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This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.

The purpose of this study was to investigate the inhibitory effect of ZH-1 ((6S,9aS,6aR,9bR)-6-(phenylcarbonyl)-6,6a,9a,9b-tetrahydro-8H-azolidino[3,4-a]b enzo [e]indolizine-7,9-dione) and its potential interaction with gemcitabine in A549 cells. MTT assay showed that the combined use of gemcitabine and ZH-1 presented a significant inhibition effect on A549 cell growth with the cell viability from 82.3 ± 5.6% to 51.0 ± 6.6%. The CI value was 0.60 suggesting a synergistic effect between these two drugs. HPLC-MS/MS data indicated that combined treatment with gemcitabine and ZH-1 induced a significant decrease in deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and deoxythymidine triphosphate levels compared with use of gemcitabine alone. Five RNs as well as seven dRNs were considered to be significantly contributive to the discrimination of samples. Furthermore, western blot analysis revealed that the combination treatment caused A549 cell apoptosis via the intrinsic pathway by up-regulating Bax/Bcl-2 ratio, activating caspase-9, caspase-3 and poly-ADP-ribose polymerase, and promoting caspase-7, caspase-9 and poly-ADP-ribose polymerase cleavage. Collectively, the combined treatment with gemcitabine and ZH-1 exerted a strong synergistic action on anticancer activity through growth inhibition, perturbations in ribonucleotides and deoxyribonucleotides and the activation of intrinsic apoptotic signaling pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

This article reviews the different possibilities towards progression of the formation of DNA/RNA in the chemical world, before life, in enzyme-free conditions. The advent of deoxyribo- and ribopentose-sugars, nucleosides, nucleotides and oligonucleotides in the prebiotic soup is briefly discussed. Further, the formation of early single stranded oligomers, base-pairing possibilities and information transfer based on the stability parameters of the derived duplexes is reviewed. Each theory has its own merits and demerits which we have elaborated upon. Lastly, using clues from this literature, a possible explanation for the specific 3'-5'-linkages in RNA is proposed.

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor-donor-acceptor pattern complementary to the acceptor-acceptor-donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor-acceptor-donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be "fixed" by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2'-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio "seen" by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ∼92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ∼86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic "alphabets", major targets in synthetic biology.

Nucleoside analogs are an important class of anticancer agent that historically show better efficacy against hematological cancers versus solid tumors. This report describes the development and characterization of a new class of nucleoside analog that displays anticancer effects against both hematological and adherent cancer cell lines. These new analogs lack canonical hydrogen-bonding groups yet are effective nucleotide substrates for several high-fidelity DNA polymerases. Permutations in the position of the non-hydrogen-bonding functional group greatly influence the kinetic behavior of these nucleosides. One particular analog designated 4-nitroindolyl-2'-deoxynucleoside triphosphate (4-NITP) is unique as it is incorporated opposite C and T with high catalytic efficiencies. In addition, this analog functions as a nonobligate chain terminator of DNA synthesis, since it is poorly elongated. Consistent with this mechanism, the corresponding nucleoside, 4-nitroindolyl-2'-deoxynucleoside (4-NIdR), produces antiproliferative effects against leukemia cells. 4-NIdR also produces cytostatic and cytotoxic effects against several adherent cancer cell lines, especially those that are deficient in mismatch repair and p53. Cell death in this case appears to occur via mitotic catastrophe, a specialized form of apoptosis. Mass spectroscopy experiments performed on nucleic acid isolated from cells treated with 4-NIdR validate that the non-natural nucleoside is stably incorporated into DNA. Xenograft mouse studies demonstrate that administration of 4-NIdR delays tumor growth without producing adverse side effects such as anemia and thrombocytopenia. Collectively, the results of in vitro, cell-based, and animal studies provide evidence for the development of a novel nucleoside analog that shows enhanced effectiveness against solid tumors.

Enzymatic synthesis of short (10-22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.

