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DNA synthesis is a fundamental requirement for cell proliferation and DNA repair, but no single method can identify the location, direction and speed of replication forks with high resolution. Mammalian cells have the ability to incorporate thymidine analogs along with the natural A, T, G and C bases during DNA synthesis, which allows for labeling of replicating or repaired DNA. Here, we demonstrate the use of the Oxford Nanopore Technologies MinION to detect 11 different thymidine analogs including CldU, BrdU, IdU as well as EdU alone or coupled to Biotin and other bulky adducts in synthetic DNA templates. We also show that the large adduct Biotin can be distinguished from the smaller analog IdU, which opens the possibility of using analog combinations to identify the location and direction of DNA synthesis. Furthermore, we detect IdU label on single DNA molecules in the genome of mouse pluripotent stem cells and using CRISPR/Cas9-mediated enrichment, determine replication rates using newly synthesized DNA strands in human mitochondrial DNA. We conclude that this novel method, termed Replipore sequencing, has the potential for on target examination of DNA replication in a wide range of biological contexts., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sensing of nucleic acids for molecular discrimination between self and non-self is a challenging task for the innate immune system. RNA acts as a potent stimulus for pattern recognition receptors including in particular human Toll-like receptor 7 (TLR7). Certain RNA modifications limit potentially harmful self-recognition of endogenous RNA. Previous studies had identified the 2'-O-methylation of guanosine 18 (Gm18) within tRNAs as an antagonist of TLR7 leading to an impaired immune response. However, human tRNALys3 was non-stimulatory despite lacking Gm18. To identify the underlying molecular principle, interferon responses of human peripheral blood mononuclear cells to differentially modified tRNALys3 were determined. The investigation of synthetic modivariants allowed attributing a significant part of the immunosilencing effect to the 2'-O-methylthymidine (m5Um) modification at position 54. The effect was contingent upon the synergistic presence of both methyl groups at positions C5 and 2'O, as shown by the fact that neither Um54 nor m5U54 produced any effect alone. Testing permutations of the nucleobase at ribose-methylated position 54 suggested that the extent of silencing and antagonism of the TLR7 response was governed by hydrogen patterns and lipophilic interactions of the nucleobase. The results identify a new immune-modulatory endogenous RNA modification that limits TLR7 activation by RNA.

There are five canonical bases in DNA and RNA. Each base has its particular molecular recognition properties and base pairing strength. Thymine and uracil form only two hydrogen bonds when pairing with adenine, and duplexes rich in A:T base pairs are more labile than duplexes rich in C and G, making some sequences difficult to detect via hybridization in a genomic context. Here we report the synthesis of an ethynylmethylpyridone C-nucleoside, abbreviated 'W', that presents a similar recognition surface as thymidine in the major groove but pairs with A about as strongly as C pairs with G. A phosphoramidite building block was synthesized that allows for incorporation of W residues via automated synthesis in high yield. Melting point increases over duplexes containing T:A pairs of up to 17.5°C, or up to 5.8°C per residue were measured for oligonucleotides containing W. Further, the new base shows excellent fidelity, with a single mismatched G opposite W causing a melting point depression of up to 20.5°C. The strongly pairing replacement for thymidine is only slightly larger than its natural counterpart and performs well in different sequence contexts. It can be used to target weakly pairing A-rich sequences in biological studies.

Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C-T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C-T mismatches (kon ≈ 105 M-1 s-1, koff ≈ 10-3 s-1) as compared to T-T mismatches (kon ≈ 104 M-1 s-1, koff ≈ 10-4 s-1), resulting in very similar equilibrium binding affinities for both types of 'all natural' metallo base pairs (Kd ≈ 10-150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T-T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6-19°C), as compared to those containing C-T mismatches (ΔTm = 1-4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C-HgII-T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs - even in situations where metal binding has little or no impact on the thermal stability of the duplex.

