NUCLEASE-RESISTANT EXTERNAL GUIDE SEQUENCE-INDUCED CLEAVAGE OF TARGETRNA BY HUMAN RIBONUCLEASE-P

External guide sequences (EGSs) are short oligoribonucleotides, which are designed to bind to a given RNA target and form a precursor tRNA-l ike complex. This complex can be recognized by ribonuclease P (RNase P ), resulting in specific cleavage of the RNA target. To explore the po tential of this class of compounds as therapeutic agents and valuable tools for gene function analysis, various chemical modifications were introduced into an all-RNA EGS molecule to confer nuclease resistance. In particular, 2'-O-methyl substitutions were incorporated into the e ntire sequence (i.e., A-stem, D-stem, and T-stem) except the T-loop re gion without loss of cleavage-inducing activity. Replacement of rU (po sition 54) and rC (position 56) in the T-loop with their 2-O-methyl co unterparts caused pronounced decrease in activity. Moreover, phosphoro thioate backbone modification of the T-loop did not provide sufficient protection against endonucleolytic attack at the ribopyrimidine resid ues, Systematic modification of the T-loop with a variety of modified nucleosides and the addition of a 3'-3' inverted T at the 3'-end have generated several lead EGS prototypes, which not only exhibit wild-typ e activity in inducing RNase P-mediated target cleavage as compared wi th the all-RNA control but also remain intact in human serum for more than 24 hours. These results should provide useful insights into the d esign and development of oligonucleotide-based EGSs as potential regul ators of gene expression.