Figure 4.

Purified Drosophila Dicer-2 discriminates duplex termini. (A) PhosphorImage of products separated by 17% denaturing PAGE after a 30 min incubation of 0.8 nM ³²P-end-labeled (bottom strand) 40/42 dsRNAs (as in Figure 2C) in cleavage buffer without Dicer (−) or with 30 nM wildtype Dicer-2 or helicase mutants as indicated. All reactions contained 8 mM ATP except those lacking ATP (−ATP). (B) Data from multiple northern blot analyses (probes as in Figure 3A) from cleavage reactions of 106 3'ovr-3'ovr and BLT-BLT with WT Dicer-2 and incubation times as indicated, were quantified to determine ratio of siRNAs derived from internal cleavage to those from 5' and 3' termini. % siRNA, as in Figure 2D; error bars, standard error of the mean (n≥ 3). (C) PhosphorImage of northern blot showing products electrophoresed as in (A), produced by incubation of 1 nM 82 BLT-BLT or 3'ovr-3'ovr dsRNAs in cleavage buffer (30 min), without (−) or with 10 nM WT or K34N Dicer-2, without ATP (−ATP) or with 5 mM ATP (+ATP). Left blot, internal probe; Right blot, 3' end probe; asterisks, cleavage intermediates; FL, full length 82 dsRNA. siRNA sizes were determined by comparing to AH ladder. See also Figure S3.