Many studies have shown that protonated nucleic acid base pairs are involved in a wide variety of nucleic acid structures. However, little information is available on relative stability of hemiprotonated self- and non-self-dimers at monomer level. We used electrospray ionization mass spectrometry (ESI-MS) to evaluate the relative stability under various concentrations of hydrogen ion. These enable conjecture of the formation of protonated non-Watson-Crick base pairs based on DNA and RNA base sequence. In the present study, we observed that ESI-MS peaks corresponded to respective self-dimers for all examined nucleosides except for adenosine. Peak heights depended on the concentration of hydrogen ion. The ESI-MS peak heights of the hemiprotonated cytidine dimers and the hemiprotonated thymidine dimer sharply increased with increased concentration of hydrogen ion, suggesting direct participation of hydrogen ion in dimer formations. In ESI-MS measurements of the solutions containing adenosine, cytidine, thymidine and guanosine, we observed protonated cytidine-guanosine dimer (CH+-G) and protonated cytidine-thymidine dimer (CH+-T) in addition to hemiprotonated cytidine-cytidine dimer (CH+-C) with following relative peak height, (CH+-C) > (CH+-G) ≈ (CH+-T) > (CH+-A). Additionally, in the ESI-MS measurements of solutions containing adenosine, thymidine and guanosine, we observed a considerable amount of protonated adenosine-guanosine (AH+-G) and protonated adenosine-thymidine (AH+-T).

The alkylation of the specific higher-order nucleic acid structures is of great significance in order to control its function and gene expression. In this report, we have described the T-T mismatch selective alkylation with a vinyldiaminotriazine (VDAT)-acridine conjugate. The alkylation selectively proceeded at the N3 position of thymidine on the T-T mismatch. Interestingly, the alkylated thymidine induced base flipping of the complementary base in the duplex. In a model experiment for the alkylation of the CTG repeats DNA which causes myotonic dystrophy type 1 (DM1), the observed reaction rate for one alkylation increased in proportion to the number of T-T mismatches. In addition, we showed that primer extension reactions with DNA polymerase and transcription with RNA polymerase were stopped by the alkylation. The alkylation of the repeat DNA will efficiently work for the inhibition of replication and transcription reactions. These functions of the VDAT-acridine conjugate would be useful as a new biochemical tool for the study of CTG repeats and may provide a new strategy for the molecular therapy of DM1.

