Hybridization and Mismatch Discrimination Abilities of 2',4'-Bridged Nucleic Acids Bearing 2-Thiothymine or 2-Selenothymine Nucleobase.

Oligonucleotides modified with 2'- O,4'- C-spirocyclopropylene-bridged nucleic acid (scpBNA) exhibit excellent duplex-forming ability with their complementary single-stranded RNA (ssRNA). Here, we demonstrate that scpBNA bearing a 2-thiothymine (scpBNA-S2T) or 2-selenothymine (scpBNA-Se2T) nucleobase provides robust mismatch discrimination capabilities to oligonucleotides without compromising their high binding affinities toward the full complementary ssRNA. X-ray crystallographic analysis of a self-assembling oligonucleotide featuring 2',4'-BNA/LNA-2-thiothymine (2',4'-BNA/LNA-S2T, where 2',4'-BNA and LNA stand for "2'- O,4'- C-methylene-bridged nucleic acid" and "locked nucleic acid", respectively), a prototype of scpBNA-S2T, revealed that the 2-thiocarbonyl moiety plays a crucial role in the destabilization of thymine-guanine mismatched wobble base pairs.

Thioamide-Bridged Nucleic Acid (thioAmNA) Containing Thymine or 2-Thiothymine: Duplex-Forming Ability, Base Discrimination, and Enzymatic Stability.

Oligonucleotides containing bridged nucleic acids (BNAs) show high duplex-forming ability towards target single-stranded RNA, so many BNAs have been developed for antisense applications. Amide-bridged nucleic acids (AmNAs), which are BNA analogues bearing an amide bond at the bridge, exhibit high duplex-forming ability, enzymatic stability, and antisense activity; thus, the AmNA motif represents a promising BNA scaffold. The high enzymatic stability of the AmNA motif is presumably attributable to the bulky amide structure, because it inhibits the access of nucleases to the phosphodiester linkage. Here, to improve enzymatic stability further, we designed thioAmNAs: thioamide-bridged nucleotides that have a bulkier bridge structure than AmNA. The synthesis of thioAmNAs bearing either thymine (thioAmNA-T) or 2-thiothymine (thioAmNA-S2 T) bases was successful, and the obtained monomers were introduced into designed oligonucleotides without noticeable by-product generation. The thioAmNA-T- and thioAmNA-S2 T-modified oligonucleotides showed strong binding affinity toward complementary single-stranded RNA, with the thioAmNA-S2 T-modified oligonucleotide displaying excellent base-discrimination capability. Moreover, both thioAmNA-T and thioAmNA-S2 T endowed oligonucleotides with higher resistance to enzymatic degradation than AmNA-T. These results indicate that thioAmNAs are potentially useful chemical modifications for oligonucleotide-based therapeutics.