In RNA synthesis without base protection, a new method for O-selective condensation with more than 99% selectivity was developed by 6-nitro-HOBt-mediated cleavage of undesired P(III)-N bonds on nucleobase moieties. Moreover, we for the first time succeeded in synthesizing oligoRNAs without base protection.
Oligodeoxynucleotide (ODN) synthesis, which avoids the formation of side products, is of great importance to biochemistry-based technology development. One side reaction of ODN synthesis is the cyanoethylation of the nucleobases. We suppressed this reaction by synthesizing ODNs using fully protected deoxynucleoside 3′-phosphoramidite building blocks, where the remaining reactive nucleobase residues were completely protected with acyl-, diacyl-, and acyl-oxyethylene-type groups. The detailed analysis of cyanoethylation at the nucleobase site showed that N3-protection of the thymine base efficiently suppressed the Michael addition of acrylonitrile. An ODN incorporating N3-cyanoethylthymine was synthesized using the phosphoramidite method, and primer extension reactions involving this ODN template were examined. As a result, the modified thymine produced has been proven to serve as a chain terminator.
To clarify the biochemical behavior of 2′-deoxyribonucleoside 5′-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (Co) and adenine N-oxide (Ao), we examined their base recognition ability in DNA duplex formation using melting temperature (Tm) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the Tm 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 Co and Ao 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 Ao 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.
The main products obtained by oxidation of cytosine and adenine bases with hydrogen peroxide are cytosine and adenine N-oxide derivatives. There is a possibility that these N-oxide derivatives are mutagenic in genomic DNA, such as 8-oxoguanine or thymine glycol. Although the chemical synthesis and properties of 2'-deoxynucleoside N-oxide derivatives have been well established, little has been reported about the chemical and biochemical behavior of DNA oligomers containing these modified 2'-deoxynucleoside. In this study, we examined their base recognition ability by T(m) experiments and computer modeling, and their substrate specificity in enzyme reaction. It was found that the T(m) values of in DNA-DNA, DNA-RNA duplexes incorporating 2'-deoxynucleoside N-oxide derivatives were significant low, while the one-point incorporation of these modified derivatives into the 3'-terminal site of a DNA oligomer by DNA polymerase occurred accurately selecting the complementary G or T base on a template DNA oligomer.
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