Environmental exposure, endogenous metabolism and cancer chemotherapy can give rise to alkylation of DNA, and the resulting alkylated thymidine (alkyldT) lesions were found to be poorly repaired and persistent in mammalian tissues. Unrepaired DNA lesions may compromise genomic integrity by inhibiting DNA replication and inducing mutations in these processes. In this study, we explored how eight O 4 -alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, (R)-sBu and (S)-sBu, are recognized by DNA replication machinery in HEK293T human embryonic kidney cells. We found that the O 4 -alkyldT lesions are moderately blocking to DNA replication, with the bypass efficiencies ranging from 20 to 33% in HEK293T cells, and these lesions induced substantial frequencies T→C transition mutation. We also conducted the replication experiments in the isogenic cells where individual translesion synthesis (TLS) DNA polymerases were depleted by the CRISPR/Cas9 genome editing method. Our results showed that deficiency in Pol η or Pol ζ, but not Pol κ or Pol ι, led to pronounced drops in bypass efficiencies for all the O 4 -alkyldT lesions except O 4 -MedT. In addition, depletion of Pol ζ resulted in significant decreases in T→C mutation frequencies for all the O 4 -alkyldT lesions except O 4 -MedT and O 4 -nBudT. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O 4 -alkyldT lesions and defined the roles of TLS polymerases in bypassing these lesions in human cells., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O(4)-alkylthymidine (O(4)-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O(4)-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O(4)-alkyldT lesions and examined their impact on DNA replication in Escherichia coli cells. We showed that the replication past all the O(4)-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of the O(4)-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O(4)-alkyldT lesions, Ogt was able to repair O(4)-MedT and, to a lesser extent, O(4)-EtdT and O(4)-nPrdT, but not other O(4)-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O(4)-alkyldT lesions and their recognition by the E. coli replication machinery., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here we report investigations, based on circular dichroism, nuclear magnetic resonance spectroscopy, molecular modelling, differential scanning calorimetry and prothrombin time assay, on analogues of the thrombin binding aptamer (TBA) in which individual thymidines were replaced by 5-fluoro-2'-deoxyuridine residues. The whole of the data clearly indicate that all derivatives are able to fold in a G-quadruplex structure very similar to the 'chair-like' conformation typical of the TBA. However, only ODNs TBA-F4: and TBA-F13: have shown a remarkable improvement both in the melting temperature (ΔTm ≈ +10) and in the anticoagulant activity in comparison with the original TBA. These findings are unusual, particularly considering previously reported studies in which modifications of T4 and T13 residues in TBA sequence have clearly proven to be always detrimental for the structural stability and biological activity of the aptamer. Our results strongly suggest the possibility to enhance TBA properties through tiny straightforward modifications., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O(4)-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription. Our results revealed that both N3-CMdT and O(4)-CMdT substantially inhibited DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II in vitro and in human cells. In addition, we found that N3-CMdT and O(4)-CMdT were miscoding lesions and predominantly directed the misinsertion of uridine and guanosine, respectively. Our results also suggested that these carboxymethylated thymidine lesions may constitute efficient substrates for transcription-coupled nucleotide excision repair in human cells. These findings provided important new insights into the biological consequences of the carboxymethylated DNA lesions in living cells., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Alkylative damage to DNA can be induced by environmental chemicals, endogenous metabolites and some commonly prescribed chemotherapeutic agents. The regioisomeric N3-, O(2)- and O(4)-ethylthymidine (N3-, O(2)- and O(4)-EtdT, respectively) represent an important class of ethylated DNA lesions. Using nonreplicative double-stranded vectors containing an N3-EtdT, O(2)-EtdT or O(4)-EtdT at a defined site in the template strand, herein we examined the effects of these lesions on DNA transcription mediated by single-subunit T7 RNA polymerase or multisubunit human RNA polymerase II in vitro and in human cells. We found that O(4)-EtdT is highly mutagenic and exclusively induces the misincorporation of guanine opposite the lesion, whereas N3-EtdT and O(2)-EtdT display promiscuous miscoding properties during transcription. In addition, N3-EtdT and O(2)-EtdT were found to inhibit strongly DNA transcription in vitro and in certain human cells. Moreover, N3-EtdT, but not O(2)-EtdT or O(4)-EtdT, is an efficient substrate for transcription-coupled nucleotide excision repair. These findings provide new important insights into how these alkylated DNA lesions compromise the flow of genetic information, which may help to understand the risk of these lesions in living cells., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson-Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 5'-side of the opposing uridine. In the absence of a 5'-adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research.

Eukaryotic chromosome ends are protected from illicit DNA joining by protein-DNA complexes called telomeres. In most studied organisms, telomeric DNA is composed of multiple short G-rich repeats that end in a single-stranded tail that is protected by the protein POT1. Mammalian POT1 binds two telomeric repeats as a monomer in a sequence-specific manner, and discriminates against RNA of telomeric sequence. While addressing the RNA discrimination properties of SpPot1, the POT1 homolog in Schizosaccharomyces pombe, we found an unanticipated ssDNA-binding mode in which two SpPot1 molecules bind an oligonucleotide containing two telomeric repeats. DNA binding seems to be achieved via binding of the most N-terminal OB domain of each monomer to each telomeric repeat. The SpPot1 dimer may have evolved to accommodate the heterogeneous spacers that occur between S. pombe telomeric repeats, and it also has implications for telomere architecture. We further show that the S. pombe telomeric protein Tpz1, like its mammalian homolog TPP1, increases the affinity of Pot1 for telomeric single-stranded DNA and enhances the discrimination of Pot1 against RNA.

