FIGURE 4.

Influence of divalent cations on the cleavage activities of human Ago2 containing antisense RNA with deoxyribonucleotide substitutions. A, denaturing PAGE of 40-nucleotide sense RNA digested with GST-Ago2 containing antisense RNAs with various deoxyribonucleotide substitutions in the presence of Mg²⁺. B, denaturing PAGE of 40-nucleotide sense RNA digested with GST-Ago2 containing antisense RNAs with various deoxyribonucleotide substitutions in the presence of Mn²⁺. Antisense RNA was incubated with Ago2 for 1 h prior to incubation with the 40-nucleotide sense RNA for an additional 1 h. Arrows indicate the position of the human Ago2 cleavage site. C, sequences of the antisense oligonucleotides with the corresponding lane designations. The antisense oligonucleotides contained either a 5′-phosphate (p) or 5′-hydroxyl (OH) and DNA substitutions (underlined). D, percent cleaved of the 40-nucleotide sense RNA by GST-Ago2 containing the antisense sequences shown in C in the presence of either Mg²⁺ (solid bar) or Mn²⁺ (hatched bar). The errors reported for the Ago2 cleavage activities are based on three trials.