Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3\u27-CH2-CO-NH-5\u27 amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5\u27-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications

Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides

Phosphorus-modified phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides were chemically synthesized and their biochemical properties evaluated. Under physiological pH, these DNA analogs possess negative charge and form stable, complementary A-like DNA:RNA heteroduplexes when analyzed via circular dichroism spectroscopy. Phosphonoacetate and thiophosphonoacetate oligomers were found to stimulate RNase H activity and to be completely resistant to degradation by snake venom phosphodiesterase, DNase I and HeLa cell nuclear extract. Further research has demonstrated that neutral, esterified forms of these analogs can be taken up by cells. Phosphonoacetate and thiophosphonoacetate oligomers therefore represent a new class of oligodeoxyribonucleotide analogs having phosphorus– carbon bonds with considerable potential for use in biological research