Tobacco smoke contains many alkylating agents that can react with DNA to produce O(4)-ethylthymidine (O(4)-etT) and several other types of promutagenic base modifications. Our aims were (i) to confirm results of a pilot study (Godschalk, R., Nair, J., Schooten, F. J., Risch, A., Drings, P., Kayser, K., Dienemann, H. and Bartsch, H. (2002) Comparison of multiple DNA adduct types in tumor adjacent human lung tissue: effect of cigarette smoking. Carcinogenesis, 23, 2081-2086) on the formation of O(4)-etT in smokers' lung; (ii) to explore associations between levels of O(4)-etT and smoking status and (iii) to investigate whether a correlation exists between levels of O(4)-etT and bulky (polycyclic aromatic hydrocarbons-derived) DNA adducts. Archived DNA samples originated from histologically normal peripheral lung tissues of 64 Hungarian lung cancer patients, who underwent lung resection. O(4)-etT was determined by an immunoenriched (32)P-postlabelling-high-performance liquid chromatography method. Levels of bulky DNA adducts were determined by the nuclease P1 adduct-enriched (32)P-postlabelling. O(4)-etT levels ranged from 0.01 to 3.91 adducts/10(8) thymidines. In the combined group of subjects who smoked until surgery or gave up smoking at most 1 year before it, the mean level of O(4)-etT was 1.7-fold (P = 0.015) and of bulky DNA adducts 2.2-fold (P < 0.0001) higher than in long-term ex-smokers (LES) and never-smokers (NS) combined. We found no significant correlation between the individual levels of the two DNA adduct types. No dose-response was detected between O(4)-etT formation and smoking dose. In one-third of LES, O(4)-etT levels were above the 2.0-fold mean level of adducts found in NS, indicating its high persistence. Our results confirm the smoking-related formation of O(4)-etT in human lung DNA that should be explored as biomarker. Its long persistence in target tissue implicates a role of this potentially miscoding lesion in tobacco smoking-associated cancers.

We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface.

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C -->T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C -->T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37 degrees C were 37.4 +/- 1.6 and 27.4 +/- 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2'-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H(2)O(2)/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2'-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C -->T transition mutation at mCpG dinucleotide site.

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C*C(+) pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TC(n)](4) tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TC(n) association results in the formation of several tetramers differing by the number of intercalated C*C(+) pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C*C(+) pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power -2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting.

Humans are exposed to N-nitroso compounds from both endogenous and exogenous sources. Many N-nitroso compounds can be metabolically activated to give diazoacetate, which can result in the carboxymethylation of DNA. The remarkable similarity in p53 mutations found in human gastrointestinal tumors and in shuttle vector studies, where the human p53 gene-containing vector was treated with diazoacetate and propagated in yeast cells, suggests that diazoacetate might be an important etiological agent for human gastrointestinal tumors. The O(6)-carboxymethyl-2'-deoxyguanosine was previously detected in isolated DNA upon exposure to diazoacetate and in blood samples of healthy human subjects. The corresponding modifications of thymidine and 2'-deoxyadenosine have not been assessed, though significant mutations at A:T base pairs were found in the p53 tumor suppressor gene in human gastrointestinal tumors and in shuttle vector studies. To understand the implications of the carboxymethylation chemistry of thymidine in the observed mutations at A:T base pairs, here we synthesized authentic N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT), incorporated them into DNA, and demonstrated, for the first time, that they were the major carboxymethylated derivatives of thymidine formed in calf thymus DNA upon exposure to diazoacetate. The demonstration of the formation of N3-CMdT and O(4)-CMdT in isolated DNA upon treatment with diazoacetate, together with the preparation of authentic oligodeoxyribonucleotide substrates housing these two lesions, laid the foundation for investigating the replication and repair of these lesions and for understanding their implications in the mutations observed in human gastrointestinal tumors.

With an aim to develop a new approach to synthesize 4'-substituted nucleosides, reactions of thymidine derivatives having a benzenesulfonyl leaving group at the 4'-position with organosilicon and organoaluminum reagents were investigated. Two substrates 4alpha (alpha-L-isomer) and 4 beta (beta-D-isomer) were prepared for this purpose. Although reaction of 4alpha with organosilicon reagents gave preferentially the 4'-substituted (allyl and N(3)) beta-D-nucleoside, its reaction with AlMe(3) gave the 4'-methyl-alpha-L-thymidine as the major product. On the other hand, the substrate 4beta, upon reacting with AlMe(3), furnished the desired 4'-methylthymidine exclusively in high yield.

Oligonucleotide that can reversibly alter the duplex-forming ability in response to an external factor has potential to be applied to various genome technologies, e.g. antisense and gene diagnosis technology. A novel bridged nucleoside which is capable of sensing an oxidant and reductant was designed and synthesized. The nucleoside formed a disulfide bridge between C2' and C4' positions, which would enhance its duplex-forming ability under oxidative conditions. Contrastingly, the disulfide bridge was cleaved under reductive conditions; it could cause decrease of duplex-forming ability.

