Conformation of formacetal and 3'-thioformacetal nucleotide linkers and stability of their antisense RNA DNA hybrid duplexes

This study focuses on the characterization of the stability and conformation of the antisense oligodeoxyribonucleotides, d[CGCGTT x TTGCGC] [x = phosphodiester (-O-P(O)(2)-O-), formacetal (FMA, -O-CH2-O-), or 3'-thioformacetal linkage (TFMA, -S-CH2-O-)], in DNA . DNA and RNA . DNA duplexes (designated DI-III and RI-III, respectively). NMR analysis of two RNA . DNA hybrid duplexes containing a single FMA (the RII duplex) or 3'-TFMA (the RIII duplex) modification has been carried out. The conformations of these duplexes are compared with that of the unmodified hybrid duplex RI and with those of the DI-III duplexes. These analyses and comparisons indicate that the residue containing a 3'-FMA linker has a preference for the C2'-endo sugar pucker and adopts a canonical backbone conformation. In contrast, the residue containing a 3'-TFMA linker has a much increased preference for the C3'-endo sugar pucker and adopts different backbone conformations in the DNA . DNA and RNA . DNA duplexes. UV and NMR melting studies of the six duplexes demonstrate that the DNA . DNA duplexes are more stable than the corresponding RNA . DNA hybrid duplexes and that both FMA and 3'-TFMA destabilize the duplex. The 3'-TFMA modified duplex is less stable than the FMA duplex in the context of DNA . DNA recognition and is slightly more stable than the FMA hybrid duplex in the context of RNA . DNA recognition. These results suggest a correlation between the conformational preference of backbone modifications and the stability of antisense duplexes. The implications of these studies for optimized incorporation of FMA and 3'-TFMA linkers into oligonucleotides and for better design of antisense oligonucleotide analogs are discussed.