Investigating a New Generation of Ribozymes in Order to Target HCV

For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+) RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA) to the ribozyme domain derived from the Hepatitis Delta Virus (HDV). The former switches cleavage activity “on” solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA-HDV ribozymes directed against HCV (−) strands

RiboSubstrates: a web application addressing the cleavage specificities of ribozymes in designated genomes

BACKGROUND: RNA-dependent gene silencing is becoming a routine tool used in laboratories worldwide. One of the important remaining hurdles in the selection of the target sequence, if not the most important one, is the designing of tools that have minimal off-target effects (i.e. cleaves only the desired sequence). Increasingly, in the current dawn of the post-genomic era, there is a heavy reliance on tools that are suitable for high-throughput functional genomics, consequently more and more bioinformatic software is becoming available. However, to date none have been designed to satisfy the ever-increasing need for the accurate selection of targets for a specific silencing reagent. RESULTS: In order to overcome this hurdle we have developed RiboSubstrates . This integrated bioinformatic software permits the searching of a cDNA database for all potential substrates for a given ribozyme. This includes the mRNAs that perfectly match the specific requirements of a given ribozyme, as well those including Wobble base pairs and mismatches. The results generated allow rapid selection of sequences suitable as targets for RNA degradation. The current web-based RiboSubstrates version permits the identification of potential gene targets for both SOFA-HDV ribozymes and for hammerhead ribozymes. Moreover, a minimal template for the search of siRNAs is also available. This flexible and reliable tool is easily adaptable for use with any RNA tool (i.e. other ribozymes, deoxyribozymes and antisense), and may use the information present in any cDNA bank. CONCLUSION: RiboSubstrates should become an essential step for all, even including "non-RNA biologists", who endeavor to develop a gene-inactivation system

A Modern Mode of Activation for Nucleic Acid Enzymes

Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process

Schematic representation of the 3-step procedure used for the identification of the sites possessing the greatest targeting potential in the HCV 5′-UTR.

<p>Step 1 involved a bioinformatic analysis that included both the prediction of the secondary structure and the identification of the 7 nt streches most likely to be bound by the ribozyme's P1 region using both the RNA structure 3.7 and Oligowalk softwares. Step 2 involved the selection of the sequences that fulfill the HDV ribozyme requirements. Step 3 involved the RNase H hydrolysis assay. The autoradiogram shown corresponds to a typical 5% polyacrylamide gel performed for the analysis of 6 potential sites. The positions of proposed cleavage sites are identified at the top of the gel. The negative control performed in the absence of any oligonucleotide is indicated by the letter C.</p

Compilation of the in vitro cleavage of HCV (+) strand by the SOFA-HDV ribozyme data.

<p>The sequences for each SOFA-HDV Rz corresponding to their P1 and biosensor recognition domains are listed. The upper section includes the SOFA-HDV Rz targetable sites as identified by bioinformatic and biochemical procedures, while the lower section includes those based on the reported secondary and crystal structures.</p

Analysis of the inhibition of the HCV replicon by SOFA-HDV ribozymes.

<p>(A) Schematic representation of the HCV replicon used (described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009627#pone.0009627-Vrolijk1" target="_blank">[38]</a>). (B) and (C). Histograms of the relative luciferase activities detected for the HCV (+) and (−) strands, respectively. Luciferase activity was detected in the presence of all SOFA-HDV ribozymes tested, and was reported relative to that determined for an irrelevant SOFA-HDV ribozyme whose level was arbitrarily set at 100%. The latter SOFA-HDV ribozyme was designed to target the hepatitis B virus and does not possess the sequences required in order to recognize the HCV strands of either the (+) or the (−) polarity. The characterization of this SOFA-HDV ribozyme was previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009627#pone.0009627-Bergeron1" target="_blank">[29]</a>.</p

Typical autoradiogram of an 8% polyacrylamide gel performed in order to analyze SOFA-HDV ribozyme cleavage <i>in vitro</i>.

<p>The experiments were performed using 5′-end-labeled HCV transcripts in the presence of an excess of SOFA-HDV ribozyme. The SOFA-HDV ribozymes are identified at the top of the gel. The negative control performed in the absence of ribozyme is indicated by the letter C. The positions of the xylene cyanol (XC) and bromophenol blue (BPB) marker dyes are indicated.</p

Compilation of the <i>in vitro</i> cleavage of the HCV (−) strand by SOFA-HDV ribozyme data.

<p>The sequences for each SOFA-HDV Rz corresponding to their P1 and biosensor recognition domains are listed. Nucleotide positions are numbered from 3′ to 5′ in order to facilitate concordance with the data obtained for the HCV (+) strand.</p

Compilation of the data from the RNase H hydrolysis of HCV (+) RNA.

<p>Compilation of the data from the RNase H hydrolysis of HCV (+) RNA.</p