Smart conferring of nuclease resistance to DNA by 3′-end protection using 2′,4′-bridged nucleoside-5′-triphosphates

Incorporation of 2′,4′-bridged nucleotides into the 3′-end of oligodeoxyribonucleotide (ODN) was examined using terminal deoxynucleotidyl transferase (TdT). The three types of 2′,4′-bridged nucleoside-5′-triphospates with different bridging structures used were incorporated efficiently into the 3′-end of DNA by TdT, although only single nucleotide incorporation was observed. Nuclease resistance was conferred on DNA, depending on the types of bridging nucleotides added.     

Insight into the recognition mechanism of DNA cytosine-5 methyltransferases (DNMTs) by incorporation of acyclic 5-fluorocytosine (FC) nucleosides into DNA

DNA cytosine-5 methyltransferase (DNMT) catalyzes methylation at the C5 position of cytosine in the CpG sequence in double stranded DNA to give 5-methylCpG (mCpG) in the epigenetic regulation step in human cells. The entire reaction mechanism of DNMT is divided into six steps, which are scanning, recognition, flipping, loop locking, methylation, and releasing. The methylation and releasing mechanism are well-investigated; however, few reports are known about other reaction steps. To obtain insight into the reaction mechanism, we planned the incorporation of acyclic nucleosides, which make it easy to flip out the target nucleobase, into oligodeoxynucleotides (ODNs) and investigated the interaction between the ODN and DNMT. Here, we describe the design and synthesis of ODNs containing new acyclic 5-fluorocytosine nucleosides and their physiological and biological properties, including their interactions with DNMT. We found that the ODNs containing the acyclic 5-fluorocytosine nucleoside showed higher flexibility than those that contain 5-fluoro-2′-deoxycytidine. The observed flexibility of ODNs is expected to influence the scanning and recognition steps due to the decrease in helicity of the B-form.     

3′-Modified oligodeoxyribonucleotides for the study of 2-deoxyribose damage in DNA

Well-defined substrates for the study of oxidative processes are important for the elucidation of the role of DNA damage in the etiology of diseases such as cancer. We have synthesized 3′-modified oligodeoxyribonucleotides (ODNs) using 5′ → 3′ ‘reverse’ DNA synthesis for the study of 2-deoxyribose oxidative damage to DNA. The modified monomers designed for these studies all share a common feature, they lack the naturally occurring 3′-hydroxyl group found in 2-deoxyribonucleosides. Modified H-phosphonates containing 3′-phenyl selenides as well as saturated and unsaturated sugars were obtained and incorporated in ODNs. These ODNs were used to investigate the fate of C3′-dideoxyribonucleotide radicals in DNA.     

Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase

Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.     

The Behaviour of 2′-Deoxy-2′-Fluorouridine Incorporated into Oligonucleotides by the Phosphoramidite Approach

Abstract

2′-Deoxy-2′-fluorouridine has been chemically incorporated into an oligodeoxynucleotide of the structure 5′ACGGAX 3′ (X=U(2′-F)) using the phosphoramidite method and the behaviour of the product has been studied. 5′-O-Monomethoxytrityl-2′-deoxy-2′-fluorouridine was fixed on silica gel at the 3′-end and the chain elongated on a DNA-synthesizer using nucleoside methoxyphosphoramidites. After alkaline work-up two products were observed. One was found to be the desired fluoro containing hexamer, whereas the other corresponds to an araU-hexamer (X=arabino-furanosyluridine). The latter compound is supposed to be a product of alkaline hydrolysis of the C-2′-F-bond. The oligomers containing 2′-fluoro- and ara-U at their 3′-end were chemically sequenced by a solid phase method on CCS-paper which confirmed the right primary structure.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

Preparation and enzymatic recognition of guanine lesions induced by nitrogen oxides.

Xanthine (Xan) and oxanine (Oxa) are the major deamination products of guanine formed by the treatment with nitrogen oxides (e.g., NO and HNO2). In this study, 2'-deoxyribonucleoside 5'-triphosphates of Xan and Oxa were prepared by the NaNO2 treatment of dGTP. These modified nucleotides were incorporated into oligonucleotides by DNA polymerase reactions. The repair activities of various DNA N-glycosylases for Xan and Oxa were examined using these substrates.     

