as effective as native oligonucleotides, (d) the conjugates with fusion peptides showed largely enhanced cellular uptake, (e) the conjugates with NLS could be predominantly delivered into cell nucleus, (f) the conjugates with NES could be localized in cytoplasm. As a result, antisense oligonucleotides conjugated with NLS could inhibit human telomerase in human leukemia cells ! much more strongly than phosphorothioate oligonucleotides.
• Single-Molecule Accommodation of Streptavidin in Nanometer-Scale Wells Formed in DNA Nanostructures
  - Nucleic Acids Symp Ser (Oxf) 52(1):681-682 (2008)
  Tape-like DNA nanostructures of 26 nm width with regularly arranged nanometer-scale wells have been prepared by bundling nine DNA helices. Just one streptavidin tetramer (d = 5 nm) is size-selectively captured in each 6.8 x 10 x 2.0 nm well when two biotins are attached to two opposite edges of the well. Accordingly, precise streptavidin nanoarray of 28 nm periods is constructed. The tetramers captured in the wells showed remarkable stability under repetitive AFM scanning. On the other hand, double-sized wells accommodated two tetramers inside, presumably because the distance between the two biotins is too large to cooperatively "anchor" a tetramer.
• Addressable Molecular Node Assembly - High Information Density DNA Nanostructures
  - Nucleic Acids Symp Ser (Oxf) 52(1):683-684 (2008)
  The inherent self-assembly properties of DNA make it ideal in nanotechnology. We present a fully addressable DNA nanostructure with the smallest possible unit cell, a hexagon with a side-length of only 3.4 nm.2,3 Using novel three-way oligonucleotides, where each side has a unique double-stranded DNA sequence that can be assigned a specific address, we will build a non-repetitive two-dimensional grid.
• Self-Assembled DNA Photonic Wire
  - Nucleic Acids Symp Ser (Oxf) 52(1):685 (2008)
  DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several nanometer regime and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. Our results show that it is possible to create two component DNA-based photonic wires capable of long range energy transfer using a straightforward self-assembly approach.
• Programmed regulation of conformational switching in diagnostic biomolecular automata using error control molecules
  - Nucleic Acids Symp Ser (Oxf) 52(1):687-688 (2008)
  We aim to present novel biomolecular automata for transcriptome diagnosis. We already proposed several biomolecular probes for the prediction of oral squamous cell carcinoma as a model case and evaluated the system noise and sensitivity. Here we introduce an error control element and discuss the specificity of the system.
• Functionalization of DNA G-Wires for patterning and nanofabrication
  - Nucleic Acids Symp Ser (Oxf) 52(1):689-690 (2008)
  DNA structures made of guanine tetrads present remarkable properties and are thus first choice candidates for applications in nanofabrication. Starting from the work of Kotlyar et al., we report here that the klenow exo- fragment of DNA polymerase I can extend poly(dG)-poly(dC) from various 5'-modified (dG)10- (dC)10 templates. This allows the production of end-functionalized four-stranded wires (G-Wires) assembled from the folding of poly(dG) strands. G-Wires bearing thiol moieties can be easily combed on Au and Pt surfaces, whereas a 5' single-stranded overhang of a random sequence provides the unique possibility to assemble complex structures for nanoconstruction purposes.
• A Membrane Anchored DNA-based Energy/Electron Transfer Assembly
  - Nucleic Acids Symp Ser (Oxf) 52(1):691 (2008)
  In this work the trapping and conversion of visible light energy into chemical energy is examined using a supramolecular assembly. This consists of a light absorbing antenna and a porphyrin redox centre both covalently attached to a DNA strand, which in turn is bound to a lipid membrane. The excitation energy is finally trapped as a benzoquinone radical anion that could potentially be used in subsequent chemical reactions.
• Properties of Superparamagnetic Iron Oxide Nanoparticles Assembled on Nucleic Acids
  - Nucleic Acids Symp Ser (Oxf) 52(1):693-694 (2008)
  We report the direct modification of SPIOs with a biomolecule, and the sequence-specific assembly of the modified SPIOs was achieved with the aptamer-small molecule interaction. In addition, the transverse relaxation rate of the aqueous solutions containing the modified SPIOs was altered by the dispersion state.
• DX DNAs as Templates for Multiple Arrangement of Zinc Fingers
  - Nucleic Acids Symp Ser (Oxf) 52(1):695-696 (2008)
  The present paper reveals that double crossover-DNAs (DX) serve as scaffolds for the multiple arrangement of a [Ru(bpy)3]2+-bound zinc finger (ZF) protein, Ru-ZF. This series of results would lead to the realization of the two-dimensional arrangement of functional molecules and nanomaterials on DX-tiles.
