siRNA Specificity: RNAi Mechanisms and Strategies to Reduce Off-Target Effects

Abstract

Short interfering RNAs (siRNAs) are processed from long double-stranded RNA (dsRNA), and a guide strand is selected and incorporated into the RNA-induced silencing complex (RISC). Within RISC, a member of the Argonaute protein family directly binds the guide strand and the siRNA guides RISC to fully complementary sites on-target RNAs, which are then sequence-specifically cleaved by the Argonaute protein-a process commonly referred to as RNA interference (RNAi). In animals, endogenous microRNAs (miRNAs) function similarly but do not lead to direct cleavage of the target RNA but to translational inhibition followed by exonucleolytic decay. This is due to only partial complementarity between the miRNA and the target RNA. SiRNAs, however, can function as miRNAs, and partial complementarity can lead to miRNA-like off-target effects in RNAi applications. Since siRNAs are widely used not only for screening but also for therapeutics as well as crop protection purposes, such miRNA-like off-target effects need to be minimized. Strategies such as RNA modifications or pooling of siRNAs have been developed and are used to reduce off-target effects.

Keywords: RISC; RNAi; microRNAs; off-target effects; siRNAs.

FIGURE 1

(A) Schematic representation of human Ago2. Argonaute proteins contain four conserved domains: the N domain (red), the PAZ domain (green), the MID domain (blue), and the PIWI domain (yellow). The PIWI domain contains the four catalytic residues D597, E637, D669, and H807 that are required for mRNA target cleavage. (B) Crystal structure of human Ago2 loaded with a siRNA (PDB ID 5JS1) (Schirle et al., 2016). The four domains are colored as in panel (A), the catalytic residues are highlighted in pink, and the 5′ region of the loaded siRNA is shown in orange. (C) Detailed view of catalytic center of the Ago2 structure from panel (B).

FIGURE 2

The guide strand of the siRNA or a miRNA is loaded into an Argonaute protein. In case of a perfect complementarity through siRNAs or miRNAs (which is often observed in plants), a catalytically active Argonaute protein cleaves the mRNA as part of RISC (left). As miRNAs in animals are only partially complementary to their target RNAs, Slicer-facilitated cleavage is impaired. In this case, Argonaute recruits a member of the GW protein family. These proteins mediate the interaction with further downstream acting factors like poly-(A)-binding proteins (PABPs) or the deadenylase complexes PAN2/PAN3 and CCR4/NOT. This leads to translational repression, deadenylation, decapping, and 5′–3′ exonucleolytic decay of the mRNA. Translational repression by miRNAs has also been observed for plant miRNAs.