5-(α-Thyminyl)-5,6-dihydrothymine, also called spore photoproduct or SP, is commonly found in the genomic DNA of UV-irradiated bacterial endospores. Despite the fact that SP was discovered nearly 50 years ago, its biochemical impact is still largely unclear due to the difficulty of preparing SP-containing oligonucleotide in high purity. Here, we report the first synthesis of the phosphoramidite derivative of dinucleotide SP TpT, which enables successful incorporation of SP TpT into oligodeoxyribonucleotides with high efficiency via standard solid-phase synthesis. This result provides the scientific community a reliable means to prepare SP-containing oligonucleotides, laying the foundation for future SP biochemical studies. Thermal denaturation studies of the SP-containing oligonucleotide found that SP destabilizes the duplex by 10-20 kJ/mol, suggesting that its presence in the spore-genomic DNA may alter the DNA local conformation.

A system for delivery of analogues of 2'-deoxyribonucleoside triphosphate (dNTP) based on SiO(2) nanoparticles was proposed. A simple and versatile method was developed for the preparation of SiO(2)-dNTP conjugates using the 'click'-reaction between premodified nanoparticles containing the azido groups and dNTP containing the alkyne-modified γ-phosphate group. The substrate properties of SiO(2)-dNTP were tested using Klenow fragment and HIV reverse transcriptase. Nucleoside triphosphates being a part of the SiO(2)-dNTP nanocomposites were shown to be incorporated into the growing DNA chain. The rate of polymerization with the use of SiO(2)-dNTP or common dNTP in case of HIV reverse transcriptase differed insignificantly. It was shown by confocal microscopy that the proposed SiO(2)-dNTP nanocomposites bearing the fluorescent label penetrate into cells and even into cellular nuclei., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Nucleoside triphosphates are moldable entities that can easily be functionalized at various locations. The enzymatic polymerization of these modified triphosphate analogues represents a versatile platform for the facile and mild generation of (highly) functionalized nucleic acids. Numerous modified triphosphates have been utilized in a broad palette of applications spanning from DNA-tagging and -labeling to the generation of catalytic nucleic acids. This review will focus on the recent progress made in the synthesis of modified nucleoside triphosphates as well as on the understanding of the mechanisms underlying their polymerase acceptance. In addition, the usefulness of chemically altered dNTPs in SELEX and related methods of in vitro selection will be highlighted, with a particular emphasis on the generation of modified DNA enzymes (DNAzymes) and DNA-based aptamers.

Deletion of the substituted pyrimidine ring in purine-2'-deoxynucleoside 5'-monophosphates leads to the artificial nucleotide analog dImMP(2-). This analog can be incorporated into DNA to yield, upon addition of Ag(+) ions, a molecular wire. Here, we measured the acidity constants of H(2)(dImMP)(±) having one proton at N(3) and one at the PO(3)(2-) group by potentiometric pH titrations in aqueous solution. The micro acidity constants show that N(3) is somewhat more basic than PO(3)(2-) and, consequently, the (H·dImMP)(-) tautomer with the proton at N(3) dominates to ca. 75%. The calculated micro acidity constants are confirmed by (31)P- and (1)H-NMR chemical shifts. The assembled data allow many quantitative comparisons, e.g., the N(3)-protonated and thus positively charged imidazole residue facilitates deprotonation of the P(O)(2)(OH)(-) group by 0.3 pK units. Information on the intrinsic site basicities also allows predictions about metal-ion binding; e.g., Mg(2+) and Mn(2+) will primarily coordinate to the phosphate group, whereas Ni(2+) and Cu(2+) will preferably bind to N(3). Macrochelate formation for these metal ions is also predicted. The micro acidity constant for N(3)H(+) deprotonation in the (H·dImMP·H)(±) species (pk(a) 6.46) and the M(n+)-binding properties are of relevance for understanding the behavior of dImMP units present in DNA hairpins and metalated duplexes., (Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.)