• Light driven open/close operation of an azobenzene-modified DNA nano-pincette
  - Nucleic Acids Symp Ser (Oxf) 52(1):697-698 (2008)
  A photoresponsive DNA nano-pincette was constructed by using azobenzene-modified DNA as materials. When the azobenzene-modified part hybridizes with its complementary sequence on the pincette, the duplex formation closes it. On the contrary, the pincette is opened after the formed duplex dissociates. Based on reversible photoswitching of this DNA hybridization, the pincette involving non-substituted azobenzene can be opened simply by UV light irradiation and closed by visible light irradiation. Interestingly, the operation can be reversed by using para-isopropyl group substituted azobenzene: visible light opens the pincette, and UV light closes it. In both cases, the azobenzene-modified part was attached to the pincette throughout the open/close operation, which makes single molecular operation possible. Furthermore, the operation can be repeated many times without any decrease of the cycling efficiency and no DNA waste was produced.
• Preparation of coherent hetero clusters with threoninol scaffold
  - Nucleic Acids Symp Ser (Oxf) 52(1):699-700 (2008)
  New hetero aggregates where different dyes stacked alternately were prepared by hybridizing two DNAs, each of which tethered a different dye in the centre of strand. Spectral changes due to exciton coupling between different kinds of dyes were observed. Especially, hetero aggregates of Methyl Red and 2- nitro-4'-dimethylaminoazobenzene showed substantial narrowing of the band, demonstrating coherent coupling occurred in these aggregates.
• Incorporation of cationic dyes into DNA for distinct stabilization of duplex
  - Nucleic Acids Symp Ser (Oxf) 52(1):701-702 (2008)
  In this study, cationic dyes (methylstilbazole) were introduced into ODN. When two complementary ODNs, both of which tethered this dye, were hybridized, the melting temperature drastically increased. The duplex was further stabilized by introducing multiple dyes.
• RNA aptamers that reversibly bind to photoresponsive peptide
  - Nucleic Acids Symp Ser (Oxf) 52(1):703-704 (2008)
  Modulation of biological network constituted by diverse interactions among biologically active molecules has provided innovative biotechnologies. Here, we report RNA aptamers that bind to photoresponsive peptide (KRAzR; Lys-Arg-Azobenzene-Arg) containing azobenzene chromophore, which can change its geometrical structure by phohtoirradiation. Aptamers were identified by 10 cycles of in vitro selection procedure from DNA library containing 70 nt random region. Surface plasmon resonance (SPR) analysis demonstrated that interactions between aptamers and KRAzR were fully controlled by appropriate photoirradiation. Upon irradiation of 360 nm light over the KRAzR-immobilized surface, the binding of each aptamer to the surface was significantly decreased. Subsequent photoirradiation of the same surface with 430 nm light restored the aptamer binding ability of the surface. We also observed that direct photoirradiation of aptamer-peptide complex on a gold surface actively promot! ed dissociation of the complex.
• Efficient quenching of the excimer fluorescence derived from pyrene arrays on RNA duplexes
  - Nucleic Acids Symp Ser (Oxf) 52(1):705-706 (2008)
  We studied the RNA oligomers having pyrenylmethyl substituents at the 2'-O-sugar residues. It has been shown that the pyrene-modified RNAs can form duplexes with complementary RNA sequences without loss of thermal stability. Absorption, fluorescence, and circular dichroism spectra revealed that the multiply incorporated pyrenes are projected toward the outside of RNA duplexes and assembled in helical aromatic arrays along the minor grooves of the RNA duplexes. The helical pyrene arrays exhibited remarkably strong excimer fluorescence. In the present work, we carried out the fluorescence quenching of the excimer derived from the pyrene-arrays assembled on RNA duplexes in order to evaluate the mobility of the exciton along the pyrene array.
• Zipper-like assembly of multi-pyrenes covalently attached to RNA sequences via duplex formation
  - Nucleic Acids Symp Ser (Oxf) 52(1):707-708 (2008)
  We describe a new strategy for multi-pyrene-modification of RNA sequences to form a unique structure of pyrene arrays on duplex RNA that exhibits remarkably strong excimer fluorescence.
• Selective recovery of target cells employing a bi-functional aptamer
  - Nucleic Acids Symp Ser (Oxf) 52(1):709-710 (2008)
  Aptamers are single-stranded nucleic acids with high and specific affinity to a target molecule. Recently, target cell-binding aptamers are studied as molecular tools for the detection and the isolation of desired cells. Here, I developed a method for the selective recovery of target cells employing a bi-functional aptamer. As a model experiment, I constructed the bi-functional aptamer by fusing a resin-binding aptamer and a target cell-binding aptamer. The bi-functional aptamer enabled the selective recovery of the target cells simply by mixing of the cells with the bi-functional aptamer and the resin. This simple method would be applied for a variety of cell types and resins.
