Engineering high-speed allosteric hammerhead ribozymes.
Full-length hammerhead ribozymes were subjected to in vitro selection to identify variants that are allosterically regulated by theophylline in the presence of a physiologically relevant concentration of Mg(2+). The population of allosteric ribozymes resulting from 15 rounds of in vitro selection yielded variants with observed rate constants (k (obs)) as high as 8 min(-1) in the presence of theophylline and maximal k (obs) increases of up to 285-fold compared to rate constants measured in the absence of effector. The selected ribozymes have kinetic characteristics that are predicted to be sufficient for cellular gene control applications, but do not exhibit any activity in reporter gene assays. The inability of the engineered RNAs to control gene expression suggests that the in vitro and in vivo folding pathways of the RNAs are different. These results provide several key pieces of information that will aid in future efforts to engineer allosteric ribozymes for gene control applications.
Link
Kristian H
KH
Howard Hughes Medical Institute, Yale University, New Haven, CT 06520-8103, USA.
Guo
Lixia
L
Ames
Tyler D
TD
Yen
Laising
L
Mulligan
Richard C
RC
Breaker
Ronald R
RR
D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D001483:Base Sequence; D002110:Caffeine / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D002999:Clone Cells; D005786:Gene Expression Regulation / Q000187:drug effects; D005818:Genetic Engineering; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid; D052156:SELEX Aptamer Technique; D012550:Schistosoma mansoni / Q000201:enzymology*; D017423:Sequence Analysis, RNA; D013806:Theophylline / Q000737:chemistry / Q000494:pharmacology
D016337:RNA, Catalytic; D038621:Regulatory Sequences, Ribonucleic Acid; C427204:hammerhead ribozyme; D002110:Caffeine; D013806:Theophylline