Copper(II) complexes with a new chelator-type nucleoside-histidine modified 2'-deoxyriboadenosine (N-[(9-β-D-2'-deoxyribofuranosylpurin-6-yl)-carbamoyl]histidine) were studied by potentiometric and spectroscopic (UV-visible, CD, EPR) techniques, in conjunction with computer modeling optimization. The ligand can act as bidentate or tridentate depending on pH range. In acidic pH a very stable dimeric complex Cu(2)L(2) predominates with coordination spheres of both metal ions composed of oxygen atoms from carboxylic groups, one oxygen atom from ureido group and two nitrogen atoms derived from purine base and histidine ring. Above pH 5, deprotonation of carbamoyl nitrogens leads to the formation of CuL(2), Cu(2)L(2)H(-1) and Cu(2)L(2)H(-2) species. The CuL(2)H(-1) and CuL(2)H(-2) complexes with three or four nitrogens in Cu(II) coordination sphere have been detected in alkaline medium. Our findings suggest that N-[(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)-carbamoyl]histidine chelates copper(II) ions very efficiently. The resulting complex might be used as an alternative base-pairing mode in which hydrogen-bonded base pairs present in natural DNA are replaced by metal-mediated ones., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.

This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2'-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

Template-directed primer extension usually requires a polymerase, nucleoside triphosphates, and magnesium ions as cofactors. Enzyme-free, chemical primer extensions are known for preactivated nucleotides at millimolar concentrations. Based on a screen of carbodiimides, heterocyclic catalysts, and reactions conditions, we now show that near-quantitative primer conversion can be achieved at submillimolar concentration of any of the four deoxynucleotides (dAMP, dCMP, dGMP and dTMP). The new protocol relies on in situ activation with EDC and 1-methylimidazole and a magnesium-free buffer that was tested successfully for different sequence motifs. The method greatly simplifies chemical primer extension assays, further reduces the cost of such assays, and demonstrates the potential of the in situ activation approach.

Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.

Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.

We previously reported the synthesis of a borononucleotide analogue of thymidine monophosphate and its association towards the formation of a new borono-linked dinucleotide. Here we describe the completion of the set of four 2'-deoxyborononucleotide analogues of natural nucleotide monophosphates, namely the previously unknown dCbn, dGbn and dAbn. These analogues were all prepared from the respective 5'-aldehydic nucleosides through a homologation/reduction sequence. The borononucleotides were subsequently obtained by either borylation (dCbn and dGbn) or cross-metathesis (CM) in the presence of the Hoveyda-Grubbs catalyst (dAbn). The reversible formation of the corresponding dinucleotides between these new analogues and uridine was studied by (1)H NMR, and semi-empirical calculations were carried out to provide bond length and electrostatic information that assess the structural similarities existing between these bioisosteres and their natural counterparts.

We report synthesis and characterization of a complete set of alpha,beta-methylene-2'-dNTPs (alpha,beta-m-dNTP; N = A, C, T, G, 12-15) in which the alpha,beta-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2'-deoxynucleoside 5'-diphosphate precursors, followed by an enzymatic gamma-phosphorylation. All four synthesized alpha,beta-m-dNTPs were found to be potent inhibitors of polymerase beta, with K i values ranging 1-5 microM. During preparation of the dG and dT derivatives of alpha,beta-methylene diphosphate, we also isolated significant amounts of 3,5'-cyclo-dG (16) and 2,5'-cyclo-dT (17), respectively. These novel 2'-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 --> C5' and O2 --> C5', respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5'- cyclo-2'-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.

Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators enabling array-based DNA sequencing by synthesis (SBS) approaches. Herein, we describe the synthesis of a new family of 3'-OH unprotected cleavable fluorescent 2'-deoxynucleotides and their evaluation as reversible terminators for high-throughput DNA SBS strategies. In this first version, all four modified nucleotides bearing a cleavable disulfide Alexa Fluor(R) 594 dye were assayed for their ability to act as a reversible stop for the incorporation of the next labeled base. Their use in SBS leaded to a signal-no signal output after successive addition of each labeled nucleotide during the sequencing process (binary read-out). Solid-phase immobilized synthetic DNA target sequences were used to optimize the method that has been applied to DNA polymerized colonies or clusters obtained by in situ solid-phase amplification of fragments of genomic DNA templates.

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 occurrence 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.

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).

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.

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.

