Synthetic Riboswitches: From Plug and Pray toward Plug and Play

Abstract

In synthetic biology, metabolic engineering, and gene therapy, there is a strong demand for orthogonal or externally controlled regulation of gene expression. Here, RNA-based regulatory devices represent a promising emerging alternative to proteins, allowing a fast and direct control of gene expression, as no synthesis of regulatory proteins is required. Besides programmable ribozyme elements controlling mRNA stability, regulatory RNA structures in untranslated regions are highly interesting for engineering approaches. Riboswitches are especially well suited, as they show a modular composition of sensor and response elements, allowing a free combination of different modules in a plug-and-play-like mode. The sensor or aptamer domain specifically interacts with a trigger molecule as a ligand, modulating the activity of the adjacent response domain that controls the expression of the genes located downstream, in most cases at the level of transcription or translation. In this review, we discuss the recent advances and strategies for designing such synthetic riboswitches based on natural or artificial components and readout systems, from trial-and-error approaches to rational design strategies. As the past several years have shown dramatic development in this fascinating field of research, we can give only a limited overview of the basic riboswitch design principles that is far from complete, and we apologize for not being able to consider every successful and interesting approach described in the literature.

Applications and limitations of regulatoryRNAelements in synthetic biology and biotechnology

“…So far, synthetically engineered riboswitches have been employed mostly for proof‐of‐concept studies, using reporter genes like green fluorescent protein (GFP) in model microbes like E. coli, Bacillus subtilis and Saccharomyces cerevisiae to demonstrate functionality (Topp et al . ; Etzel and Morl ; Hallberg et al . ).…”

“…Instead, they have mostly been used in proof‐of‐concept studies or as fluorescently tagged biosensors (Topp et al . ; Etzel and Morl ; Hallberg et al . ).…”

“…Practical applications of synthetic biology are often limited to relatively simple tasks that may involve only model organisms (Topp et al . ; Etzel and Morl ; Hallberg et al . ).…”

“…Although riboswitches can be bioengineered with relative ease, and they hold great promise in various applications (e.g. bioenergetics, biofuels, therapy, bioremediation and biochemical engineering) (Etzel and Morl ; Hallberg et al . ; Villa et al .…”

RNA-based dynamic genetic controllers: development strategies and applications

“…These methods rely on structure-prediction algorithms to identify structural requirements and calculate free energies. However, current algorithms are limited in accurately calculating the contributions of pseudoknots and non-Watson-Crick base pairings to the overall structure [40]. Additionally, binding of a ligand to an RNA molecule can induce substantial structural rearrangements.…”