Figure 5.

5΄ -(E)-vinylphosphonate infers resistance against phosphatases in vivo and 5΄-3΄ exonuclease in vitro. (A) HPLC traces of Cy3-PNA/hsiRNA hybrids in liver lysates from mice harvested 1 week after intravenous (IV) or subcutaneous (SC) injection with 5΄-hydroxyl (5΄-OH), 5΄-phosphate (5΄-P), or and 5΄-(E)-vinylphosphonate (5΄-VP) hsiRNA. The ‘spike-in’ (left) panels show control traces of Cy3-PNA/hsiRNA hybrids after full-length guide strands were spiked into liver lysates from untreated mice. 5΄-P spike-in guide strands elute more slowly than 5΄-OH guide strands, corresponding to the difference of one charge (phosphate). Metabolite profile in liver lysates clearly show dephosphorylation of 5΄-P hsiRNA but partially intact 5΄ end of 5΄-VP hsiRNA. (B) Urea-PAGE of 5΄-hydroxyl (5΄-OH), 5΄-phosphate (5΄-P), or and 5΄-(E)-vinylphosphonate (5΄-VP) hsiRNAs resolved on an 7 M urea/24% polyacrylamide gel after 12 h incubation in the absence (–) or presence (+) of Terminator™ enzyme. 5΄-P and 5΄-VP (no PS) compound had the same nucleotide modification pattern (2΄-F, 2΄-O-Me) as 5΄-P, 5΄-OH and 5΄ -VP, but did not contain phosphorothioate (PS) internucleotide linkages. 5΄-VP hsiRNA is protecting against degradation by Terminator™ enzyme, whereas 5΄-P hsiRNA is degraded, and phosphorothioate internucleotide linkage interferes with enzyme processivity.