A variant of the semipinacol rearrangement that was observed in our laboratory has been applied to the synthesis of several furanose and pyranose derivatives. The process consists of an "orchestrated" [1,2]-hydride shift with departure of a leaving group from the opposite face. Transient formation of a C=O group is followed by rapid transfer of a hydride-equivalent from the same face from which the leaving group departed, which results in double inversion of stereochemistry at the two vicinal carbon atoms. Treatment of 2'-O- and 3'-O-tosyladenosine with lithium triethylborohydride in DMSO/THF gave the respective 2'- and 3'-deoxynucleoside analogues with beta-D-threo configurations. Identical treatment of 5'-O-TPS-2'-O-tosyladenosine gave 9-(5-O-TPS-2-deoxy-beta-D-threo-pentofuranosyl)adenine. The same [1,2]-hydride shift and stereochemistry with the 5'-OH and 5'-O-TPS compounds demonstrated the absence of remote hydroxyl-group participation. Application of this process to other nucleoside 2'-O-tosyl derivatives gave the 2'-deoxy-threo compounds in good yields. The reaction-rate order was OTs approximately Br >> Cl for 2'-O-tosyladenosine, 2'-bromo-2'-deoxyadenosine, and 2'-chloro-2'-deoxyadenosine (all with beta-d-ribo configurations). Analogous results were obtained with mannopyranoside derivatives with either 4,6-O-benzylidene protection or a free OH group at C4. Deuterium labeling clearly defined the stereochemical course as a cis-vicinal [1,2]-hydride shift on the face opposite to the original cis OH and OTs groups followed by hydride transfer from the face opposite to the [1,2]-hydride shift. Synthetic and mechanistic considerations are discussed.

UV light causes the formation of thymine dimers that can be misreplicated to induce mutagenesis and carcinogenesis. This report describes the use of a series of non-natural indolyl nucleotides in probing the ability of the high-fidelity bacteriophage T4 DNA polymerase to replicate this class of DNA lesion. Kinetic data reveal that indolyl analogues containing large pi-electron surface areas are incorporated opposite the thymine dimer almost as effectively as an abasic site, a noninstructional lesion. However, there are notable differences in the kinetic parameters for each DNA lesion that indicate distinct mechanisms for their replication. For example, the rate constants for incorporation opposite a thymine dimer are considerably slower than those measured opposite an abasic site. In addition, the magnitude of these rate constants depends equally upon contributions from pi-electron density and the overall size of the analogue. In contrast, binding of a nucleotide opposite a thymine dimer is directly correlated with the overall pi-electron surface area of the incoming dXTP. In addition to defining the kinetics of polymerization, we also provide the first reported characterization of the enzymatic removal of natural and non-natural nucleotides paired opposite a thymine dimer through exonuclease degradation or pyrophosphorolysis activity. Surprisingly, the exonuclease activity of the bacteriophage enzyme is activated by a thymine dimer but not by an abasic site. This dichotomy suggests that the polymerase can "sense" bulky lesions to partition the damaged DNA into the exonuclease domain. The data for both nucleotide incorporation and excision are used to propose models accounting for polymerase "switching" during translesion DNA synthesis.

Isodeoxynucleic acid (INA) is one of the most attractive sugar modified nucleic acids for therapeutic use. We report here the enzymatic synthesis and properties of INA. All four isodeoxynucleoside triphosphates (INTPs) were synthesized for use in a single nucleotide insertion reaction. K(m) and V(max) values showed the almost same order as natural dNTPs. Primer extension reactions were also carried out to investigate the function of INTPs as substrates for several DNA polymerases. INTPs were incorporated into N + 2 position. One INTP and three dNTPs were able to generate a full-length product (27 mer). These results suggested that template-primer duplex containing one isodeoxynucleoside could be recognized by DNA polymerases. However, duplexs containing more than two isodeoxynucleosides could not be recognized by DNA polymerases because of conformational changes by isodeoxynucleosides.

Novel triphosphate derivatives bearing bulky or small groups at alpha-position attached to the triphosphate residue through linkers of different structures and lengths were synthesized and studied as substrates toward terminal deoxynucleotidyltransferase. The substrate efficacy depends on the structure of substituents, linker length, and nature of metal activator. The replacement of hydrophobic groups by small substituents decreased the substrate efficacy by about 20 times in respect to hydrophobic residues. The dependence on metal activator is the following: Co(2+) > Mn(2+) >> Mg(2+). The model of interaction of alkyl triphosphates with linkers of different lengths bearing TdT active site is presented.

