Shana Topp

Emerging Applications of Riboswitches in Chemical Biology

Living systems use RNA sequences known as riboswitches to detect the concentrations of small-molecule metabolites within cells and to regulate the expression of genes that produce these metabolites. Like their natural counterparts, synthetic riboswitches also regulate gene expression in response to small molecules. Because synthetic riboswitches can be engineered to respond to nonendogenous small molecules, they are powerful tools for chemical and synthetic biologists interested in understanding and reprogramming cellular behavior. In this review, we present an overview of natural riboswitches, highlight recent studies toward developing synthetic riboswitches and provide an overview of emerging applications of these RNA switches in chemical biology.

Random Walks to Synthetic Riboswitches—A High‐Throughput Selection Based on Cell Motility

A major goal of chemical and synthetic biologists is to create ligand-dependent genetic-control systems to report on cellular metabolism, to construct synthetic gene circuits, or to reprogram cellular behavior. Because designing genetic switches de novo is challenging, many groups have employed directedevolution strategies to achieve these goals. 2] Our laboratory recently reported a high-throughput robotic screen that was used to identify synthetic riboswitches that displayed low background levels of gene expression in the absence of a ligand, and strongly activated gene expression in the presence of the ligand. We subsequently demonstrated that these riboswitches could control bacterial motility in a ligand-dependent fashion. Because the differences in cell motility at different ligand concentrations were easy to distinguish by using only a ruler, we asked whether motility differences could be the basis of a high-throughput selection to discover new synthetic riboswitches from large libraries. We envisioned that this method could equal, if not exceed, the throughput of our previously reported robotic screen, and could be performed at a fraction of the cost. Here we report an inexpensive and operationally simple selection method based on cell motility that not only approaches the throughput of a genetic selection, but also provides the quantitative nature of a genetic screen. We further show that this selection quickly identifies synthetic riboswitches that display low background levels of gene expression in the absence of a ligand, and robust increases in the presence of a ligand. We anticipate that motility-based selections will be generally useful in the discovery of rare events from large genetic libraries. There is good precedent for using motility to select for rare events. For decades, microbiologists have identified rare mutants by spotting cells at the center of a Petri dish containing semisolid media, and manually selecting cells that migrate abnormally. Capitalizing on these successes, Goulian and coworkers recently developed a motility-based selection to discover mutant chemoreceptors that could recognize a new ligand. Because a riboswitch displays two different phenotypes, depending on whether or not the ligand is present, a selection process must be able to quantitatively assay both the “on” and “off” states. Such counter-selections, which select for one phenotype and against another, are powerful, but can be challenging to implement. We envisioned that a motility-based selection could be used to discover “on” switches by selecting for mutants that do not move in the absence of the ligand, and then further selecting for mutants that move in the presence of the ligand. Of course, there are many ways that one can imagine setting up the experiment (for example, looking for “off” switches), but the key is to be able to rapidly and ACHTUNGTRENNUNGinexpensively determine the phenotype under two sets of conditions. To compare our motility selection to a more traditional screening technique, we assayed combinatorial libraries that were similar to those previously reported by our laboratory, which were comprised of four to eight randomized base pairs flanked by the mTCT8-4 theophylline aptamer and a fixed ribosome-binding site upstream of the b-galactosidase reporter gene. E. coli expressing these libraries were assayed by using a multistep process that used a robotic colony picker and an ACHTUNGTRENNUNGautomated liquid-handling system. While our previous assay identified several outstanding synthetic riboswitches, it relied on expensive robotics, in addition to large quantities of consumables. We anticipated that a motility-based selection could eliminate the need for specialized capital equipment, and identify synthetic riboswitches with only standard consumables. To select for riboswitches by using motility as the readout, we used cheZ as a reporter gene. CheZ plays a critical role in E. coli chemotaxis by dephosphorylating the CheY-P protein, which binds to the flagellar motor and causes cells to tumble. Optimal levels of CheZ are necessary for E. coli cells to migrate on semisolid media. If too little CheZ is present, the level of CheY-P will increase, and the cells will tumble incessantly and not migrate. If cells have excess CheZ, they will swim very smoothly and rarely tumble. Because cells that swim extremely smoothly can become embedded in the semisolid media, they cannot migrate. Thus it is critical to ensure that CheZ is not over-expressed in these assays. Since the strength of the promoter will ultimately dictate the maximum expression level of the cheZ gene, we began with two different promoters: a “weak” IS10 promoter and the tac promoter, which is 60to 100-fold stronger (S.T. unpublished results). We anticipated that the motility selections would readily reveal which promoter provides the appropriate CheZ expression level. Using cassette-based PCR mutagenesis, we constructed a library in which the mTCT8-4 theophylline aptamer was followed by ten consecutive randomized base pairs (N10) in the 5’ untranslated region (5’ UTR) of cheZ (Figure 1). We chose to assay an N10 library that lacked a preset ribosome-binding site for these experiments rather than our previously described “N8” library, for two reasons. First, we anticipated that this selection could more effectively sample the additional sequence space; and second, having the additional [a] S. Topp, Prof. J. P. Gallivan Department of Chemistry and Center for Fundamental and Applied Molecular Evolution Emory University 1515 Dickey Drive, Atlanta, GA 30322 (USA) Fax: (+1)404-727-6586 E-mail : justin.gallivan@emory.edu Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

