Engineering ligand-responsive gene-control elements: lessons learned from natural riboswitches

Abstract

In the past two decades, remarkable advances have been made in the development of technologies used to engineer new aptamers and ribozymes. This has encouraged interest among researchers who seek to create new types of gene control systems that can be made to respond specifically to small molecule signals. Validation that RNA molecules can exhibit the characteristics needed to serve as precision genetic switches has come from the discovery of numerous classes of natural ligand-sensing RNAs called riboswitches. While a great deal of progress has been made towards engineering useful designer riboswitches, considerable advances are needed before the performance characteristics of these RNAs match those of protein systems that have been co-opted to regulate gene expression. In this review, we will evaluate the potential for engineered RNAs to regulate gene expression and lay out possible paths to designer riboswitches based upon currently available technologies. Furthermore, we will discuss some technical advances that would empower RNA engineers who seek to make routine the production of designer riboswitches that can function in eukaryotes.

Synthetic Botany

“…The thiamine pyrophosphate riboswitch is an endogenous example in plants: It was identified in the 3 0 end of the untranslated region of the Arabidopsis thiC gene, and exerts control by affecting pre-mRNA splicing of the transcript (Sudarsan et al 2003;Wachter et al 2007). Methods have been developed to tune the sensitivities of riboswitches for their cognate ligands (Beisel and Smolke 2009), and to expand their chemical diversity (reviewed in Link and Breaker 2009).…”

Reengineering orthogonally selective riboswitches

“…Notably, insertion of aptamers into the 5′-UTRs of bacterial or eukaryotic mRNAs (9)(10)(11)(12), splice sites within introns of eukaryotic pre-mRNA (13,14), or coupling of aptamers to ribozymes (15,16) have shown significant potential for small-molecule modulation of gene expression. However, there are currently very few aptamers, generated by in vitro selection, that are selective for ligands with desirable physicochemical and pharmacokinetic properties (8). Moreover, in vitro selection approaches can be laborious (8), and it is not clear if some of the existing aptamers are completely selective for the desired ligand over structurally related molecules present within the cell.…”

“…However, there are currently very few aptamers, generated by in vitro selection, that are selective for ligands with desirable physicochemical and pharmacokinetic properties (8). Moreover, in vitro selection approaches can be laborious (8), and it is not clear if some of the existing aptamers are completely selective for the desired ligand over structurally related molecules present within the cell. An alternative approach, which has been little explored to date, would be to reengineer existing natural riboswitches (17).…”

“…In eukaryotes, riboswitches present within introns have also been shown to affect alternative splicing pathways (7). These simple protein-free mechanisms make the engineering of natural or synthetic riboswitches an attractive strategy for developing new small-molecule responsive regulatory systems (8).…”

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

“…Hence, they enable conditional cleavage of RNA, including mRNAs, and fulfill all of the above-mentioned criteria. Moreover, they can be engineered for ON-or OFF-switch activity by modification of the aptamer-ribozyme linking sequences (8)(9)(10). Compared with well-established inducible promoters, as used for example in the Tet system (11), aptazymes are much smaller (approximately 100 bp) and do not require the expression of recombinant, potenSignificance Riboswitches are short RNA sequences for ligand-dependent modulation of gene expression in cis.…”