A tetracycline-binding RNA aptamer

Berens, Christian; Thain, Alison; Schroeder, Renée

doi:10.1016/s0968-0896(01)00063-3

Cited by 180 publications

(167 citation statements)

References 24 publications

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“…3) . Thus, the pronounced cleavage signal at position A55 provides additional evidence for the importance of loop L3 in tc binding and is also in nice agreement with lead cleavage data that indicated an involvement of bulge B1-2 and the loop L3 in tc binding (Berens et al 2001).…”

Section: Bulge B1-2 and Loop L3 Are Involved In Tc Bindingsupporting

confidence: 85%

Molecular analysis of a synthetic tetracycline-binding riboswitch

Hanson¹,

Bauer²,

Fink³

et al. 2005

RNA

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Riboswitches are newly discovered regulatory elements that consist solely of RNA, sense their ligand in a preformed binding pocket, and perform a conformational switch in response to ligand binding, resulting in altered gene expression. Regulation by a tetracycline (tc)-binding aptamer when inserted into the 5 untranslated region (UTR) of a reporter gene exhibits all characteristics of a riboswitch. Chemical and enzymatic probing reveals that the aptamer consists of two stems, P1 and P2, which are already present in the absence of tc and form the scaffold of the aptamer. They are separated by a bulge B1-2 and an opposing stem-loop (P3-L3). Tc-dependent changes in the probing pattern only appear in the upper part of the bulge B1-2 (nucleotides 9-13) and the loop L3. Saturating mutagenesis corroborates the involvement of these two regions in regulation. Structural probing of the mutant A55U, which contains a single-nucleotide exchange in loop L3 results in a changed probing pattern of the loop, but also of the opposing bulge B1-2. This denotes that both regions cooperate and form a composite binding pocket. Thus, our model for aptamer-mediated translational regulation is that the ligand-free aptamer has only marginal influence on translational initiation. Tc then leads to an intramolecular connection in a pseudoknot-like manner and turns the aptamer into its inhibitory form. This represents a new mechanism for riboswitch action clearly distinguished from currently known naturally occurring riboswitches, which function by sequestration of the ribosomal binding site, transcriptional attenuation, and ribozyme-mediated degradation.

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Section: Bulge B1-2 and Loop L3 Are Involved In Tc Bindingsupporting

confidence: 85%

Molecular analysis of a synthetic tetracycline-binding riboswitch

Hanson¹,

Bauer²,

Fink³

et al. 2005

RNA

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“…19. toward clinically usable pharmaceuticals. To verify this critical property of our prototype T-cell proliferation control system, we replaced the theo aptamer (23) with the tetracycline (tet) aptamer (24) to construct a tet-responsive switch (L2bulge18tc; Fig. 2D).…”

Section: Ribozyme Switches Can Be Programmed To Respond To Alternativementioning

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.

242

245

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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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“…Furthermore, we observe the highest entropy difference for nucleotide G51. Experimental data reveals that G51 crosslinks to tetracycline when the complex is subjected to UV irradiation [42]. These findings suggest a strong interaction with TC and thus a dramatic, positive change in H. Nucleotides A52 and U54 show a positive entropy difference inside L3.…”

Section: Application To Molecular Synthetic Biologymentioning

confidence: 92%

StreAM- $$T_g$$ : Algorithms for Analyzing Coarse Grained RNA Dynamics Based on Markov Models of Connectivity-Graphs

Jäger

Schiller

Strufe

et al. 2016

Lecture Notes in Computer Science

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Background:In this work, we present a new coarse grained representation of RNA dynamics. It is based on adjacency matrices and their interactions patterns obtained from molecular dynamics simulations. RNA molecules are well-suited for this representation due to their composition which is mainly modular and assessable by the secondary structure alone. These interactions can be represented as adjacency matrices of k nucleotides. Based on those, we define transitions between states as changes in the adjacency matrices which form Markovian dynamics. The intense computational demand for deriving the transition probability matrices prompted us to develop StreAM-T g , a streambased algorithm for generating such Markov models of k-vertex adjacency matrices representing the RNA. Results:We benchmark StreAM-T g (a) for random and RNA unit sphere dynamic graphs (b) for the robustness of our method against different parameters. Moreover, we address a riboswitch design problem by applying StreAM-T g on six long term molecular dynamics simulation of a synthetic tetracycline dependent riboswitch (500 ns) in combination with five different antibiotics. Conclusions:The proposed algorithm performs well on large simulated as well as real world dynamic graphs. Additionally, StreAM-T g provides insights into nucleotide based RNA dynamics in comparison to conventional metrics like the root-mean square fluctuation. In the light of experimental data our results show important design opportunities for the riboswitch.

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A tetracycline-binding RNA aptamer

Cited by 180 publications

References 24 publications

Molecular analysis of a synthetic tetracycline-binding riboswitch

Molecular analysis of a synthetic tetracycline-binding riboswitch

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

StreAM- $$T_g$$ : Algorithms for Analyzing Coarse Grained RNA Dynamics Based on Markov Models of Connectivity-Graphs

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