The MTH1 protein catalyzes hydrolysis of oxidatively damaged purine nucleotides including 8-hydroxy-dGTP to the monophosphates. The MTH1 protein seems to act as an important defense system against mutagenesis, carcinogenesis, and cell death induced by oxidized purine nucleotides. We previously reported that the functional groups at the 2- and 6-positions of the purine ring affect the recognition by the human MTH1 protein. 8-Hydroxy-dGTP and 8-hydroxy-dATP are substrates of MTH1, and both have the "7,8-dihydro-8-oxo structure." In this study, three nucleotide analogs containing this motif were examined. A synthetic purine analog containing the 7,8-dihydro-8-oxo structure and the 2-amino function (dJTP) was hydrolyzed to the monophosphate with high efficiency by MTH1. On the other hand, two analogs that lack the two-ring system of their bases [formamidopyrimidine-dGTP (FAPY-dGTP) and 2-OH-dYTP] were poor substrates. FAPY-dGTP is a mixture of conformers and was hydrolyzed more than ten-fold less efficiently than 8-hydroxy-dGTP. These results clarify the effects of the 2-amino group and the two-ring system of the purine base on the recognition by the human MTH1 protein.

Enzymatic synthesis of alpha-(32)P and alpha-(33)P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either gamma-(32)P adenosine triphosphate (gamma-ATP) or gamma-(32)P guanosine triphosphate (gamma-GTP). Subsequent removal of these ribonucleotides is essential for the preparation of chemically pure deoxyribonucleotides. Agarose-phenyl boronate columns, which bind specifically to cis-diol moieties, have been used for the removal of ribonucleotide contaminants. However, this involves column losses and additional radiation exposure. In the present work we describe a chemical method for the improvement of the chemical purity, based on the preferential oxidation of ribose sugars by periodate. The cis-diol moiety of ribose is specifically oxidised to the dialdehyde. The excess periodate ions were destroyed using ethylene glycol. The phosphate group was then cleaved by beta-elimination using alkali. The product was purified using anion exchange chromatography. The efficiency of the process was validated using tracer gamma-(32)P ATP and alpha-(32)P dATP. Samples at various steps were analysed by TLC, autoradiography and HPLC. During the process ATP is oxidised whereas 2'-deoxyadenosine triphosphate (dATP) remains intact. The alpha-(32)P dATP synthesized by this process was assayed for its incorporation in lambda-DNA by the random priming method and was found to be effectively incorporated. The process developed is an efficient and convenient method for the preparation of chemically pure deoxyribonucleotides.

The triphosphates of antiviral 2',3'-dideoxynucleosides (ddNs) are the active chemical species that inhibit viral DNA synthesis. The inhibition involves incorporation of ddNMP into DNA and subsequent chain termination. A conceivable strategy for antiviral drugs is to employ nucleoside 5'-triphosphate mimics that can entirely bypass cellular phosphorylation. AZT 5'-alpha-R(P)-borano-beta,gamma-(difluoromethylene)triphosphate (5'-alphaB-betagammaCF(2)TP) has been identified as a potent inhibitor of HIV-1 reverse transcriptase (HIV-1 RT). This work was aimed at confirming that 5'-alphaB-betagammaCF(2)TP is a useful generic triphosphate moiety and can render antiviral ddNs with potent inhibitory effects on HIV-1 RT. Thus, 10 ddNs were converted to their 5'-alphaB-betagammaCF(2)TPs via a sequence (one-pot) of reactions: formation of an activated phosphite, formation of a cyclic triphosphate, boronation, and hydrolysis. Other synthetic routes were also explored. All ddN 5'-alphaB-betagammaCF(2)TPs tested exhibited essentially the same level of inhibition of HIV-1 RT as the corresponding ddNTPs. A conclusion can be made that 5'-alphaB-betagammaCF(2)TP is a generic and promising triphosphate mimic (P3M) concerning HIV-1 RT inhibition and serum stability. It is anticipated that use of 5'-alphaB-betagammaCF(2)TP as P3M moiety will lead to the discovery of a new class of anti-HIV agents.

