Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP.
RNA transcripts containing the hammerhead ribozyme have been engineered to self-destruct in the presence of specific nucleoside 3',5'-cyclic monophosphate compounds. These RNA molecular switches were created by a new combinatorial strategy termed 'allosteric selection,' which favors the emergence of ribozymes that rapidly self-cleave only when incubated with their corresponding effector compounds. Representative RNAs exhibit 5,000-fold activation upon cGMP or cAMP addition, display precise molecular recognition characteristics, and operate with catalytic rates that match those exhibited by unaltered ribozymes. These findings demonstrate that a vast number of ligand-responsive ribozymes with dynamic structural characteristics can be generated in a massively parallel fashion. Moreover, optimized allosteric ribozymes could serve as highly selective sensors of chemical agents or as unique genetic control elements for the programmed destruction of cellular RNAs.
Koizumi
M
M
Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, Connecticut 06520-8103, USA.
Soukup
G A
GA
Kerr
J N
JN
Breaker
R R
RR
D000143:Acids / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site / Q000187:drug effects; D001483:Base Sequence; D002384:Catalysis / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology*; D003563:Cyclic CMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005818:Genetic Engineering / Q000379:methods; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000143:Acids; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D003563:Cyclic CMP; D000242:Cyclic AMP; D006152:Cyclic GMP