Effect of target secondary structure on RNAi efficiency

Abstract

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. However, the levels of knockdown vary greatly. Here, we examine the effect of target disruption energy, a novel measure of target accessibility, along with other parameters that may affect RNAi efficiency. Based on target secondary structures predicted by the Sfold program, the target disruption energy represents the free energy cost for local alteration of the target structure to allow target binding by the siRNA guide strand. In analyses of 100 siRNAs and 101 shRNAs targeted to 103 endogenous human genes, we find that the disruption energy is an important determinant of RNAi activity and the asymmetry of siRNA duplex asymmetry is important for facilitating the assembly of the RNA-induced silencing complex (RISC). We estimate that target accessibility and duplex asymmetry can improve the target knockdown level significantly by nearly 40% and 26%, respectively. In the RNAi pathway, RISC assembly precedes target binding by the siRNA guide strand. Thus, our findings suggest that duplex asymmetry has significant upstream effect on RISC assembly and target accessibility has strong downstream effect on target recognition. The results of the analyses suggest criteria for improving the design of siRNAs and shRNAs.

Function-based gene identification using enzymatically generated normalized shRNA library and massive parallel sequencing

“…S2B). As presented elsewhere (27), we have also found that the activity in the cDNA dataset was significantly correlated with target disruption energy, a measure of accessibility of the target mRNA. cDNA-derived shRNAs showed lower activity when their disruption energy was <−15 kcal/mol, which we have used as the sixth filtering criterion.…”

Dual-targeting siRNAs

“…Known factors that influence siRNA efficacy include the difference in the duplex end thermodynamic stabilities (DDG) (Khvorova et al 2003;Schwarz et al 2003), sequence motifs within the guide strand (Reynolds et al 2004;Saetrom and Snove 2004), the overall duplex stability (Reynolds et al 2004), and the target site accessibility (Heale et al 2005;Shao et al 2007). Of these, only the strand's sequence similarity to highly effective standard siRNAs and DDG showed weak to strong correlations with the observed siRNA activities.…”

“…We used RNAfold (Hofacker 2003), RNAhybrid (Rehmsmeier et al 2004), and sfold (Shao et al 2007) to compute duplex end and intrastrand stability, overall duplex stability, and target site accessibility. To compute the stability of a given duplex end without overhangs, we placed 1-5 nt from each strand on opposite ends of an artificial hairpin sequence cassette (59-CCLLLL LGG-39; L denotes a loop nucleotide) and used RNAfold to predict the stability of the resulting hairpin sequence.…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…When searching for factors influencing knock-down efficiency, attention turned to the local RNA structure at siRNA target sites and it was demonstrated that local RNA target structure is an important factor for siRNA efficacy (Schubert et al, 2005). Accordingly, siRNA design tools started to accommodate not only properties of siRNAs but also properties of the target site because it strongly increased efficiency of designed siRNAs (Heale et al, , 2006Shao et al, 2007;Tafer et al, 2008).…”