Here we highlight the unusual substrate specificity of Plasmodium falciparum thymidylate kinase (PfTMK) and the validity of the enzyme as a new drug target. Furthermore, we predict that the Anaplasma marginale enzyme has attractive domain constituents and may be functionally different from other TMPKs. We postulate that thymidylate kinases could have multiple attractive functions in pathogens and may be a new drug target against numerous microorganisms.

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3'-side on a 31 nt ssDNA.

A facile, sensitive method for detecting specific sequences of oligonucleotides was developed. Detection of DNA sequences with single nucleotide discrimination is achieved by combining the selectivity of hybridization with an efficient cross-linking reaction. Readily synthesized bifunctional oligonucleotide probes containing a modified pyrimidine that is capable of forming interstrand cross-links under mild oxidative conditions internally, and biotin at their 5'-termini were used to discriminate between 16-nt long sites in plasmid DNA that differ by a single nucleotide. The target sequence was detected via fluorescence spectroscopy by utilizing conjugates of avidin and horseradish peroxidase in a microtiter plate assay. The method is able to detect as little as 250 fmol of target without using PCR and exhibits single nucleotide discrimination that approaches 200:1. In principle, this method is capable of probing any target sequence containing a 2'-deoxyadenosine.

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.

We have recently shown that peptides derived from nucleoside beta-amino acids adopt an unusual 8-helical conformation in solution(1). 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.

The synthesis of a borononucleotide analogue of thymidine and its association with various nucleosides, nucleotides and saccharides is described.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

A precise chemistry of ternary complex formation between osmium reagents, 2,2'-bipyridine (Bpy) and DNA strand has been characterized. Enzymatically digested products of ternary complexes formed for a thymidine in an oligonucleotide has given two diastereoisomers corresponding the 5S,6R- and 5R,6S-isomers of T-Os-Bpy in 70:30 ratio.

By enzymatically hydrolyzing the terminal phosphodiester bond at the 3'-ends of DNA breaks, tyrosyl-DNA phosphodiesterase (Tdp1) repairs topoisomerase-DNA covalent complexes and processes the DNA ends for DNA repair. To identify novel Tdp1 inhibitors, we developed a high-throughput assay that uses electrochemiluminescent (ECL) substrates. Subsequent to screening of 1981 compounds from the 'diversity set' of the NCI-Developmental Therapeutics Program, here we report that furamidine inhibits Tdp1 at low micromolar concentrations. Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA. Surface plasmon resonance studies show that furamidine binds both single- and double-stranded DNA, though more weakly with the single-stranded substrate DNA. Thus, the inhibition of Tdp1 activity could in part be due to the binding of furamidine to DNA. However, the inhibition of Tdp1 by furamidine is independent of the substrate DNA sequence. The kinetics of Tdp1 inhibition by furamidine was influenced by the drug to enzyme ratio and duration of the reaction. Comparison with related dications shows that furamidine inhibits Tdp1 more effectively than berenil, while pentamidine was inactive. Thus, furamidine represents the most potent Tdp1 inhibitor reported to date.

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein-DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle.

We previously synthesized bridged nucleic acid (BNA) analogues possessing an S-type sugar conformation. Recently, we achieved the synthesis of a 2'-deoxy analogue of trans-3',4'-BNA. The structure of this compound was confirmed by X-ray crystallography, which indicated that its sugar ring had an S-type conformation. The hybridization ability of the 2'-deoxy-trans-3',4'-BNA-modified oligonucleotide with DNA and RNA complements was evaluated, and it was found that 2'-deoxy-trans-3',4'-BNA showed superior hybridization ability compared with other BNA analogues with S-type sugar conformation.

Nucleoside H-boranophosphonates were synthesized via the condensation reactions of appropriately protected nucleosides with monopyridinium H-boranophosphonate. The condensation reactions gave only the mono-esterified products under the optimized conditions without formation of di-esterified byproducts. Deprotection of the condensation products was achieved under basic conditions to afford the fully-deprotected nucleoside H-boranophosphonates in excellent yields.

