In vitro selection of allosteric ribozymes.
In vitro selection techniques offer powerful and versatile methods to isolate nucleic acid sequences with specific activities from huge libraries. The present protocol describes an in vitro selection strategy for the de novo selection of allosteric self-cleaving ribozymes responding to virtually any drug of choice. We applied this method to select hammerhead ribozymes inhibited specifically by doxycycline or pefloxacin in the sub-micromolar range. The selected ribozymes can be converted into classical aptamers via insertion of a point mutation in the catalytic center of the ribozyme.
Piganeau
Nicolas
N
Department of Chemistry, Institute for Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany.
D000494:Allosteric Regulation / Q000235:genetics; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D004318:Doxycycline / Q000378:metabolism*; D015366:Pefloxacin / Q000378:metabolism*; D017354:Point Mutation; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D052156:SELEX Aptamer Technique / Q000379:methods*
D052157:Aptamers, Nucleotide; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D015366:Pefloxacin; D004318:Doxycycline