title: Targeting hammerhead ribozymes against Hepatitis B virus
Creators: name: Smith, Richard
Publisher: name: University of Glasgow
Year: 1998

description: Hammerhead ribozymes are RNA motifs found in a number of plant viroids which possess an autocatalytic function for cleaving intramolecular phosphodiester bonds. The hammerhead motif consists of a conserved catalytic core of 22 nucleotides which is flanked by sequences of varying length which hybridise to the target region of the substrate RNA through Watson-Crick base-pair interactions. The flanking sequences can be manipulated to enable the hammerhead motif to function in a trans-conformation and cleave any RNA molecule after a triplet motif with the consensus sequence 'NUX', where N can be any nucleotide and X any nucleotide with the exception of guanosine. There has been considerable interest in the potential of hammerhead ribozymes as therapeutic gene silencing agents for the treatment of dominant-negative genetic disorders, tumours and chronic viral infections. The current study has focused on hammerhead ribozymes targeted against hepatitis B virus (HBV). The ribozyme-mediated cleavage reaction has been split into two components: hybridisation and phosphodiester bond cleavage. The influence of substrate RNA secondary structure on both of these steps has been investigated. A potential stem-loop in the HBV core protein open reading frame was identified by computer modelling. Five hammerhead ribozymes with total flanking sequences of 16 bases were targeted against NUX triplets in this stem-loop. Ribozyme-substrate hybridisation was studied by gel-shift assay utilising radiolabelled ribozymes with an unlabelled excess of either a 60-base truncated substrate corresponding to the stem-loop, or a 639-base substrate corresponding to the entire core protein open reading frame. The ribozymes were shown to bind more strongly to the 639-base substrate. This was thought to be due to the 60-base substrate forming aggregates in which the ribozyme binding sites were inaccessible. Increasing the length of the ribozyme flanking sequences from 16 to 59 bases increased the strength of binding. The strength of ribozyme-substrate hybridisation was not related to the predicted extent of double-stranded RNA in the target region of the substrate. The rate of ribozyme-mediated cleavage was determined under single-turnover conditions, with radiolabelled 60-base substrate and an excess of unlabelled ribozyme. Strong hybridisation did not necessarily confer a rapid rate of cleavage. Two ribozymes, RZ572 and RZ572B, targeted against the same NUX triplet, had different flanking sequences (RZ572, total flanking sequence of 16 bases; RZ572B, total flanking sequence of 59 bases). Although RZ572B has a seven-fold higher affinity for the substrate than RZ572, RZ572 has a five-fold higher cleavage rate. It is thought that this is due to changes in the substrate secondary structure influenced by the binding of the ribozyme flanking sequences. It is proposed that a highly structured substrate RNA promotes rapid ribozyme-mediated cleavage as a result of increased torsional stress within the phosphodiester bonds. However, the substrate secondary structure should not prevent the ribozyme and substrate from hybridising. The activity of the ribozymes in the rabbit reticulocyte lysate cell-free translation system was determined. Only one ribozyme, RZ534, when transcribed in situ, induced a decrease of 20% in core protein production relative to a luciferase control. This ribozyme did not demonstrate any cleavage activity in the earlier in vitro studies. Primer extension analysis of RNA extracted from the reticulocyte lysate reactions showed that no cleavage products were generated by any of the ribozymes used, suggesting that the inhibition seen with RZ534 was the result of an antisense mechanism. Repeating the experiment with in vitro synthesized ribozyme RNA did not result in any inhibition of protein synthesis, suggesting that the antisense inhibition previously seen was caused by the RZ534 DNA transcription template. The disparity between the results obtained with the 60-base and 639-base substrates, and between the purified RNA and cell-free translation experiments, and the large inaccuracies seen in the techniques used raises concerns about the validity of this and many other earlier in vitro studies, and their relevance to in vivo conditions. It may be better to discard such methodologies, replacing them with intracellular screening and in vitro evolution of RNA libraries.