Structural and thermodynamic insights into the PAZ/3¿-overhang interaction to improve siRNA specificity

Abstract

The discovery of the RNA interference (RNAi) pathway and the identification of the small interfering RNA (siRNA) molecule as RNAi trigger,1 have simplified the study of gene function paving the way to the treatment of any disease-related gene.2 The siRNA molecules are short double-stranded RNAs, of approximately 21 nucleotides (nt) in length, bearing 2-nt overhangs at both 3-ends. The siRNA molecule is initially incorporated into the RISC (RNA-Induced Silencing Complex) in double stranded fashion, then a process called RISC maturation results in the dissociation of siRNA passenger strand from the guide strand, permitting the recognition of target messenger RNA (mRNA). The process of siRNA loading requires the action of the RISCLoading Complex (RLC), which includes DICER, Argonaute2 (Ago2) and TRBP (HIV-1 TAR RNA Binding Protein) proteins. The Ago2 protein is a cradle-shaped protein of four main domains: MID (middle), PIWI (Pelement-induced wimpy testes), PAZ (Piwi/Argonaute/Zwille) and N-terminal. Herein, we conduct a systematic study of Ago2 response by introducing chemical modifications at siRNA 3’-overhang by both computational and experimental techniques. Atomistic simulations and free energy calculations allow us to propose a robust and self-contained procedure for studying the factors governing PAZ/siRNA interactions. Results from calculations based on two different crystal structures allow direct comparison and consistency checks. An overall description of the systems is thus achieved, which is followed by a comprehensive experimental study on the 3’-overhang structural requirements for the design of more specific siRNA molecules.