A family of synthetic riboswitches adopts a kinetic trapping mechanism.

Riboswitches are sequences of RNA that control gene expression via RNA-ligand interactions, without the need for accessory proteins. Riboswitches consist of an aptamer that recognizes the ligand and an expression platform that couples ligand binding to a change in gene expression. Using in vitro selection, it is possible to screen large (∼ 10(13) members) libraries of RNA sequences to discover new aptamers.

Riboswitches for intracellular study of genes involved in Francisella pathogenesis.

Unlabelled: The study of many important intracellular bacterial pathogens requires an understanding of how specific virulence factors contribute to pathogenesis during the infection of host cells. This requires tools to dissect gene function, but unfortunately, there is a lack of such tools for research on many difficult-to-study, or understudied, intracellular pathogens. Riboswitches are RNA-based genetic control elements that directly modulate gene expression upon ligand binding.

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.

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.

Synthetic riboswitches that induce gene expression in diverse bacterial species.

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

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.

Reprogramming bacteria to seek and destroy an herbicide.

A major goal of synthetic biology is to reprogram cells to perform complex tasks. Here we show how a combination of in vitro and in vivo selection rapidly identifies a synthetic riboswitch that activates protein translation in response to the herbicide atrazine. We further demonstrate that this riboswitch can reprogram bacteria to migrate in the presence of atrazine.

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.

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.

A flow cytometry-based screen for synthetic riboswitches.

Riboswitches regulate gene expression through direct, small molecule-mRNA interactions. The creation of new synthetic riboswitches from in vitro selected aptamers benefits from rapid, high-throughput methods for identifying switches capable of triggering dramatic changes in gene expression in the presence of a desired ligand. Here we present a flow cytometry-based screen for identifying synthetic riboswitches that induce robust increases in gene expression in the presence of theophylline.

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.

Toward reprogramming bacteria with small molecules and RNA.

A major goal of synthetic biology is to reprogram bacteria to carry out complex tasks, such as synthesizing and delivering drugs, and seeking and destroying environmental pollutants. Advances in molecular biology and bacterial genetics have made it straightforward to modify, insert, or delete genes in many bacterial strains, and advances in gene synthesis have opened the door to replacing entire genomes. However, rewriting the underlying genetic code is only part of the challenge of reprogramming cellular behavior.

Guiding bacteria with small molecules and RNA.

Chemotactic bacteria navigate their chemical environment by coupling sophisticated information processing capabilities to molecular motors that propel the cells forward. The ability to reprogram bacteria to follow entirely new chemical signals would create powerful new opportunities in bioremediation, bionanotechnology, and synthetic biology. However, the complexities of bacterial signaling and limitations of current protein engineering methods combine to make reprogramming bacteria to follow novel molecules a difficult task.

A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function.

Riboswitches are RNA-based genetic control elements that regulate gene expression in a ligand-dependent fashion without the need for proteins. The ability to create synthetic riboswitches that control gene expression in response to any desired small-molecule ligand will enable the development of sensitive genetic screens that can detect the presence of small molecules, as well as designer genetic control elements to conditionally modulate cellular behavior. Herein, we present an automated high-throughput screening method that identifies synthetic riboswitches that display extremely low background levels of gene expression in the absence of the desired ligand and robust increases in expression in its presence.

A genetic toolbox for creating reversible Ca2+-sensitive materials.

A major goal of polymer science is to develop "smart" materials that sense specific chemical signals in complex environments and respond with predictable changes in their mechanical properties. Here, we describe a genetic toolbox of natural and engineered protein modules that can be rationally combined in manifold ways to create reversible self-assembling materials that vary in their composition, architecture, and mechanical properties. Using this toolbox, we produced several materials that reversibly self-assemble in the presence of Ca2+ and characterized these materials using particle-tracking microrheology.

Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation.

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.