In vitro selection of allosteric ribozymes: theory and experimental validation.
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.
Piganeau
N
N
Kekule Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms Universität Bonn, Gerhard-Domagk-Strasse 1, Bonn, 53121, Germany.
Thuillier
V
V
Famulok
M
M
D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D001483:Base Sequence; D003001:Cloning, Molecular; D008957:Models, Genetic; D016296:Mutagenesis / Q000235:genetics; D009690:Nucleic Acid Conformation; D015366:Pefloxacin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D016337:RNA, Catalytic; D015366:Pefloxacin