CLEAVAGE OF HIGHLY STRUCTURED VIRAL-RNA MOLECULES BY COMBINATORIAL LIBRARIES OF HAIRPIN RIBOZYMES - THE MOT EFFECTIVE RIBOZYMES ARE NOT PREDICTED BY SUBSTRATE SELECTION-RULES

Combinatorial libraries of hairpin ribozymes representing all possible cleavage specificities (>10(5)) were used to evaluate all ribozyme cl eavage sites within a large (4.2-kilobase) and highly structured viral mRNA, the 26 S subgenomic RNA of Sindbis virus, The combinatorial app roach simultaneously accounts for target site structure and dynamics, together with ribozyme folding, and the sequences that result in a rib ozyme-substrate complex with maximal activity. Primer extension was us ed to map and rank the relative activities of the ribozyme pool agains t individual sites and revealed two striking findings. First, only a s mall fraction of potential recognition sites are effectively cleaved ( activity-selected sites). Second, nearly all of the most effectively c leaved sites deviated substantially from the established consensus sel ection rules for the hairpin ribozyme and were not predicted by examin ing the sequence, or through the use of computer-assisted predictions of RNA secondary structure. In vitro selection methods were used to is olate ribozymes with increased activity against substrates that deviat e from the GUC consensus sequence. trans-Acting ribozymes targeting ni ne of the activity-selected sites were synthesized, together with ribo zymes targeting four sites with a perfect match to the cleavage site c onsensus (sequence-selected sites). Activity-selected ribozymes have m uch higher cleavage activity against the long, structured RNA molecule s than do sequence-selected ribozymes, although the latter are effecti ve in cleaving oligoribonucleotides, as predicted. These results imply that, for Sindbis virus 26 S RNA, designing ribozymes based on matche s to the consensus sequence may be an ineffective strategy.