Gene silencing using micro-RNA designed hairpins

TLDR

RNA interference uses ~21‑nt siRNAs derived from long dsRNA to silence genes via mRNA degradation, while endogenous miRNAs and stRNAs, also ~21‑nt, are processed from hairpin precursors and repress translation. Synthetic hairpin RNAs that mimic siRNAs and miRNA precursors silence target genes through mRNA degradation, with activity dependent on sequence and structure and retained when expressed from Pol III DNA vectors, enabling stable RNAi loss‑of‑function studies.

Abstract

During RNA interference (RNAi), long dsRNA is processed to ∼21 nt duplexes, short interfering RNAs (siRNAs), which silence genes through a mRNA degradation pathway. Small temporal RNAs (stRNAs) and micro-RNAs (miRNAs) are ∼21 nt RNAs that are processed from endogenously encoded hairpin-structured precursors, and function to silence genes via translational repression. Here we report that synthetic hairpin RNAs that mimic siRNAs and miRNA precursor molecules can target a gene for silencing, and the mechanism of silencing appears to be through mRNA degradation and not translational repression. The sequence and structural configuration of these RNAs are important, and even slight modification in structure can affect the silencing activity of the hairpins. Furthermore, these RNAs are active when expressed by DNA vectors containing polymerase III promoters, opening the possibility for new approaches in stable RNAi-based loss of function studies.