Deriving a 67-Nucleotidetrans-Cleaving Ribozyme from the Hepatitis Delta Virus Antigenomic RNA

Abstract

RNAs derived from the genomic and antigenomic hepatitis delta virus are capable of self-cleavage, and thus have the potential for serving as ribozymes in a trans-cleaving reaction. Because the catalytic core of such an enzymatic RNA was not evident from phylogenetic data, we took a step-wise approach to identifying the core, reducing the RNA in size, and characterizing various properties for each size class. Thus, a 186-nucleotide antigenomic RNA (termed Ag180) was found to be capable of cleaving well in 20 M formamide (Smith and Dinter-Gottlieb, 1991), and this unusual stability in formamide was lost by reducing the 3' end of the molecule, leaving a 140-nucleotide RNA (Ag 140). Both RNAs showed only intramolecular cleavage at a wide range of concentrations, and a number of conformers could be seen in the Ag140 RNA, some of which were resistant to cleavage at 37 degrees C. Since Ag140 could not cleave in 20 M formamide, the 5' and 3' termini of Ag180 were truncated and produced Ag5-84, which cleaved to 100% at 37 degrees C in less than 0.25 min. Internal deletions of the Stem IV region resulted in Ag5-73, still capable of efficient cleavage, although with a lessened stability in formamide. A trans-cleaving enzyme-substrate pair was finally derived from this RNA, and it consisted of a 67-nucleotide enzyme that cleaved a 13-nucleotide RNA substrate.

Evidence That Alternate Foldings of the Hepatitis .delta. RNA Confer Varying Rates of Self-Cleavage

“…Third and finally, since the RNAs can form numerous conformers in solution (Prasad et al, 1992a), the establishment of one definitive form for the RNA is at present beyond our technical abilities. We have sought in our experimental approach to present a reasonable model for the refolding, based upon the free energy measurements, and to take the global approach of preventing the proposed interaction by deletion mutagenesis.…”

“…Earlier it was proposed that the biphasic nature of cleavage profiles at a given temperature might be due to the necessity of some RNAs within the population to reassort into the catalytically active conformation (Prasad et al, 1992a;Been et al, 1992). The possible exception to this was the Ag5-89 molecule, in which the entire population cleaved in under 15 s. While a structural rearrangement to achieve the active form may still be one of the rate-limiting steps in the cleavage reaction, this work suggests that other self-cleaving conformations may coexist with the most rapid form.…”

“…The Alternate Folding May Define an Alternate Ribozyme. We have previously described a irc/is-cleavage reaction in which a 67-nt ribozyme derived from the antigenomic RNA of hepatitis virus cleaved a 13-nt substrate RNA, containing 5 nts 5' and 8 nts 3' of the cleavage site (Prasad et al, 1992a). Similarly, Perrotta and Been cut an 8-nt substrate with a 73-nt ribozyme (1992), while Branch and Robertson (1991) and Wu et al (1992) designed enzymes which were smaller in size than the substrate molecule, with ribozymes of 26 and 16 nts and substrates of 65 and 80 nts, respectively.…”

Future Directions in the Treatment of Patients with Chronic Hepatitis C Virus Infection

“…Drug delivery and the nonspecific nature of antisense strategies will limit the application of this technology for the near future. Ribozymes and nonfunctioning ribozymes: Ribozymes are catalytic RNA molecules that serve as enzymes able to break RNA molecules at specific sites (63)(64)(65). Ribozymes designed for specific sites in the HCV genome may lead to decreased viral protein production.…”