RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture.
An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.
Kilani
A F
AF
Program of Infectious Diseases and Immunity, Program of Comparative Biochemistry, School of Public Health, University of California, Berkeley, California 94720, USA.
Trang
P
P
Jo
S
S
Hsu
A
A
Kim
J
J
Nepomuceno
E
E
Liou
K
K
Liu
F
F
D000818:Animals; D001483:Base Sequence; D002999:Clone Cells; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005818:Genetic Engineering; D014644:Genetic Variation; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D043262:Ribonuclease P; D016415:Sequence Alignment; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D013937:Thymidine Kinase; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli