In vitro selection of allosteric ribozymes: theory and experimental validation
Identifieur interne : 003548 ( Main/Exploration ); précédent : 003547; suivant : 003549In vitro selection of allosteric ribozymes: theory and experimental validation
Auteurs : Nicolas Piganeau [Allemagne] ; Vincent Thuillier [États-Unis] ; Michael Famulok [Allemagne]Source :
- Journal of Molecular Biology [ 0022-2836 ] ; 2001.
English descriptors
- KwdEn :
- Teeft :
- Academic press, Allosteric, Allosteric ribozyme, Allosteric ribozymes, Allosteric ribozymes figure, Allosteric selection, Apparent cleavage rate, Aptamers, Catalytic rate, Chem, Cleavage, Cleavage rate, Cleavage rates, Cleavage reaction, Cleavx drug, Clone, Derivative, Different derivatives, Dissociation constants, Doxycycline, Doxycycline selection, Gene expression, Hammerhead, Hammerhead ribozyme, Incubation, Incubation step, Incubation time, Inhibitor, Inhibitor molecule, Inhibitory drug, Initial pool, Kapp, Kinetics, Ligand, Lled circles, Manganese ions, Mathematical model, Molecule, Mutagenic, Natl acad, Negative control, Next selection cycle, Nmol, Open circles, Optimal conditions, Other quinolone derivatives, Parasitic, Parasitic ribozymes, Parasitic sequences, Piperazine group, Primer, Reaction rates, Regulatory drug, Ribozyme, Ribozymes, Room temperature, Same time, Second incubation, Second step, Secondary structure, Selection buffer, Selection conditions, Selection cycle, Selection cycles, Selection parameters, Selection procedure, Selection process, Selection scheme, Selection strategy, Single clone analysis, Sodium acetate, Such ribozymes, Theoretical analysis, Theoretical fraction, Theoretical model, Transcription, Transcription reaction, Uncleaved, Uncleaved fraction, Uncleaved ribozymes, Unmutagenized molecules, Various concentrations.
Abstract
Abstract: In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. We describe an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to pefloxacin and other quinolone derivatives. Within 16 selection cycles, highly sensitive clones responding to drug levels in the sub-micromolar range were obtained. The morpholine moiety of the quinolone derivatives was required for inhibition of the self-cleavage of the selected ribozymes: modifications of the aromatic system were tolerated better than modifications of the morpholine ring. We also present a theoretical model that analyzes the predicted fraction of ribozymes with a given binding constant and cleavage rate recovered after each selection cycle. This model precisely predicts the actual experimental values obtained with the selection procedure. It can thus be used to determine the optimal conditions for an in vitro selection of an allosteric ribozyme with a desired dissociation constant and cleavage rate for a given application.
Url:
DOI: 10.1006/jmbi.2001.4981
Affiliations:
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conditions</term><term>Selection cycle</term><term>Selection cycles</term><term>Selection parameters</term><term>Selection procedure</term><term>Selection process</term><term>Selection scheme</term><term>Selection strategy</term><term>Single clone analysis</term><term>Sodium acetate</term><term>Such ribozymes</term><term>Theoretical analysis</term><term>Theoretical fraction</term><term>Theoretical model</term><term>Transcription</term><term>Transcription reaction</term><term>Uncleaved</term><term>Uncleaved fraction</term><term>Uncleaved ribozymes</term><term>Unmutagenized molecules</term><term>Various concentrations</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. We describe an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to pefloxacin and other quinolone derivatives. Within 16 selection cycles, highly sensitive clones responding to drug levels in the sub-micromolar range were obtained. The morpholine moiety of the quinolone derivatives was required for inhibition of the self-cleavage of the selected ribozymes: modifications of the aromatic system were tolerated better than modifications of the morpholine ring. We also present a theoretical model that analyzes the predicted fraction of ribozymes with a given binding constant and cleavage rate recovered after each selection cycle. This model precisely predicts the actual experimental values obtained with the selection procedure. 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