Frameworks for Programming Biological Function through RNA Parts and Devices

Win, Maung Nyan; Liang, Jack; Smolke, Christina D.

doi:10.1016/j.chembiol.2009.02.011

Cited by 109 publications

(92 citation statements)

References 127 publications

(154 reference statements)

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“…They are potential agents for disease diagnosis and treatment (Famulok et al 2007) and for biotechnological applications like biosensors . Aptamers are also used as riboswitches for conditional gene regulation (Suess and Weigand 2008;Win et al 2009). Such RNA molecules are isolated by SELEX (Systematic Evolution of Ligands by EXponential Enrichment) from a random pool of RNA molecules (Ellington and Szostak 1990;Tuerk and Gold 1990).…”

Section: Introductionmentioning

confidence: 99%

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

Wunnicke

Strohbach²,

Weigand³

et al. 2010

RNA

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RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 29-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.

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Section: Introductionmentioning

confidence: 99%

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

Wunnicke

Strohbach²,

Weigand³

et al. 2010

RNA

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“…Recently, the diverse roles of RNA-mediated regulation have become important tools for synthetic biology applications ranging from detecting metabolic state (1), balancing metabolic pathway expression (2), tightly regulating toxin genes (3), and detecting environmentally harmful chemicals (4). In particular, RNA-based genetic parts have been engineered that regulate transcription through RNA-mediated transcription factor recruitment (5,6), transcript stability through small-molecule-mediated ribozyme cleavage (1,7) and siRNA targeted degradation (8), and translation through cis-acting mRNA conformational changes (9) and trans-acting antisense RNA-mRNA interactions (10,11).…”

mentioning

confidence: 99%

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

Lucks

Mutalik

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

234

354

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The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.gene networks | regulatory systems | orthogonal regulators N oncoding RNA has been found to play a central role in regulating gene expression in both prokaryotes and eukaryotes. Recently, the diverse roles of RNA-mediated regulation have become important tools for synthetic biology applications ranging from detecting metabolic state (1), balancing metabolic pathway expression (2), tightly regulating toxin genes (3), and detecting environmentally harmful chemicals (4). In particular, RNA-based genetic parts have been engineered that regulate transcription through RNA-mediated transcription factor recruitment (5, 6), transcript stability through small-molecule-mediated ribozyme cleavage (1, 7) and siRNA targeted degradation (8), and translation through cis-acting mRNA conformational changes (9) and trans-acting antisense RNA-mRNA interactions (10,11).This wide array of RNA function is beginning to be used to engineer programmable genetic circuitry required for the next level of synthetic biology applications (12). By interfacing with protein-based transcription factors and repressors, hybrid RNA∕ protein cascades have been made that perform sophisticated logic evaluation (8) and even count extracellular events (13). Much like previous work on protein-based cascades (14), protein regulators propagate the signal between different levels of the hybrid cascades. This makes the inner workings of these cascades complicated by the many interconversions between mRNA and protein that must take place. This not only incre...

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“…Alternative strategies with unregulated expression of growth-related genes pose the risk of uncontrolled lymphoproliferation and leukemic transformation (6). Thus, the ability to integrate growth-stimulatory gene expression with tightly controlled regulatory systems has the potential to greatly improve the safety and efficacy of adoptive T-cell therapy.Inspired by the diverse functional roles exhibited by regulatory RNAs in natural systems (12-14) and the relative ease by which RNA can be modeled and designed (15), researchers have begun developing synthetic RNA-based regulatory systems that integrate discrete gene-regulatory and sensing functions as genetic control strategies (16)(17)(18). However, the absence of successful adaptations of these earlier genetic devices to the regulation of functional responses in mammalian cells highlights remaining difficulties in translating designs that regulate reporter gene expression to functional control.…”

mentioning

confidence: 99%

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

Chen

Jensen

Smolke

2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

237

242

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RNA molecules perform diverse regulatory functions in natural biological systems, and numerous synthetic RNA-based control devices that integrate sensing and gene-regulatory functions have been demonstrated, predominantly in bacteria and yeast. Despite potential advantages of RNA-based genetic control strategies in clinical applications, there has been limited success in extending engineered RNA devices to mammalian gene-expression control and no example of their application to functional response regulation in mammalian systems. Here we describe a synthetic RNA-based regulatory system and its application in advancing cellular therapies by linking rationally designed, drug-responsive, ribozymebased regulatory devices to growth cytokine targets to control mouse and primary human T-cell proliferation. We further demonstrate the ability of our synthetic controllers to effectively modulate T-cell growth rate in response to drug input in vivo. Our RNAbased regulatory system exhibits unique properties critical for translation to therapeutic applications, including adaptability to diverse ligand inputs and regulatory targets, tunable regulatory stringency, and rapid response to input availability. By providing tight gene-expression control with customizable ligand inputs, RNA-based regulatory systems can greatly improve cellular therapies and advance broad applications in health and medicine. T he ability to control functional responses in mammalian cells with customizable and compact regulatory systems in vivo addresses a critical need in diverse clinical applications, particularly in cellular therapies (1). As an example, adoptive T-cell therapy seeks to harness the precision and efficacy of the immune system against diseases that escape the body's natural surveillance. The adoptive transfer of antigen-specific T cells can reconstitute immunity to viruses and mediate tumor regression (2-4). T cells engineered to express tumor-specific T-cell antigen receptors can achieve highly refined target recognition (5), thus minimizing toxic off-target effects associated with conventional chemotherapy. However, considerable research has shown that the persistence of transferred T cells in vivo is both central to therapeutic success and elusive to current technology (6, 7). The efficacy of adoptive immunotherapy in humans is often limited by the failure of transferred T cells to survive in the host (8, 9).Clonal expansion of T cells is a critical component of T-cell activation mediated by cytokines such as IL-2 and IL-15, which activate JAK-STAT signaling pathways and lead to the expression of genes involved in growth modulation (10) (Fig. 1A). Sustaining the survival and proliferation of Tcells following adoptive transfer is challenging because of the limited availability of homeostatic cytokines (IL-15/IL-7) and stimulatory antigen presenting cells. State-of-the-art strategies for improving the persistence of adoptively transferred lymphocytes require that patients be subjected to myeloablative total body irradiation/chemother...

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Frameworks for Programming Biological Function through RNA Parts and Devices

Cited by 109 publications

References 127 publications

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

Versatile RNA-sensing transcriptional regulators for engineering genetic networks

Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems

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