Potent RNAi by short RNA triggers

Abstract

RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3'-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an approximately 42 A A-form helix with approximately 1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene, suggesting that 16-nt siRNA is a more potent RNAi trigger. In vitro kinetic analysis of RNA-induced silencing complex (RISC) programmed in HeLa cells indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that RISC assembly and activation during RNAi does not necessarily require a 19-nt duplex siRNA and that 16-nt duplexes can be designed as more potent triggers to induce RNAi.

FIGURE 1.

(A) siRNAs with passenger-strand (PS) deletions as triggers for RNAi. Each GFP siRNA construct shown and reporter plasmids were co-transfected into HeLa cells and RNAi activity was quantified 48 h post-transfection. Relative RNAi activity represents the percentage of GFP knockdown induced by 50 nM siRNA with passenger-strand deletions relative to the inhibition induced by 50 nM 19-nt wild-type siRNA (designated 100%). Each siRNA was tested for knockdown in duplicate in two independent experiments. (B) Effects of guide-strand and two-strand deletions on RNAi activity. The relative RNAi activity of each GFP siRNA construct shown was evaluated as described in (A).

FIGURE 2.

The 16-nt siRNA targeting CDK9 efficiently induces endogenous gene silencing in HeLa cells. (A) The wild-type and 16-nt CDK9 siRNAs used to program RISC are shown. Arrowheads mark target cleavage sites defined by the 5′-end of the guide strand. siRNA sequences are shown in Supplemental Figure 1. (B) The 16-nt CDK9 siRNA more efficiently knocks down CDK9 mRNA than 19-nt WT siRNA. HeLa cells were transfected with 50 nM CDK9 siRNA (19-nt or 16-nt), harvested 48 h post-transfection, and 1 μg total RNA was reverse-transcribed. CDK9 mRNA levels quantified by quantitative PCR and normalized to GAPDH mRNA are presented relative to mRNA levels in mock-transfected cells. Data represent three independent experiments. (C) The 16-nt CDK9 siRNA more efficiently decreases CDK9 protein expression than 19-nt WT siRNA. Immunoblot analysis of CDK9 knockdown by 19-nt and 16-nt siRNAs. HeLa cells were transfected and harvested as in (B), and 120 μg total protein was analyzed by immunoblot using anti-CDK9 and anti-CycT1 antibodies. (D) The 16-nt CDK9 siRNA more efficiently programs RISC to cleave target CDK9 mRNA than 19-nt WT siRNA. CDK9 siRISC was programmed by transfecting HeLa cells with 19-nt or 16-nt siRNA. Target cleavage reaction was performed at 37°C for 90 min. See Materials and Methods for details. The arrow designated “³²P-cap-labeled target” points to full-length ³²P-cap-labeled CDK9 target mRNA. The arrows designated “cleavage product (19-nt)” and “cleavage product (16-nt)” point to products of target mRNA cleavage by CDK9 siRISC programmed with 19-nt and 16-nt siRNA, respectively.

FIGURE 3.

Kinetic analysis of CDK9 16-nt RISC. (A) CDK9 RISC cleavage of target mRNA depends on substrate concentration. Efficiency of target mRNA cleavage by CDK9 RISC programmed by 16-nt siRNA at various substrate concentrations. (B) CDK9 RISC cleavage of target mRNA shows Michaelis–Menten kinetics. Michaelis–Menten analysis of 16-nt RISC. The initial velocity of cleavage product formation was determined from the slope of a curve fit to the cleavage reactions at 0–30 min as shown in (A). (C) The 16-nt siRNA programs more RISC than 19-nt siRNA or 29-nt shRNA. Concentrations of active CDK9 RISC programmed by 16-nt RNA, 19-nt siRNA, or 29-nt shRNA were determined by 2′-O-Me inhibition in vitro cleavage assay. (D) Kinetic analysis of 16-nt CDK9 RISC. K _m and V _max were determined by Michaelis–Menten analysis as shown in (B).