Figure 5.

Cleavage site versatility of the X motif. (A) Three 43-nt RNAs carrying the conserved features of the X motif ribozyme but differing in base pairing potential of arms I and IV were generated by in vitro transcription and targeted to cleave a 51-nt RNA. The lines represent nts within binding arms I and IV that are complementary to the X-E1 target site (arrow). Likewise, ribozymes X-E2 and X-E3 carry the arms that allow unique base pairing (dashes) with their corresponding target sites. (B) Cleavage of the RNA substrate (sub) upon incubation with three engineered ribozymes. Substrate (trace, 5′ ³²P-labeled) is not cleaved when incubated under in vitro selection conditions for 1 h in the absence of ribozyme (lane 1), but undergoes site-specific cleavage (clv) at the expected locations when incubated with 500 nM ribozymes X-E1, X-E2, and X-E3 (lanes 2–4, respectively).