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2'-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.

Sugar nucleotidyltransferases, also known as sugar pyrophosphorylases, catalyze the formation of a phosphate linkage to produce sugars activated for use by Leloir pathway glycosyltransferases and are subjects of protein engineering for chemoenzymatic synthesis strategies. Herein we present evidence that differences in substrate binding affinity do not primarily account for substantial contrasts in deoxysugar nucleotide product yields with this class of enzymes. Prokaryotic and eukaryotic glucose-1-phosphate uridylyltransferases (EC 2.7.7.9) can exercise kinetic discrimination in choosing carbohydrates of comparable binding affinity for catalytic turnover. These findings have implications for the in vivo and in vitro function and use of these enzymes.

Synthetic pathways to a mononucleotide prodrug of cytarabine (Ara-C) bearing S-pivaloyl-2-thioethyl (tBuSATE) groups, as biolabile phosphate protections, are reported. Using a common phosphoramidite approach, two different kinds of nucleoside protecting groups have been investigated. During this study, we observed an intermolecular migration of the Boc protecting group in the course of the tert-butyldimethylsilyl ether cleavage using tetrabutyl ammonium fluoride.

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.

Several methods for the chemical synthesis of gamma-32P-labeled and unlabeled nucleoside 5(')-triphosphates and thiamine triphosphate (ThTP) have been described. They often proved unsatisfactory because of low yield, requirement for anhydrous solvents, procedures involving several steps or insufficient specific radioactivity of the labeled triphosphate. In the method described here, all these drawbacks are avoided. The synthesis of [gamma-32P]ThTP was carried out in one step, using 1,3-dicyclohexyl carbodiimide as condensing agent for thiamine diphosphate and phosphoric acid in a dimethyl sulfoxide/pyridine solvent mixture. Anhydrous solvents were not required and the yield reached 90%. After purification, [gamma-32P]ThTP had a specific radioactivity of 11Ci/mmol and was suitable for protein phosphorylation. The method can also be used for the synthesis of [gamma-32P]ATP of the desired specific radioactivity. It can easily be applied to the synthesis of unlabeled ThTP or ribo- and deoxyribonucleoside 5(')-triphosphates. In the latter case, inexpensive 5(')-monophosphate precursors can be used as reactants in a 20-fold excess of phosphoric acid. Deoxyribonucleoside 5(')-triphosphates were obtained in 6h with a yield of at least 70%. After purification, the nucleotides were found to be suitable substrates for Taq polymerase during polymerase chain reaction cycling. Our method can easily be scaled up for industrial synthesis of a variety of labeled and unlabeled triphosphoric derivatives from their mono- or diphosphate precursors.

Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming oligo-2'-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-2'-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the parent phosphodiester sequences. Incorporating the modifications into the third oligonucleotide strands has little effect on the structure of the triplexes. At pH 7, the incorporation of two, three or four modified phosphate groups into the third strands stabilizes the triplexes, as compared to the unmodified oligonucleotide. Stronger stabilization was observed with compounds containing linkers ending with amino functions. Stability increases with the number of modifications without being fully additive. This might be due to the different environments of the phosphate groups inside the sequence.

Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In addition, we attached novel fluorophore derivatives. The labeled products were obtained in at least 96% purity after HPLC purification. Enzymatic incorporation into DNA and subsequent extension of the modified DNA chain were studied. Vent(R) exo- DNA polymerase and a defined template-primer system were used to analyze each dye-labeled dNTP derivative. Our data suggest that the incorporation efficiency depends on the selected dye, the nucleobase or a combination of both.

