Internally Deleted Antigenomic Hepatitis Delta RNA Self‐Cleaves Even in 18 M Formamide

Abstract

Self-splicing and self-cleaving RNAs have been engineered to function as antisense RNAs by defining separate enzyme and substrate moieties.'-' Although these RNAs are efficient in cleaving the substrate RNAs in vitro, none has yet been shown to work enzymatically in vivo. The hepatitis delta RNAs offer higher efficiency of cleavage and stability than do any other ribozymes.Hepatitis delta virus is a unique mammalian virus, a single-stranded, circular RNA of only 1679 nucleotides (nts). We detected self-cleavage activity of longer-thanunit-length genomic and antigenomic R N A S .~.~ Subsequently, both RNAs were truncated to 86 genomic nts6 and 89 antigenomic nts, without loss of activity. In fact, the antigenomic RNA cleaved more efficiently at 37°C than has any other selfcleaving RNA yet reported, cleaving to completion in less than 15 seconds.'Although the computer-derived structure of the genomic RNA generated a central core in RNAs ranging from 154 to 86 nts (FIG. 1A),8 the structure of the antigenomic RNA, even in the same size class, differed greatly. As the RNAs are 70% similar in sequence, however, we generated an analogous antigenomic structure by analysis of possible conserved base pairs (FIG. 1B). A striking feature in both molecules was the extended stem 4 structure. To determine if this stem region was required for catalytic activity, deletions were introduced into the cloned cDNA. The resulting RNAs transcribed in vitro9 were assayed for self-cleavage at 37°C in 50 mM Tris buffer, p H 7.5, and 10 mM MgClz for 5 minutes.When the self-cleavage of the internally truncated genomic RNA G73 was assayed (TABLE 1) and compared with the extended stem 4 parent RNA G86 in the absence of formamide, cleavage was reduced sixfold, from 59 to 9%. To test the effect of formamide on the cleavage reaction, first the RNAs were incubated in the presence of formamide at 37°C for 5 minutes, and then 10 mM magnesium was added, followed by an additional 5 minutes of incubation. The formamide enhanced the cleavage of (386, and complete cleavage was seen in 5 minutes. This presumably was due to the denaturing of inactive RNA conformers, allowing formation of the active RNA and self-cleavage. The truncated G73 completely lost activity in the presence of formamide, and no cleavage was seen at all. Rather than allow structure to form, the formamide appeared to denature the active site of the molecule, preventing cleavage. Thus, an extended stem 4 is essential to the active core of the genomic RNA in the presence of formamide.The antigenomic RNA had previously been noted to be unusually resistant to formamide denaturation even in comparison with the genomic RNA.7J0,L' We identified a sequence element, GGAGA, at the 3' terminus of the 89 nucleotide 277

Chemical probing studies of variants of the genomic hepatitis delta virus ribozyme by primer extension analysis

“…The results suggest that strong interactions exist in these basepairing regions because the base-pairings were not disrupted under the conditions tested. Thus, several shorter forms of the HDV ribozyme, which had previously been shown to possess self-cleavage activity under denaturing conditions (Perrotta & Been, 1990;Prasad et al, 1992), appeared to persist as active structures under our denaturing conditions.…”