The siRNA Non-seed Region and Its Target Sequences Are Auxiliary Determinants of Off-Target Effects.

Yuko Nakano,Ilya Ioshikhes,Paul A Wilson,Piotr J Kamola,Tomoko Takahashi,Kumiko Ui-Tei

doi:10.1371/journal.pcbi.1004656

Abstract

RNA interference (RNAi) is a powerful tool for post-transcriptional gene silencing. However, the siRNA guide strand may bind unintended off-target transcripts via partial sequence complementarity by a mechanism closely mirroring micro RNA (miRNA) silencing. To better understand these off-target effects, we investigated the correlation between sequence features within various subsections of siRNA guide strands, and its corresponding target sequences, with off-target activities. Our results confirm previous reports that strength of base-pairing in the siRNA seed region is the primary factor determining the efficiency of off-target silencing. However, the degree of downregulation of off-target transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential. Here, we demonstrate that both the melting temperature (Tm) in a subsection of siRNA non-seed region, and the GC contents of its corresponding target sequences, are negatively correlated with the efficiency of off-target effect. Analysis of experimentally validated miRNA targets demonstrated a similar trend, indicating a putative conserved mechanistic feature of seed region-dependent targeting mechanism. These observations may prove useful as parameters for off-target prediction algorithms and improve siRNA ‘specificity’ design rules.

Highlights

RNA interference (RNAi) is a highly regulated, evolutionarily conserved mechanism of posttranscriptional gene regulation
The function is exerted through the RNA interference (RNAi) pathway and has revolutionised biological research due to its ease-of-use and high potency
The approach allowed us to distinguish three, well separated clusters which sequence composition correlated with RNAi effects (Fig 2): [1] positively correlated cluster in the siRNA seed region, [2] negatively correlated cluster in the non-seed region, [3] positively correlated 3’ termini position

Summary

Introduction

RNA interference (RNAi) is a highly regulated, evolutionarily conserved mechanism of posttranscriptional gene regulation. When siRNAs are transfected into a cell, one of the siRNA strands (guide strand) is incorporated into the RNA-induced silencing complex (RISC) while the opposite strand (passenger strand) is degraded [2,3]. The activated siRNA-containing RISC (siRISC) recognises and binds to the target transcript in a sequence-specific manner (Fig 1B). The perfectly complementary region within the target transcript is cleaved between the 10th and 11th nucleotide relative to the 5’ end of the guide strand [4]. This elegant, endogenous process has been extensively utilised in functional genomics studies and shows potential as a therapeutic platform [5]

Methods

Results

Discussion

Conclusion