An unnatural hydrophobic base, pyrrole-2-carbaldehyde (denoted as Pa), was developed as a specific pairing partner of 9-methylimidazo[(4,5)-b]pyridine (Q). The Q base is known to pair with 2,4-difluorotoluene (F) as an isostere of the A-T pair, and F also pairs with A efficiently in replication. In contrast, the Q-Pa pair showed specific selectivity in replication, and the five-membered-ring base Pa paired efficiently with Q but paired poorly with A. In addition, the interaction of Pa with DNA polymerases was superior, in comparison to that of F. The aldehyde group of Pa was recognized well by the Klenow fragment of Escherichia coli DNA polymerase I and the reverse transcriptase of Avian myeloblastosis virus. The structural features of the Q-Pa pair in a DNA duplex were analyzed by NMR, showing the shape complementarity of the Pa fitting with Q. The structurally unique base Pa provides valuable information for the development of unnatural base pairs toward the expansion of the genetic alphabet.

A four-color set of negatively charged, single dye as well as energy transfer dye labeled-ddNTPs were synthesized and evaluated in combination with a novel polymerase in a "direct-load" DNA sequencing, obviating the laborious and time consuming post-reaction work-up.

Reaction mechanisms for the diastereoselective synthesis of deoxyribonucleoside 3'-cyclic phosphoramidites as well as dinucleoside phosphite intermediates by the oxazaphospholidine approach were analyzed by means of ab initio molecular orbital calculations at the HF/6-31G* level. These reactions are essential for the diastereoselective synthesis of phsophorothioate DNA.

We report here the synthesis and binding studies of oligo-2'-deoxyribonucleotides (ODNs) containing 2'-deoxyuridines, modified at the 5-position by linkers ending with either one or two guanidinium groups. Comparison was made with ODNs containing 2'-deoxyuridines modified at the 5-position with linkers ending with either two or one amino groups. One or two modified 2'-deoxyuridines were incorporated into pyrimidine strands, and their influence on the stability of duplex (with both DNA and RNA targets) and triplex structures was studied. The strongest stabilization was obtained with modified ODNs containing guanidinium groups. This result confirms that the reduction of the global negative charge number on one strand is an important parameter in the stability of duplex and triplex structures.

The low redox potential of 8-oxo-7,8-dihydroguanine (OG), a molecule regarded as a marker of oxidative damage in cells, makes it an easy target for further oxidation. Using a temperature-dependent method of synthesis, the oxidation products of OG, guanidinohydantoin (Gh) and/or its isomer iminoallantoin (Ia) as well as spiroiminodihydantoin (Sp), have been site-specifically incorporated into DNA oligomers. Single nucleotide insertion and primer extension experiments using Escherichia coli Kf exo(-) DNA polymerase were carried out under "standing start" and "running start" conditions in various sequence contexts. dAMP and dGMP were found to be inserted opposite these OG oxidation products. Steady-state kinetic studies show that the Gh/Ia.G base pair yields a lower K(m) value compared to the Sp.G pair or X.A (X = Gh/Ia or Sp). Running start experiments using oxidized and unoxidized OG-containing templates showed enhanced full extension in the presence of all four dNTPs. A sequence preference for efficiency of extension was found when Gh/Ia and Sp are present in the DNA template, possibly leading to primer misalignment. Full extension is more efficient for the templates containing two Gs immediately 3' to the lesions compared to two As. Although these lesions cause a significant block for DNA elongation, results show that they are more easily bypassed by the polymerase when situated in the appropriate sequence context. UV melting studies carried out on duplexes mimicking the template/primer systems were used to characterize thermal stability of the duplexes. These experiments suggest that both Gh/Ia and Sp destabilize the duplex to a much greater extent than OG, with Sp being most severe.

An efficient procedure is described for the preparation of ribonucleotides and deoxyribonucleotides with deuterium incorporated at the 1', 4', or 5' position. Three intermediates-[1-2H]-D-ribose, [4-2H]-D-ribose, and [5-2H(2)]-D-ribose-were prepared by chemical synthesis and subsequently converted to ribonucleotides and deoxyribonucleotides via enzymatic reactions. Milligram quantities of the desired products were obtained with an average deuterium content of 96+/-1%.