Efficient and specific targeting of DNA sequences by synthetic ligands is a major goal in chemical biology. Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but are limited to homopurine/homopyrimidine target sequences. We have previously reported two useful analogues (WNA: W-shaped nucleoside analogues), WNA-bT and WNAbC, which recognize a TA and a CG interrupting site forming triplexes with high stability and selectivity, respectively. However, their ability to form triplexes depended on their neighbouring bases in the TFO. Subsequent studies have shown that the sequence-dependency of the WNA analogues, for the formation of triplexes, has been partially solved by use of a WNA analogue bearing a substituted aromatic ring. Investigations into the effects of the substituted aromatic ring of WNA derivatives on the stability of triplexes led to the discovery of strand invasion by the TFO incorporating the new WNA analogue to form a highly stable duplex.

BNA We designed and synthesized a novel bridged nucleic acid (BNA) with N-type sugar conformation, possessing a seven-membered cyclic carbamate structure (2',4'-(CNC) monomer). The monomer shows interesting chemical properties, such as prompt migration of the carbamate bridge from the 2'-to the 3'-position and consequent conformational change of the furanose moiety. These chemical behaviors of 2',4'-BNA(CNC) were investigated by (1)H-NMR analysis, and the structure of the compound after migration was confirmed by X-ray crystallographic analysis.

AONs containing aza-ENA (1), 5-membered (2) and 6-membered (3) carbocyclic analogs of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) are both nuclease resistant and capable of eliciting RNase H response, very similar to that of the native.

The specific 1,3 dipolar Hüisgen cycloaddition reaction known as 'click-reaction' between azide and alkyne groups is employed for the synthesis of peptide-oligonucleotide conjugates. The peptide nucleic acids (PNA)/DNA and peptides may be appended either by azide or alkyne groups. The cycloaddition reaction between the azide and alkyne appended substrates allows the synthesis of the desired conjugates in high purity and yields irrespective of the sequence and functional groups on either of the two substrates. The versatile approach could also be employed to generate the conjugates of peptides with thioacetamido nucleic acid (TANA) analog. The click reaction is catalyzed by Cu (I) in either water or in organic medium. In water, approximately 3-fold excess of the peptide-alkyne/azide drives the reaction to completion in 2 h with no side products.

Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.

We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine.

The (6-4) photoproduct, which is a major UV light-induced lesion formed between adjacent pyrimidine bases, is isomerized to its Dewar valence isomer by exposure to longer wavelengths. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'-5')thymidine site, and incorporated it into oligonucleotides on a DNA synthesizer, aiming to use them for biological studies. An alternative activator, benzimidazolium triflate, gave better results, while by-products were detected at longer retention time in the ordinary synthesis. We characterized the synthetic oligonucleotides by UV conversion, nuclease digestion and mass spectrometry. Their use in the study of the (6-4) photolyase, a DNA repair enzyme, revealed its different recognition modes between the (6-4) photoproduct and the Dewar isomer.

We synthesized oligodeoxynucleotides (ODN) possessing 2-oxoalkyl group on thymidine (d(oxo)T) to characterize the radiolytic reduction in aqueous solution. Corresponding unmodified ODNs were generated from ODNs containing d(oxo)T upon hypoxic irradiation. Enzymatic digestion of the duplex consisting of the hypoxically irradiated ODN containing d(oxo)T and its complementary strand resulted in an efficient cleavage, but no cleavage was observed when a control duplex was digested without irradiation.

Cold shock proteins (CSP) belong to the family of single-stranded nucleic acid binding proteins with OB-fold. CSP are believed to function as 'RNA chaperones' and during anti-termination. We determined the solution structure of Bs-CspB bound to the single-stranded DNA (ssDNA) fragment heptathymidine (dT7) by NMR spectroscopy. Bs-CspB reveals an almost invariant conformation when bound to dT7 with only minor reorientations in loop beta1-beta2 and beta3-beta4 and of few aromatic side chains involved in base stacking. Binding studies of protein variants and mutated ssDNA demonstrated that Bs-CspB associates with ssDNA at almost diffusion controlled rates and low sequence specificity consistent with its biological function. A variation of the ssDNA affinity is accomplished solely by changes of the dissociation rate. 15N NMR relaxation and H/D exchange experiments revealed that binding of dT7 increases the stability of Bs-CspB and reduces the sub-nanosecond dynamics of the entire protein and especially of loop beta3-beta4.