The Hairpin Ribozyme: Discovery, Two-Dimensional Model, and Development for Gene Therapy

Arnold Hampel    Departments of Biological Sciences and Chemistry, Northern Illinois University DeKalb, Illinois 60115

RNA catalysis was co-discovered by S. Altman, who found the M1 RNA of RNAse P could catalytically cleave and process the 5' terminus of the tRNA precursor (1), and by T. Cech, who found the tetrahymena ribosomal RNA intron had autocatalytic activity (2). The term “ribozyme” was coined to describe catalytic RNA. Since then, four other catalytic RNAs, all self-cleaving, have been discovered: the hepatitis delta ribozyme, the neurospora ribozyme, the hammerhead ribozyme, and the hairpin ribozyme. The latter two are from plant viral satellite, virusoid, and viroid RNAs (see Ref. 3 for review).

This review focuses on the hairpin ribozyme (4, 5). Specifically, I describe its discovery–followed by the many facets of development required to bring it to the point of being tested in clinical trials as a drug for human use as an AIDS therapeutic. (For previous reviews of this work, see 6, 7, and for a detailed description of many aspects, see 8. We have used the HIV-1 system as an initial test model for determining the utility of the hairpin ribozyme in the down-regulation of gene expression. Based on our excellent success with that system, we are very optimistic that the hairpin ribozyme may have more general utility for a wide variety of applications in other systems as well.

The hairpin ribozyme was found as the catalytic center of three known plant satellite RNAs. These were the negative strands of the satellite RNAs from tobacco ringspot virus (sTRSV), chicory yellow mottle virus type 1 (sCYMV1), and arabis mosaic virus (sArMV) (9, 10). Initial studies identified the hairpin ribozyme first in the negative strand of sTRSV. Using molecular modeling of the negative strand of sTRSV as a first approximation, we made substrate and ribozymes of various lengths and sequences in order to determine the minimum catalytic center. A 50-nt ribozyme sequence was found to be capable of cleaving a 14-nt substrate sequence in a trans reaction. It proceeded without depletion of the 50-nt RNA component, and therefore was catalytic. It followed true Michaelis–Menten kinetics, allowing determination of Km, kcat, energy of activation, Mg2 + dependence, temperature dependence, and pH optima (4).

The two-dimensional structure was determined by making an extensive collection of site-specific mutants for both the ribozyme and the substrate. The location of individual base pairs was determined by comparison of catalytic activity for these mutant sequences with that of the native sequence. That is, if the site of a predicted base pair lost activity with a mismatch in this position, and if the activity was restored with an alternate base pair, then a base pair at this site has been identified. This method identified four helices and five loops for the ribozyme–substrate complex. The overall structure was hairpin-like, so I named it the hairpin ribozyme (5, 11). Of the five helices, helices 1 and 2 occurred between the ribozyme and the substrate, and helices 3 and 4 were within the ribozyme itself. Single-stranded loops 1, 2, 3, and 4 were in the ribozyme sequence, and loop 5 was in the substrate sequence (Fig. 1).

Following its discovery, its biochemical characterization, and determination of its two-dimensional structure, the hairpin ribozyme was engineered to cleave heterologous substrate RNAs (5, 11). This led to development of the hairpin ribozyme system for human gene therapy and other applications for down-regulation of gene expression. Targeting rules for cleavage of heterologous substrates were determined. The substrate had a sequence preference of BN*GUC where the * is the site of cleavage. The nucleotide B is G, U, or C but not A. With these targeting rules in hand, we now had the possibility of specifically cleaving target mRNA or viral RNA molecules, resulting in inhibition of gene expression or viral replication.

Sequence searches were done for a number of systems, including HIV-1, to identify sequences containing BN*GUC for use as possible target sites (5, 8, 12, 13). Using HIV-1 as an example, ribozymes were made to a number of potential targets and in vitro cleavage efficiency of the ribozymes to these targets carried out. Optimization was done by varying the length of helix 1 to determine its optimal length for maximum catalytic efficiency. In general the optimal length of helix 1 varied between 6 and 12 bp, with 8 bp being a useful first approximation. Helix 2 was fixed at 4 bp.

The catalytic activity of the ribozyme was improved by making specific sequence changes in regions of the ribozyme containing nonessential nucleotides. Certain of these changes...