Targeted cleavage of RNA molecules by human RNase P using minimized external guide sequences

The endoribonuclease RNase P processes tRNA-Iike structures that are assemb led out of two separate strands. In these bimolecular constructs, one of th e strands is cleaved by the enzyme, and the other one is called the externa l guide sequence (EGS). A number of EGS with different mutations and deleti ons were tested for the ability to induce cleavage with human RNase P. Diff erent domains of the original tRNA(tyr)-like structure were deleted or modi fied. The anticodon stem and loop and the variable loop could be deleted wi thout a detrimental effect on recognition by RNase P. Modifications in the lengths of T stem and aminoacyl acceptor stem led to a decrease in the rela tive amount of cleavage, whereas modifications of the D stem were more perm issible. Single nucleotide deletions in the T loop reduced cleavage to diff erent extents, depending on the position. Values for the K-d of complex for mation of bimolecular constructs with annealing arms of varying lengths ran ged from 0.2 nM to 28 nM. A cleavage rate of 1 min(-1) was measured for bot h the bimolecular target-EGS complex and tRNA precursor.