Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch.

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.

Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells.

The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.

Design of Artificial Riboswitches as Biosensors.

RNA aptamers readily recognize small organic molecules, polypeptides, as well as other nucleic acids in a highly specific manner. Many such aptamers have evolved as parts of regulatory systems in nature. Experimental selection techniques such as SELEX have been very successful in finding artificial aptamers for a wide variety of natural and synthetic ligands. Changes in structure and/or stability of aptamers upon ligand binding can propagate through larger RNA constructs and cause specific structural changes at distal positions. In turn, these may affect transcription, translation, splicing, or binding events. The RNA secondary structure model realistically describes both thermodynamic and kinetic aspects of RNA structure formation and refolding at a single, consistent level of modelling. Thus, this framework allows studying the function of natural riboswitches in silico. Moreover, it enables rationally designing artificial switches, combining essentially arbitrary sensors with a broad choice of read-out systems. Eventually, this approach sets the stage for constructing versatile biosensors.

Synthetic Riboswitches: From Plug and Pray toward Plug and Play.

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.