Synthesis, biophysical properties and biological activities of non-ionic RNA analogues having triazole and amide internucleoside linkages

The potential of RNA interference to become a new therapeutic approach has prompted interest in chemical modifications of RNA. Nonionic backbone modifications may increase nuclease resistance, enhance cellular uptake and improve pharmacokinetic and pharmacodynamic properties of short interfering RNAs. This study focuses on synthesis, biophysical and biological studies of triazole and amide backbone modified RNA analogues.;RNAs having extended five-atom triazole linkage were synthesized via the Huisgen [3+2] dipolar cyclo-addition (the "click" reaction). Thermal stability using UV melting studies showed that triazole linkages strongly destabilized RNA duplexes (-7 to -4 °C per mod). These results were further confirmed by the solution NMR and CD data that showed formation of triazole modified RNA duplexes that are more flexible and less stable than unmodified RNA duplexes. In conclusion, our data strongly suggest that the extended triazole is not well tolerated as an internucleosidic linkage in RNA.;Previous studies have pointed out that AM1 amide modification does not disturb the thermal stability, overall helical geometry and hydration of the modified RNAs.1 Our crystallography studies reported herein confirm these observations. Measurements of the torsional angles around the AM1 amide modified sugars show that the carbonyl group (C=O) of amide is inclined towards the major groove of RNA duplex aligning in similar orientation as one of the non-bridging oxygens of the phosphate group. Slight conformational deviations as a result of densely packed amide modified duplexes were observed. This resulted in slight differences in the hydration pattern of modified RNA with two water molecules connecting the phosphate oxygens 5&feet; and 3&feet;- of the amide carbonyl oxygen as opposed to a single water molecule in the native phosphate group. In the minor groove, 2&feet;-OH groups of sugars flanking amide group, appear to be well hydrated while no water molecules are observed linking amide N-H and adjacent phosphate groups.;RNA interference activity of amide modified short interfering RNAs showed that amide group is well tolerated near the 3&feet; end of guide strand. This is consistent with earlier results by Iwase's group. In addition, we showed that AM1 amide linkages are not only tolerated at the internal positions of both guide and passenger strands of siRNAs, but may also increase the silencing activity when placed near the 5&feet;-end of the passenger strand. Taken together, our crystallography and RNA interfering studies suggest that AM1 amide group may be excellent mimic of RNA phosphate linkage.