An Allosteric Ribozyme Regulated by Doxycyline

Piganeau, Nicolas; Jenne, Andreas; Thuillier, Vincent; Famulok, Michael

doi:10.1002/1521-3773(20001201)39:23<4369::aid-anie4369>3.0.co;2-n

Cited by 51 publications

(45 citation statements)

References 23 publications

(11 reference statements)

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“…In addition, two of these-namely, the theophylline and the tetracycline aptamer-have been used as a binding domain for the engineering of more complex riboswitches with broadened functions and applicability (Buskirk et al 2004;Desai and Gallivan 2004;Isaacs et al 2004;Suess et al 2004;Bayer and Smolke 2005). In vitro selection techniques to isolate small molecule-binding aptamers (Ellington and Szostak 1990;Tuerk and Gold 1990) or regulatable ribozymes (Koizumi et al 1999;Piganeau et al 2001) have been well established in the past. However, there is a noticeable lack of methods for the detection of those in vitro selected molecules that in addition show regulatory activity within the cell.…”

Section: Introductionmentioning

confidence: 99%

Screening for engineered neomycin riboswitches that control translation initiation

Weigand

Sanchez²,

Gunnesch³

et al. 2007

RNA

187

214

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Riboswitches are genetic control elements that regulate gene expression in a small molecule-dependent way. We developed a two-stage strategy of in vitro selection followed by a genetic screen and identified several artificial small molecule-binding riboswitches that respond to the aminoglycoside neomycin. Structure-function relationships and structural probing revealed that they adopt the general neomycin-binding motif. They display no sequence similarities to in vitro selected neomycin aptamers but contain parts of the decoding site that is the binding site for neomycin on the ribosomal RNA. We propose a model of a composed binding pocket of an internal loop as primary docking site and a terminal flaplike loop structure fixing neomycin in a sandwich-like manner. Such binding pockets characterized by multiple contacts between ligand and RNA are described for both natural and engineered riboswitches. We anticipate that combination of in vitro selection and in vivo screening is a useful strategy to identify RNA molecules with a desired functionality.

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Section: Introductionmentioning

confidence: 99%

Screening for engineered neomycin riboswitches that control translation initiation

Weigand

Sanchez²,

Gunnesch³

et al. 2007

RNA

187

214

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“…20-23 However, such sequence alterations usually undermine the affinity and specificity of the molecule, and a strategy for directly selecting for the desired function may therefore prove more advantageous. Toward this end, pioneering work from the Ellington, 24,25 Breaker, 26,27 and Famulok groups 28,29 has demonstrated the selection of allosteric ribozymes that exhibit enhanced catalytic activity in response to binding of various ligands. Moreover, there have been several successes in isolating allosterically-regulated aptamers that can fluorescently report binding events.…”

mentioning

confidence: 99%

In Vitro Selection of Shape-Changing DNA Nanostructures Capable of Binding-Induced Cargo Release

Plakos

Xiao

et al. 2013

ACS Nano

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Many biological systems employ allosteric regulatory mechanisms, which offer a powerful means of directly linking a specific binding event to a wide spectrum of molecular functionalities. There is considerable interest in generating synthetic allosteric regulators that can perform useful molecular functions for applications in diagnostics, imaging and targeted therapies, but generating such molecules through either rational design or directed evolution has proven exceptionally challenging. To address this need, we present an in vitro selection strategy for generating conformation-switching DNA nanostructures that selectively release a small-molecule payload in response to binding of a specific trigger molecule. As an exemplar, we have generated a DNA nanostructure that hybridizes with a separate ‘cargo strand’ containing an abasic site. This abasic site stably sequesters a fluorescent cargo molecule in an inactive state until the DNA nanostructure encounters an ATP trigger molecule. This ATP trigger causes the nanostructure to release the cargo strand, thereby liberating the fluorescent payload and generating a detectable fluorescent readout. Our DNA nanostructure is highly sensitive, with an EC50 of 30 μM, and highly specific, releasing its payload in response to ATP but not to other chemically similar nucleotide triphosphates. We believe that this selection approach could be generalized to generate synthetic nanostructures capable of selective and controlled release of other small-molecule cargos in response to a variety of triggers, for both research and clinical applications.

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“…In this approach, an entire allosteric domain of the ribozyme is randomized and an allosteric selection protocol is applied to isolate constructs whose cleavage activity is activated or deactivated in the presence of external effectors [50][51][52]. A protein dependent RNA-ligase ribozyme was also reported using an allosteric selection technique [53,54].…”

Section: Generation Of Allosteric Ribozymesmentioning

confidence: 99%

Functional Nucleic Acids in High Throughput Screening and Drug Discovery

Srivatsan¹,

Famulok²

2007

CCHTS

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In vitro selection can be used to generate functional nucleic acids such as aptamers and ribozymes that can recognize a variety of molecules with high affinity and specificity. Most often these recognition events are associated with structural alterations that can be converted into detectable signals. Several signaling aptamers and ribozymes constructed by both design and selection have been successfully utilized as sensitive detection reagents. Here we summarize the development of different types of signaling nucleic acids, and approaches that have been implemented in the screening format.

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An Allosteric Ribozyme Regulated by Doxycyline

Cited by 51 publications

References 23 publications

Screening for engineered neomycin riboswitches that control translation initiation

Screening for engineered neomycin riboswitches that control translation initiation

In Vitro Selection of Shape-Changing DNA Nanostructures Capable of Binding-Induced Cargo Release

Functional Nucleic Acids in High Throughput Screening and Drug Discovery

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