In vitro selection of external guide sequences for directing human RNase P to cleave a target mRNA.
External guide sequences (EGSs) are oligonucleotides that consist of a sequence that is complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. Recent studies indicate that increasing the targeting activity of EGSs in directing human RNase P to cleave an mRNA in vitro can lead to better efficacies of the EGSs in inducing RNase P-mediated inhibition of the expression of the target mRNA in cultured cells. This chapter will describe the procedure for the generation of highly functional EGSs by in vitro selection. We also describe protocols for in vitro evaluation of the activity of the EGSs. These methods should provide general guidelines for using in vitro selection for generating highly active EGSs for gene-targeting applications.
Raj
Stephen
S
Division of Infectious Diseases, School of Public Health, University of California, Berkeley, USA.
Liu
Fenyong
F
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D012333:RNA, Messenger; D004247:DNA; D043262:Ribonuclease P