In vitro selection of external guide sequences for directing RNase P-mediated inhibition of viral gene expression.
External guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNA in vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application.
Zhou
Tianhong
T
Program in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, California 94720, USA.
Kim
Joseph
J
Kilani
Ahmed F
AF
Kim
Kihoon
K
Dunn
Walter
W
Jo
Solomon
S
Nepomuceno
Edward
E
Liu
Fenyong
F
D001483:Base Sequence; D017931:DNA Primers; D004722:Endoribonucleases / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000378:metabolism*; D043262:Ribonuclease P; D058727:RNA, Small Untranslated
D017931:DNA Primers; D016337:RNA, Catalytic; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P; D058727:RNA, Small Untranslated