Characterization of 3′-terminal RNA sequence of ribozymes from round 6 of the selection from the degenerate pool (Fig. 2B). The locations of G residues in ribozymes having different terminal sequences were mapped to confirm that the sequence of the active RNA species corresponded to the DNA sequence in this region. Ribozymes were ligated to pRpp and reacted with 4SUra. After 3′ end-labeling with [32P]-α-CoTP and poly(A) polymerase, RNA was either lightly digested with RNase T1 to generate a G ladder (left lane of each pair) or partially hydrolyzed with sodium bicarbonate to produce a hydrolysis ladder (right lane of each pair). Uppercase letters, location of G residues; lowercase, the residues inferred from DNA sequence (the cleavage of RNA 3′ to G residues combined with the 3′ end-labeling strategy displaces the G ladder upwards by 1 nt in the gel). The lowest spot on the gel is 4SUpCo, as confirmed by APM gel analysis. No discrepancies between DNA and RNA sequence were observed.