• Regulating mRNA translation with a kiss
  - Nucleic Acids Symp Ser (Oxf) 52(1):711-712 (2008)
  Loop-loop interactions mediate the recognition between RNA hairpins leading to the formation of so-called kissing complexes. Both the size and the sequence of the loop are critical for ensuring stable interaction. Using in vitro selection we have characterized a few loop sequences that lead to the formation of highly stable kissing complexes. These sequences constitute targets of interest for the rational design of RNA stem loop ligands. Such an appropriate target sequence was identified in a sub-domain of the Internal Ribosomal Entry Site (IRES) of the Hepatitis C Virus (HCV) mRNA. We synthesized chemically-modified RNA hairpins and demonstrated that they specifically reduced the expression of a HCV IRES driven reporter gene in cultured cells.
• Improvement of transcription- and transfection-efficiency by synthetic polyampholytes
  - Nucleic Acids Symp Ser (Oxf) 52(1):713-714 (2008)
  Water-soluble PEG derivatives having both amino- and carboxyl-pendants (PEG-ACs) were synthesized, and examined for their transcription- and transfection-enhancing activity on DNA/polycation complexes. PEG-AC could be deposited onto the surface of DNA/polyethylenimine(PEI) complexes, and enhanced their transcriptional activity. Fluorescence anisotropy study showed that the amphoteric PEG-AC loosened the tightly compacted DNA/PEI complex to facilitate the approach of transcriptional factors. Transcriptional activity of the complex was in good correlation with its transgene expression efficiency, indicating the importance of improvement in transcription for effective gene transfection.
• Analysis of cationic comb-type copolymers/DNA interaction by the single molecular observation and intermolecular force measurement
  - Nucleic Acids Symp Ser (Oxf) 52(1):715-716 (2008)
  We have reported that poly(L-lysine)-graft-dextran (PLL-g-Dex) accelerates DNA hybridization and increases stability of a double stranded DNA (dsDNA). Furthermore, PLL-g-Dex was found to stimulate the DNA strand exchange reaction between dsDNA and its complementary single stranded DNA (ssDNA). In order to better understand these phenomena, we evaluate PLL-g-Dex/DNA interaction by the single molecular observation and intermolecular force measurement at single molecular level. We have been able to observe PLL-g-Dex/DNA complex at single molecular level. PLL-g-Dex was found to increases the dissociation force of a dsDNA.
• Synthesis of naphthalenediimide having two {beta}-cyclodextrins at both of its substituent termini
  - Nucleic Acids Symp Ser (Oxf) 52(1):717-718 (2008)
  Naphthalenediimide (1) carrying two {beta}-cyclodextrins ({beta}CDs) was successfully synthesized by click chemistry between pentynylnaphthalenediimide and azido {beta}CD. Absorption spectra of 1 showed hypochromic and red shifts upon addition of sonicated calf thymus DNA as a double stranded DNA. Kinetic studies showed its slow association with and dissociation from calf thymus DNA. These results suggested that 1 can bind to double stranded DNA by the threading mode, where one of the {beta}CD is required to go through adjacent base pairs of double stranded DNA. If this is proven, {beta}CD is one of the largest groups ever reported which can go through a DNA duplex for threading.
• Synthesis of new non-nucleosidic ligand building blocks for solid-phase oligonucleotide assembly
  - Nucleic Acids Symp Ser (Oxf) 52(1):719-720 (2008)
  Synthesis of new non-nucleosidic building blocks that incorporate 2,2'-biquinoline is described. Starting from readily available bicinchoninic acid, the corresponding bifunctional monomeric reagents for solid-phase oligonucleotide synthesis, phosphoramidite and H-phosphonate, have been prepared. The compounds developed may be useful for the design of sequence-specific artificial nucleases.
• Oligonucleotides direct synthesis on porous silicon chip
  - Nucleic Acids Symp Ser (Oxf) 52(1):721-722 (2008)
  A solid phase oligonucleotide (ON) synthesis on porous silicon (PSi) chip is presented. The prepared Si- OH surface were analyzed by FT-IR and the OH functions were quantified by reaction with 3'- phosphoramidite nucleotide building block. Short ONs were synthesized on the chip surface and the coupling yields evaluated.
• Microarray based oligonucleotide synthesis
  - Nucleic Acids Symp Ser (Oxf) 52(1):723 (2008)
  Type your abstract here. (Font: Times or Times New Roman, 10 pt, bold) Access to DNA sequence information on a genomic scale has enabled the design of large sets of oligonucleotides for high-throughput assays. The ability to synthesize oligonucleotides directly from sequence information has facilitated many key advances in genomics, including the development of synthetic biology. These applications would benefit from new technologies to synthesize thousands of oligos in parallel at even lower costs than possible today. To this end we are developing a novel approach for manufacturing oligonucleotides based on microarray technology. We have also developed methods to characterize pools of oligonucleotides using nextgeneration sequencing, which for the first time provides a direct measure of the relative abundance of large numbers of sequences in a complex pool of oligonucleotides as well as the frequency and distribution of mutations.
• Conformationally-2',4'-Locked Aza-ENA and Carbocyclic ribo-Thymidine
  - Nucleic Acids Symp Ser (Oxf) 52(1):729 (2008)