Many reports indicate different nonantisense yet sequence-specific effects of antisense phosphorothioate oligonucleotides. Products of enzymatic degradation of the oligonucleotides can also influence cell proliferation. The cytotoxic effects of deoxyribonucleoside-5'-phosphates (dNMPs) and their 5'-phosphorothioate analogs, deoxyribonucleoside-5'-monophosphorothioates (dNMPSs) on 4 human cell types (HeLa, HL-60, K-562, and endothelial cells) were examined, and the effects were correlated with the catabolism of these compounds. The results indicate that differences in cytotoxicity of dNMPs or dNMPSs in these cells depend upon different activity of an ecto-5'-nucleotidase. It has also been found that dNMPSs stimulate proliferation of human umbilical vein endothelial cells and HL-60 cells in a concentration-dependent manner. This stimulation might be caused by the binding of deoxynucleoside-5'-phosphorothioates to as-yet unidentified nucleotide receptor(s) at the cell surface. (Blood. 2001;98:995-1002)

In the past years, a variety of 4'-C-substituted-2'-deoxynucleosides (4'SdNs) were designed, synthesized, and examined as potential therapeutics against human immunodeficiency virus (HIV) infection and certain such analogues proved to exert potent activity against HIV-1 in vitro. Unlike currently available nucleoside reverse transcriptase (RT) inhibitors such as 3'-azido-3'-deoxythymidine (AZT), which have the 2',3'-dideoxy configuration and thereby cause DNA chain termination in the elongating proviral DNA, 4'SdNs do retain the 3'-alpha-OH moiety but also appear to work against retrovirus as proviral DNA chain terminators. Several 4'SdNs have been shown to be active against various laboratory and clinical HIV-1 strains including known drug-resistant HIV-1 variants. Among such 4'SdNs is 4'-azido-2'-deoxythymidine (4'-AZT) which exerts potent antiviral activity against wild type and AZT-resistant clinical HIV strains. More anti-HIV 4'SdNs have recently been reported including 4'-ethynylthymidine, 4'-ethynyl-2'-deoxy-D-ribofuranosyl-2,6-diaminopurine (4'-E-dDAP), 4'-ethynyl-2'-deoxyguanosine (4'-E-dG) and 4'-ethynyl-2'-deoxyadenosine (4'-E-dA). The latter three analogues are highly potent against HIV-1 and HIV-2 with EC50 values ranging from 0.0003 to 0.01 microM and have favorable cytotoxicity profiles with selective index (SI) values ranging from 975 to 2600. These 4'-ethynyl-2'-deoxynucleosides also exert potent activity against all known drug-resistant HIV-1 variants including the multi-dideoxynucleoside-resistant HIV and the variants with the 6-base pair inserts. Some of these compounds have favorable pharmacological properties and further development as potential therapeutics against HIV-1 infection is warranted.

The first ribo- and deoxyribo-nucleoside (alpha-P-borano, alpha-P-thio)triphosphates have been synthesized. The chemical and biochemical properties of adenosine (alpha-P-borano, alpha-P-thio)triphosphate and thymidine (alpha-P-borano, alpha-P-thio) triphosphate have been investigated.

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N8-(2'-deoxyribonucleoside) (2) and the 7-deazaguanine (pyrrolo[3,4-d]pyrimidine-2-amin-(3H)-4-one) C8-(2'-deoxyribonucleoside) (4) were synthesized and incorporated in oligonucleotides employing phosphoramidite chemistry. Oligonucleotides carrying compound 2 are able to form base pairs with the four canonical DNA constituents without significant structural discrimination. The nucleoside 4 was obtained from the corresponding ribonucleoside by deoxygenation. Oligonucleotides containing compound 4 showed similar base pairing properties as those with 2'-deoxyisoguanosine.

A novel base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and the counter part, pyridin-2-one (denoted y) were designed. The bulky 6-dimethylamino group of x is expected to eliminate base pairing with all natural bases. The phosphoramidite of x for DNA templates and the 2'-deoxyribonucleoside triphosphate of y (dyTP) for a substrate were synthesized, and the selectivity of the enzymatic incorporation of dyTP opposite x in the templates was examined. dyTP was preferentially incorporated opposite x than canonical dNTPs by Klenow fragment of Escherichia coli DNA polymerase I. While dyTP was also incorporated opposite A and G, the misincorporation was suppressed in the presence of dTTP and dCTP, respectively.