Genetic Screens and Selections for Small Molecules Based on a Synthetic Riboswitch That Activates Protein Translation

Abstract

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.

Riboswitches in unexpected places—A synthetic riboswitch in a protein coding region

“…As expected, gene expression remained unrepressed for all caffeine concentrations tested (0-2 mM). To further demonstrate the ligand dependence of the riboswitch, we introduced a previously verified point mutation (C27A) to the aptamer that significantly reduces its affinity for theophylline but permits binding of 3-methylxanthine (Soukup et al 2000;Desai and Gallivan 2004). The mutated repressor exhibited high levels of gene expression when cells were grown in the absence of ligand or in the presence of 1 mM theophylline, while gene expression was reduced to one tenth of the unrepressed levels when cells were grown in the presence of 1 mM 3-methylxanthine (11,000, 11,000, and 910 Miller Units, respectively).…”

“…Plasmid DNA was extracted from each culture and digested with KpnI and HindIII. Each library of inserts was cloned in front of a lacZ reporter gene in a previously reported pUC18-based vector (Desai and Gallivan 2004) that was digested with the same enzymes. TOP10F9 cells were transformed with these libraries, and cells were plated on media containing ampicillin, X-gal, and 1 mM theophylline.…”

“…Previous work has revealed that bacterial riboswitches that act on a translational level often regulate gene expression through ligand-dependent structural changes in the 59-UTR that provide or limit access to the ribosome binding site (RBS) of an RNA transcript (Nudler and Mironov 2004). This insight has inspired efforts to convert synthetic aptamers into riboswitches through rational engineering (Werstuck and Green 1998;Desai and Gallivan 2004;Suess 2005) or genetic screens and selections (Lynch et al 2007;Topp and Gallivan 2008;Weigand et al 2008). Here, we report that synthetic riboswitches that modulate translation in bacteria can be located within the coding region of a gene and that opportunities for developing new riboswitches may be enhanced by placing fewer restrictions on the expected mechanisms of RNA-based genetic regulation.…”

FRET-Based Optical Assay for Monitoring Riboswitch Activation

“…23 The aptamer moiety of this riboswitch has been well-characterized and displays a 10000-fold higher affinity for theophylline when compared to structurally related caffeine. 24 Desai and Gallivan were the first to demonstrate the theophylline-dependent activation of a reporter gene -galactosidase in E. coli, utilizing an externally added substrate X-gal to produce a color change in cells.…”

Synthetic circuits, devices and modules

“…Upon binding, the riboswitch can affect the conformation of its adjacent mRNA, resulting in either the premature termination of transcription, inhibition of translation initiation, or mRNA self-cleavage (Winkler, 2005). Once discovered in nature, riboswitches have been quickly applied to control gene expression (Desai and Gallivan, 2004;Suess et al, 2004). Recently Dixon et al designed riboswitches that are selective for synthetic "nonnatural" small molecules rather than the natural intracellular ligands (Dixon et al, 2010).…”