Figure 4.
Selection of the sites with the greatest potential for delta ribozyme cleavage sites based on a ribozyme cleavage assay performed in the presence of a ribozyme library. (A) Scheme illustrating the procedure. HBV RNA and cDNA are represented by the full and dashed lines, respectively. RT is reverse transcriptase. The PCR strategy used to synthesize the library is shown in the inset in which oligonucleotides A and C served as sense and antisense primers, respectively, to amplify the randomized sequences of the templates (oligonucleotide B). (B) Autoradiogram of a sequencing gel revealing the cleavage site at position 303. Lane 1 is the reaction performed in the presence of ribozymes and MgCl2, while MgCl2 and the ribozyme were omitted in the reactions in lanes 2 and 3, respectively. Adjacent to the gel is a DNA sequencing reaction, and the corresponding sequence is shown on the right side. The arrow indicates the cleavage site.