The Mechanism of Dna Repair by Uracil-Dna Glycosylase: Studies Using Nucleotide Analogues

Abstract

2′,4′-Dideoxy-4′-methyleneuridine incorporated into oligodeoxynucleotides forms regular B-DNA duplexes as shown by T_m and CD measurements. Such oligomers are not cleaved by the DNA repair enzyme, UDG, which cleaves the glycosylic bond in dU but not in dT nor in dC nucleosides in single stranded and double stranded DNA. Differential binding of oligomers containing carbadU, 4′-thiodU, and dU residues to wild type and mutant UDG proteins identify an essential role for the furanose 4′-oxygen in recognition and cleavage of dU residues in DNA.     

Synthesis and properties of PNA oligomers containing orotic acid derivatives

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.     

Synthesis and Evaluation of Oligodeoxynucleotides Containing 3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine: Introduction of a Novel Class of Phosphodiester Internucleoside Linkages

Abstract

3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine (5) was synthesized and converted into the phosphoramidite building block 8. Novel oligodeoxynucleotide analogues containing 4′-C-hydroxymethyl phosphodiester internucleoside linkages were synthesized on an automated DNA-synthesizer. The hybridization properties and enzymatic stability were studied on oligomers with one to four modifications. The 3′-end modified oligodeoxynucleotides were resistent towards 3′-exonuclease degradation and showed only moderate lowered affinity towards complementary DNA compared with oligodeoxynucleotides bearing modifications in the middle.     

Synthesis and recognition by DNA polymerases of a reactive nucleoside, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide

We report the synthesis of a new nucleoside, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH2)) as a reactive monomer for DNA diversification. The 5'-triphosphate derivative (dY(NH2)TP, 1) was evaluated in vitro as a substrate for several DNA polymerases. Primer extension reactions showed that dYNH2TP was well tolerated by KF (exo(-)) and Vent (exo-) DNA polymerases. One dYNH2MP was incorporated opposite each canonical base with an efficiency depending on the template base (A approximately T > G > C). Significant elongation after YNH2 incorporation was observed independently of the YNH2:N base pair formed. When the nucleobase YNH2 was incorporated into synthetic oligodeoxynucleotides via the phosphoramidite derivative 11, it directed the insertion of natural bases as well as itself. The mutagenicity of dYNH2TP was evaluated by PCR amplification using Vent (exo-) DNA polymerase. The triphosphate dY(NH2)TP was preferentially incorporated as a dATP or dGTP analogue and led to misincorporations at frequencies of approximately 2 x 10(-2) per base per amplification. A high proportion of transversions with a large distribution of all possible mutations was obtained. The reactivity of the nucleobase YNH2 within a template with several aldehydes was demonstrated.     

Quantitative parameters of the 3′–5′ exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 with nicked DNA containing dYMP or a modified dCMP analogue

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, that incises DNA 5′ to the AP-site, it possesses other weak enzymatic activities. One of them is 3′–5′ exonuclease activity, which is most effectively exhibited for DNA duplexes containing modified or mismatched nucleotides at the 3′-end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase β through the base excision repair process. We determined the quantitative parameters of the 3′–5′ exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3′ terminus of single-strand DNA and of photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined.     

SYNTHESIS AND PROPERTIES OF PNA OLIGOMERS CONTAINING OROTIC ACID DERIVATIVES

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.     

CONTROL OF DNA CONFORMATION USING 3′-S-PHOSPHOROTHIOLATE-MODIFIED LINKAGES

An in-depth study into the incorporation of multiple 3′-S-phosphorothiolate modifications into oligodeoxynucleotides (ODNs) and their subsequent effect on ODN/DNA and ODN/RNA duplex stability. 3′-S-Phosphorothiolate linkages increase the stability of ODN/RNA duplexes and decrease the stability of ODN/DNA duplexes.     

Effect of Acridine with Various Linker Arms Attached to C5 Position of 2′-Deoxyuridine on the Stability of DNA/DNA and DNA/RNA Duplexes

Abstract

Acridine-modified oligodeoxyribonucleotides (ODNs) at the C5-position of a 2′-deoxyuridine via different lengths of linker arms were synthesized. Reaction of 5-(N-aminoalkyl)carbamoylmethyl-2′-deoxyuridines with 9-phenoxyacridine gave the acridine-modified 2′-deoxyuridines which were incorporated into ODNs. The duplexes containing the acridine-modified strands and their complementary DNA or RNA were thermally more stable than that containing the unmodified strand. Thermal stability of the duplexes of the modified ODNs varied depending on the length of the linker arms.

