Conformation matters: siRNAs with antisense strands with 5'-( E )-vinyl-phosphonate-α-L-LNA elicit stronger RNAi-mediated gene silencing than those with 5'-( E )-vinyl-phosphonate-LNA.

Conformationally constrained nucleotides, LNA or α-L-LNA, at the 5' terminus of the antisense strand impeded gene silencing of small interfering RNA (siRNA) by hindering phosphorylation, thereby deterring loading into the RNA-induced silencing complex. Installation of a phosphate mimic, ( E )-vinyl phosphonate (VP), improved activity considerably. Gene silencing was more efficient when the antisense strand of the siRNA was modified with 5'-VP-α-L-LNA, which adopts a C3'- exo (south) conformation, than when the antisense strand was modified with 5'-VP-LNA, which adopts a C3'- endo (north) pucker. These data underscore the critical role of conformation of nucleotides in RNA interference.