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Oligonucleotides were inserted into the Lp3 region of a hepatitis delta virus (HDV) cis-cleaving ribozyme and the effect of Lp3 enlargement on ribozyme folding was examined by assaying the activity of each mutant. The location of insertion and the sequence of the inserted oligonucleotide had distinct effects on ribozyme activity, and the HDV ribozyme was capable of adopting the active structure for cis-cleavage when a pentanucleotide was inserted into the 3′ portion of Lp3. Furthermore, whether the insertion mutant cis-cleaved or not, the structure of Lp3 was altered by the inserted oligonucleotide. These findings disclose that the 5′ rather than the 3′ portion of Lp3 is critical for ribozyme folding and catalysis.

A mutational analysis of the helix 3 (H3) region of hepatitis delta virus (HDV) ribozymes disclosed that an AU at the first base pair of H3 elevates the catalytic activity of various cis- and trans-acting HDV ribozymes. A GC to AU substitution at this position of H3, which is located at the junction of three of the four helices of the pseudoknot-like structure model, altered the structure of HDV ribozymes. This substitution in the H3 did not change the independence of the cleavage rate to pH nor the sensitivity to formamide treatment of the ribozymes.