Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites

Substrate recognition and cleavage by the bacterial RNase P RNA requires tw o domains, a specificity domain, or S-domain, and a catalytic domain, or C- domain, The S-domain binds the T stem-loop region in a pre-tRNA substrate t o confer specificity for tRNA substrates, In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substr ate binding module. New RNA substrates are isolated by in vitro selection u sing two libraries containing random regions of 60 nt, At the end of the se lection, the cleavage rates of the substrate library are similar to 0.7 min (-1) in 10 mM MgCl2 at 37 degrees C, similar to 4-fold better than the clea vage of a pre-tRNA substrate by the wild-type RNase P RNA under the same co nditions, The contribution of the S-domain replacement to the catalytic eff iciency is from 6- to 22000-fold, Chemical and nuclease mapping of two ribo zyme product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. T hese results demonstrate the feasibility of design and isolation of RNase P -based, matching ribozyme-substrate pairs without prior knowledge of the se quence or structure of the interactive modules in the ribozyme or substrate .