Synthesis of oligonucleotides containing 5'-homo-4'-selenouridine derivative and its increased resistance against nuclease.

As technologies using RNA or DNA have been developed, various chemical modifications of nucleosides have been attempted to increase the stability of oligonucleotides. Since it is known that 2'-OMe-modification greatly contributes to increasing the stability of oligonucleotides, we added 2'-OMe to our previously developed 4'-selenonucleoside and 5'-homo-4'-selenonucleoside as the modified monomers for oligonucleotide: 2'-methoxy-4'-selenouridine (2'-OMe-4'-Se-U) and 5'-homo-2'-methoxy-4'-selenouridine (5'-homo-2'-OMe-4'-Se-U). We synthesized oligonucleotides containing the chemically modified 4'-selenouridine and evaluated their thermal stability and nuclease resistance. In conclusion, the nuclease stability of the oligonucleotide containing 5'-homo-2'-OMe-4'-selenouridine increased while its thermal stability decreased.

Synthesis and Behavior of DNA Oligomers Containing the Ambiguous Z-Nucleobase 5-Aminoimidazole-4-carboxamide.

5-Amino-1-ß-D-ribofuranosylimidazole-4-carboxamide 5'-monophosphate (ZMP) is a central intermediate in de novo purine nucleotide biosynthesis. Its nucleobase moiety, 5-aminoimidazole-4-carboxamide (Z-base), is considered an ambiguous base that can pair with any canonical base owing to the rotatable nature of its 5-carboxamide group. This idea of ambiguous base pairing due to free rotation of the carboxamide has been applied to designing mutagenic antiviral nucleosides, such as ribavirin and T-705. However, the ambiguous base-pairing ability of Z-base has not been elucidated, because the synthesis of Z-base-containing oligomers is problematic. Herein, we propose a practical method for the synthesis of Z-base-containing DNA oligomers based on the ring-opening reaction of an N1-dinitrophenylhypoxanthine (HxaDNP) base. Thermal denaturation studies of the resulting oligomers revealed that the Z-base behaves physiologically as an A-like nucleobase, preferentially forming pairs with T. We tested the behavior of Z-base-containing DNA oligomers in enzyme-catalyzed reactions: in single nucleotide insertion, Klenow fragment DNA polymerase recognized Z-base as an A-like analog and incorporated dTTP as a complementary nucleotide to Z-base in the DNA template; in PCR amplification, Taq DNA polymerase similarly incorporated dTTP as a complementary nucleotide to Z-base. Our findings will contribute to the development of new mutagenic antiviral nucleoside analogs.

Synthesis and properties of fully-modified 4'-selenoRNA, an endonuclease-resistant RNA analog.

A large number of chemically modified oligonucleotides (ONs) have been developed for RNA-based technologies. In most modified RNAs, the characteristic 2'-hydroxyl (2'-OH) groups are removed to enhance both nuclease resistance and hybridization ability. However, the importance of the 2'-OH group in RNA structure and function is well known. Here, we report the synthesis and properties of 4'-selenoRNA in which all four nucleoside units retain the 2'-OH groups but contain a selenium atom instead of an oxygen atom at the 4'-position of the furanose ring. 4'-SelenoRNA has enhanced ability to form duplexes with RNA, and high endonuclease resistance despite the presence of the 2'-OH groups. X-ray crystallography analysis showed that the 4'-selenoRNA duplex adopts an A-conformation, similar to natural RNA, although one 4'-selenocytidine residue has unusual South-type sugar puckering. Furthermore, preliminary studies using 4'-seleno-modified siRNAs suggest that 4'-selenoRNA may be applicable to RNA interference technology. Collectively, our results raise the possibility of a new class of modified RNA in which 2'-OH groups do not need to be masked.