A riboswitch-based inducible gene expression system for mycobacteria.

Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

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

In natural and engineered systems, cis-RNA regulatory elements such as riboswitches are typically found within untranslated regions rather than within the protein coding sequences of genes. However, RNA sequences with important regulatory roles can exist within translated regions. Here, we present a synthetic riboswitch that is encoded within the translated region of a gene and represses Escherichia coli gene expression greater than 25-fold in the presence of a small-molecule ligand. The ability to encode riboswitches within translated regions as well as untranslated regions provides additional opportunities for creating new genetic control elements. Furthermore, evidence that a riboswitch can function in the translated region of a gene suggests that future efforts to identify natural riboswitches should consider this possibility.

Engineering bacteria to recognize and follow small molecules

The ability to recognize and react to specific environmental cues allows bacteria to localize to environments favorable to their survival and growth. Synthetic biologists have begun to exploit the chemosensory pathways that control cell motility to reprogram how bacteria move in response to novel signals. Reprograming is often accomplished by designing novel protein or RNA parts that respond to specific small molecules not normally recognized by the natural chemosensory pathways. Additionally, cell motility and localization can be coupled to bacterial quorum sensing, potentially allowing consortia of cells to perform complex tasks.

From SELEX to cell dual selections for synthetic riboswitches.

Synthetic riboswitches have emerged as useful tools for controlling gene expression to reprogram cellular behavior. However, advancing beyond proof-of-principle experiments requires the ability to quickly generate new synthetic riboswitches from RNA libraries. In this chapter, we provide a step-by-step overview of the process of obtaining synthetic riboswitches for use in Escherichia coli, starting from a randomized RNA library.

Feline leukemia virus integrase and capsid packaging functions do not change the insertion profile of standard Moloney retroviral vectors

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.

High-throughput screens to discover synthetic riboswitches.

Synthetic riboswitches constructed from RNA aptamers provide a means to control bacterial gene expression using exogenous ligands. A common theme among riboswitches that function at the translational level is that the RNA aptamer interacts with the ribosome-binding site (RBS) of a gene via an intervening sequence known as an expression platform. Structural rearrangements of the expression platform convert ligand binding into a change in gene expression. While methods for selecting RNA aptamers that bind ligands are well established, few general methods have been reported for converting these aptamers into synthetic riboswitches with desirable properties. We have developed two such methods that not only provide the throughput of genetic selections, but also feature the quantitative nature of genetic screens. One method, based on cell motility, is operationally simple and requires only standard consumables; while the other, based on fluorescence-activated cell sorting (FACS), is particularly adept at identifying synthetic riboswitches that are highly dynamic and display very low levels of background expression in the absence of the ligand. Here we present detailed procedures for screening libraries for riboswitches using the two methods.

Characterization of engineered preQ1 riboswitches for inducible gene regulation in mycobacteria

We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from <2 to ∼10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches.IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.