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.