Three-dimensionally designed protein-responsive RNA devices for cell signaling regulation

Abstract

The three-dimensional (3D) structures of many biomacromolecules have been solved to reveal the functions of these molecules. However, these 3D structures have rarely been applied to constructing efficient molecular devices that function in living cells. Here, we demonstrate a 3D structure-based molecular design principle for constructing short hairpin RNA (shRNA)-mediated genetic information converters; these converters respond to specific proteins and trigger the desired gene expression by modulating the function of the RNA-processing enzyme Dicer. The inhibitory effect on Dicer cleavage against the shRNA designed to specifically bind to U1A spliceosomal protein was correlated with the degree of steric hindrance between Dicer and the shRNA-protein complex in vitro: The level of the hindrance was predicted based on the models. Moreover, the regulation of gene expression was achieved by using the shRNA converters designed to bind to the target U1A or nuclear factor-κB (NF-κB) p50 proteins expressed in human cells. The 3D molecular design approach is widely applicable for developing new devices in synthetic biology.

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals

“…Here, we present a robust and reversible RNA-based switching element that allows efficient control of miRNA processing. Previous work documented ‘one-way’ switching of small non-coding RNA biogenesis to switch off processing through the addition of the respective ligands to aptamer-coupled systems ( 25 , 26 , 28 , 29 , 53 ). However, for short-term interference applications, the operating system must be fully reversible to allow a recovery to the original expression level.…”

“…In a comparable approach, various aptamers together with competing strands were integrated into shRNA loop structures affecting the processing of the respective shRNAs by ligand-dependent switching between two structural conformations ( 26 ). Furthermore, protein-binding motifs for the human U1A protein and L7Ae, or the p50-binding aptamer positioned in the shRNA loop region have been shown to regulate shRNA processing by Dicer ( 27 , 28 ). In another design, small molecule or protein-binding aptamers were integrated into the basal segment of a synthetic pri-miRNA bearing an siRNA.…”

Modular construction of mammalian gene circuits using TALE transcriptional repressors

“…Sophisticated control of living cells demands synthetic gene circuits that are capable of sensing multiple endogenous molecular signals. RNA interference provides a versatile interface that allows synthetic gene circuits to modularly sense and integrate endogenous molecular inputs in mammalian cells 18 , 44 - 48 . In this work, we demonstrated that endogenous microRNAs can be used to control the state of TALER sensory switches and that the sensitivity of TALER switches to shRNAs is tunable by adjusting the ratio of the two primary components.…”

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…In another study, a novel design of aptamer-siRNA fusions enabled siRNA-processing control in mammalian cells (Fig. 2C) (Kashida et al 2012). Proteinbinding RNA aptamers (e.g., U1A protein-U1A RNA motif ) have been engineered into siRNA precursors that can replace RNA stem loops that are essential for siRNA processing.…”