The virtues of self-binding: high sequence specificity for RNA cleavage byself-processed hammerhead ribozymes

Naturally occurring hammerhead ribozymes are produced by rolling circle rep lication followed by self-cleavage. This results in monomer-length catalyti c RNAs which have self-complementary sequences that can occupy their trans- binding domains and potentially block their ability to cleave other RNA str ands. Here we show, using small self-processed ribozymes, that this self-bi nding does not necessarily inhibit trans-cleavage and can result in greatly elevated discrimination against mismatches. We utilized a designed 63 nt c ircular DNA to encode the synthesis of a self-processed ribozyme, MDR63. Ro lling circle transcription followed by self-processing produced the desired 63 nt ribozyme, which potentially can bind mdr-1 RNA with 9+9 nt of comple mentarity or bind itself with 4+5 nt of self-complementarity by folding bac k its ends to form hairpins. Kinetics of trans-cleavage of short complement ary and mismatched RNAs were measured under multiple turnover conditions, i n comparison to a standard 40 nt ribozyme (MDR40) that lacks the self-compl ementary ends. The results show that MDR63 cleaves an mdr-1 RNA target with a k(cat)/K-m almost the same as MDR40, but with discrimination against mis matches up to 20 times greater, Based on folding predictions, a second self -processed ribozyme (UG63) having a single point mutation was synthesized; this displays even higher specificity (up to 100-fold) against mismatches. The results suggest that self-binding ends may be generally useful for incr easing sequence specificity of ribozymes.