Conditional RNA Interference Mediated by Allosteric Ribozyme

Abstract

Conditional RNA interference (RNAi) enables spatial and/or temporal control over gene silencing. The currently available methods require coexpression of engineered proteins and/or modified promoters which may limit their applications. We designed a novel RNA architecture that combines a drug-inducible allosteric ribozyme with a microRNA precursor analogue that allows chemical induction of RNAi in mammalian cells. The compact and highly modular RNA design should facilitate the construction of conditional RNAi systems that can sense and respond to a variety of molecules recognized by RNA aptamers to regulate virtually any desired genes sensitive to RNAi.

Thermozymes

“…So far, aptamers binding several ligands were combined with HHRs functioning as expression platforms in order to control mRNAs, tRNAs, 16S rRNA as well as RNAi in organisms as diverse as bacteria and mammalian cells. 11,15,16,21,[23][24][25]41 The present work shows that a physical stimulus such as a change in temperature can also be used in order to regulate gene expression via a hammerhead-mediated mRNA cleavage reaction. Importantly, most natural RNAT and all previously engineered RNAT are composed of secondary structures that mask the SD sequence.…”

Artificial Ribozyme-Based Regulators of Gene Expression

“…48,50 The group of Yokobayashi even showed that HHR self-cleavage is capable of regulating RNAi substrates. 56 All these RNA parts exhibit a highly modular nature, which should simply allow for the exchange of one ligand-binding aptamer to another. However, many aptazyme switches reported by different groups described here have been shown to work only with a limited number of aptamers.…”

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…Ligand-responsive ribozymes (aptazymes) have been fused to siRNA precursors to block siRNA maturation to construct ligand-responsive siRNA-based gene switches in mammalian cells ( Fig. 2A) (Kumar et al 2009;Nomura et al 2012). The addition of the small-molecule ligand theophylline or guanine induces ribozyme self-cleavage and releases the respective siRNA precursor, which further matures into a functional siRNA molecule that can repress target gene expression.…”