A FACS‐Based Approach to Engineering Artificial Riboswitches

Nucleic acids have established themselves as valuable building blocks for the construction of synthetic elements with a wide range of uses in biological research. Indeed, nucleic acid enzymes (ribozymes and deoxyribozymes) and receptors (aptamers) have emerged as major players in the era of synthetic biology, and can be applied in such disciplines as biosensing, ACHTUNGTRENNUNGinity purification, nanodevices and therapeutic development. The success in isolating these elements can be largely attributed to the methods used to select for molecules that carry out a specific function from extremely large, random sequence libraries. While a great deal of effort and progress has been made toward advancing these protocols in vitro, synonymous methods for selections within a cellular environment are far less developed. This shortcoming is becoming more relevant as our understanding of the diverse array of functions that nucleic acids—particularly RNA—have within cells continues to grow at an astounding pace. To have the ability to produce these interesting new RNA molecules with customdesigned characteristics would be invaluable to biological research. One of the most intriguing classes of newly discovered RNAs is a group of genetic control elements called riboswitches. They reside within the untranslated regions (UTRs) of mRNA where they sense the metabolic status of the cell through directly binding a relevant ligand. The binding event results in structural changes in the RNA, which ultimately leads to modulation of the level of expression of neighbouring gene or genes. The bulk of natural riboswitches that have been uncovered have been found in bacteria where they regulate the ACHTUNGTRENNUNGexpression of a remarkable number of genes in response to a growing number of different metabolic cues. From a molecular engineering point of view, riboswitches represent a highly desirable design for artificial gene expression systems. They have a very simple composition that consists of an RNA receptor domain (aptamer domain) and a neighbouring expression platform with a simple stem–loop architecture. Riboswitches are relatively small, cis-acting elements, usually encoded by 300 bases or less. These factors simplify the design, optimisation and troubleshooting associated with their creation. RiboACHTUNGTRENNUNGswitches have shown the potential to be extremely efficient as well, and demonstrate both remarkable specificity and impressive induction levels in response to their cognate ligand. Artificial riboswitches could be designed for a wide range of applications. One such possibility is the engineering of programmable cells. This could include the production of cells that have an array of genes under the control of different inducer molecules; this would open the door to experiments for working out the interplay of numerous genes or pathways. Programmed bacteria also have great potential as tools for environmental cleanup and drug delivery. Topp and Gallivan ACHTUNGTRENNUNGrecently made progress toward this goal by employing an artificial riboswitch to create a strain of E. coli cells that migrate specifically toward the small molecule theophylline. We believe that riboswitches also hold great potential as intracellular sensors for the detection and quantification of small molecules. While these fields are still largely unexploited, the continuing development of methods to create and modify riboACHTUNGTRENNUNGswitches should make such systems possible. The goal of this work was to develop a fast, efficient and general intracellular selection method for isolating riboswitches from a library of sequences. In order to sort through a large number of sequences quickly and easily, we opted to use fluorescent proteins and fluorescence activated cell sorting (FACS). FACS can be used to sort individual cells based on their fluorescence intensity at rates that exceed 10 cells per second. Previously FACS has been employed to identify proteins with desirable properties from libraries of random mutants. Here, we apply similar thinking to identify riboswitch mediated gene regulation from a library “potential riboswitches”. Figure 1A demonstrates the principle of our selection scheme in which cells that are highly fluorescent only in response to the small-molecule inducer of our choice are isolated by using two steps of FACS. As a pilot project to demonstrate our selection scheme we chose to seek riboswitches that respond to the small molecule theophylline. We built a library of “potential riboswitches” that consisted of an RNA aptamer that binds theophylline connected to an intrinsic transcriptional terminator by a partially random linker region (Figure 1B). Transcriptional terminators are RNA elements that consist of a simple stem–loop followed by a string of uracil residues. When RNA polymerase encounters such an element it is released from the template and sequences downstream are not transcribed. We sought linker regions that were able to transduce the binding of theophylline to a disruption of the neighbouring transcriptional terminator stem; this would result in activating transcription of the downstream green fluorescent protein (GFP) gene. This riboACHTUNGTRENNUNGswitch mechanism (transcriptional termination) appears to be the most common, and has not been exploited by artificial ACHTUNGTRENNUNGriboswitches that have been created to date. Before beginning the selection process we first chose each of the elements for the construction of our riboswitch library. The TCT8-4 theophylline aptamer, which has previously been [a] C. C. Fowler, Prof. Dr. E. D. Brown, Prof. Dr. Y. Li Department of Biochemistry and Biomedical Sciences McMaster University, 1200 Main Street, W. Hamilton ON L8N 3Z5 (Canada) Fax: (+1)905-522-9033 E-mail : liying@mcmaster.ca Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.