Engineering Biological Systems with Synthetic RNA Molecules

Abstract

RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors.

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

“…116 Finally, bacteria show potential for use in the development of RNA devices that can be transferred to other cellular platforms for more relevant applications since many RNA functions are considered portable and as such can be transferred from one kingdom to the other with no considerable additional adaptation. 117,118 Additionally, the highly valuable ability of bacterial sRNAs to modulate multiple pathways in metabolic engineering applications could strengthen the potential of using bacteria as a bio-factory.…”

“…So far, we have only touched the surface of all these questions by focusing on a handful of riboswitches and small RNAs via different approaches: rational design (sometimes computationally aided), molecular evolution, and the use of natural elements found in nature. 21,34 Increased fundamental understanding in this area will continue to broaden our creativity in exploiting the many different types of non-coding RNAs that we suspect are present in nature in a much wider range of impactful applications.…”

“…20 Another advantage of using RNA-based sensing approaches is that these molecules exhibit more compact genetic footprints than their protein counterparts and, as such, are more "economical" since they generally place less of an energetic load on the cell by not requiring translation. 21 Furthermore, RNA-based post-transcriptional responses generally act at a faster time scale than transcription-based strategies 22 and RNA-based mechanisms can be evolved more rapidly than protein-based mechanisms. 23 Since 1982, when the Nobel Prize committee recognized the discovery of RNA catalytic properties 24 as groundbreaking, 25 several "big" catalytic RNAs have been exploited for various biotechnological and medical applications.…”

“…One of the most powerful examples of this has been the ability to tune riboswitch recognition to discriminate between subtle structural differences in small molecules that differ only by the presence of an H and a CH 3 groups (e.g., theophylline vs. caffeine) with nM affinity. 21 Indeed, it is the strong sensitivity intrinsic to these RNA switches that is highly exploitable in the detection of small molecules, macromolecules, and external stimuli. This extreme specificity is attributed to a modular component of riboswitches called the aptamer (Fig.…”