Engineered riboswitches: Expanding researchers' toolbox with synthetic RNA regulators

Abstract

a b s t r a c tRiboswitches are natural RNA-based genetic switches that sense small-molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within the last years, several engineered riboswitches have been developed that act on various stages of gene expression. These switches can be engineered to respond to any ligand of choice and are therefore of great interest for synthetic biology. In this review, we present an overview of engineered riboswitches and discuss their application in conditional gene expression systems. We will provide structural and mechanistic insights and point out problems and recent trends in the development of engineered riboswitches.

Regulation of Gene Expression in Diverse Cyanobacterial Species by Using Theophylline-Responsive Riboswitches

“…Aptamers that bind a ligand of choice can be obtained through in vitro evolution methods, and expression platform function can be obtained through in vivo screening. Because we observed some toxicity toward cyanobacteria at 2 mM theophylline, the potential to develop novel riboswitches responsive to alternative, less toxic ligands is a key benefit of using riboswitches to regulate gene expression (40). While positive selection markers such as antibiotic resistance genes can be used for screening of novel riboswitches, negative selection markers can also be useful.…”

Design criteria for synthetic riboswitches acting on transcription

“…1 In synthetic biology, they are often used to design complex gene regulation circuits. [2][3][4][5] In recent years there has been great effort in designing synthetic riboswitches that can in principle bind any ligand and regulate any step in gene expression of interest. 6 Regulation of transcription is of special interest, as it allows for a tight regulation in an early step of gene expression.…”

Regulatory RNAs

“…However, a major drawback in the analysis of large-scale metagenomic sequence data sets is the isolation of functional elements, given that these RNAs tend to: (1) be short (often mistaken for chemically degraded RNAs), (2) lack open reading frames, and (3) sometimes evolve rapidly at the sequence levels while still conserving structure that is integral to their function. 107,108 Need for novel in vitro RNA stabilization methods Although new sensing capabilities of natural and unnatural ligands have been successfully engineered into RNAs via in vitro and in vivo molecular evolution approaches, [109][110][111] the ability to use these molecules by incorporating them into more complex systems and devices represents a bigger practical challenge. 23 All potential RNA Synthetic Biology applications require reproducible and controlled molecular stability, especially when activity is required outside the native cellular host and in the context of other biological and inorganic materials.…”