     

Repair and replication of oxidized DNA bases using modified oligodeoxyribonucleotides

Modified oligodeoxyribonucleotides (ODNs) are powerful tools to assess the biological significance of oxidized lesions to DNA. For this purpose, we developed original synthetical pathways for the site-specific insertion of several oxidized bases into DNA fragments. Thus, the chemical solid-phase synthesis of ODNs using original strategies of protection and mild conditions of deprotection, as well as a specific post-oxidation approach of an unique nucleoside residue within the sequence have been applied. These two approaches of preparation allowed us to have access to a set of modified ODNs that contain a single modified nucleoside, i.e., N-(2-deoxy-beta-D-erythro-pentofuranosyl)formylamine (dF), 5-hydroxy-2'-deoxycytidine (5-OHdCyd), thymidine glycol (dTg), 5,6-dihydrothymidine (DHdThd), 2,2-diamino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)-amino]-5(2H)- oxazolone (dZ), N-(2-deoxy-beta-D-erythro-pentofuranosyl)cyanuric acid (dY), 5',8-cyclo-2'-deoxyguanosine (cyclodGuo) and 5',8-cyclo-2'-deoxyadenosine (cyclodAdo). The substrates were used to investigate recognition and removal of the lesions by bacterial DNA N-glycosylases, including endonuclease III (endo III) and Fapy glycosylase (Fpg). In addition, the DNA polymerase-mediated nucleotide incorporation opposite the damage was determined using modified ODNs as templates.     

Automated synthesis of new ferrocenyl-modified oligonucleotides: study of their properties in solution

We have developed new ferrocenyl-modified oligonucleotide (ODN) probes for electrochemical DNA sensors. A monofunctional ferrocene containing phosphoramidite group has been prepared, and a new bisfunctional ferrocene containing phosphoramidite and dimethoxytrityl (DMT) groups has been developed. These ferrocenyl-phosphoramidites have been directly employed in an automated solid-phase DNA synthesizer using phosphoramidite chemistry. The advantages of this method are that it allows a non-specialist in nucleotide chemistry to access labeled ODNs and that it has demonstrated good results. ODNs modified at the 3′ and/or 5′ extremities have been prepared, with the incorporation of the ferrocenyl group into the chain. The 5′ position appears to be more important due to its particular behavior. The thermal stability and electrochemical properties of these new ODN ferrocenes were analyzed before and after hybridization with different ODNs. The feasibility of using these new ferrocenyl-labeled ODNs in DNA sensors has been demonstrated.     

Practical and Reliable Synthesis of 1,2-Dideoxy-d-ribofuranose and its Application in RNAi Studies

We developed a practical and reliable method for synthesizing an abasic deoxyribonucleoside, 1,2-dideoxy-d-ribofuranose (dR^H) via elimination of nucleobase from thymidine. To synthesize oligonucleotides bearing dR^H by the standard phosphoramidite solid-phase method, dR^H was converted to the corresponding phosphoramidite derivative and linked to a solid support (controlled pore glass resin). Chemically modified small interfering RNAs (siRNAs) possessing dR^H at their 3′-overhang regions were synthesized. Introducing dR^H to the 3′-end of the antisense strand of siRNA reduced its knockdown effect.     

Hybridization-dependent fluorescence of oligodeoxynucleotides incorporating new pyrene-modified adenosine residues

We report the synthesis and properties of oligonucleotides incorporating N(6)-[N-(pyren-1-ylmethyl)carbamoyl]-deoxyadenosine (dA(pymcm)). We designed the ODN which incorporated two consecutive dA(pymcm) residues. It was revealed that on hybridization with the target DNA and RNA oligomers, the fluorescence spectra of ODNs having two consecutive dA(pymcm) molecules near the 5'-terminal position can change from the pyrene monomer emission to the excimer, depending on the chain length of the target DNA and RNA. These results indicated that dA(pymcm)-modified ODNs can be used as interesting hybridization sensors that are sensitive to the size of the target strand.