A guanine-linked end-effect is a sensitive reporter of charge flow through DNA and RNA double helices.

The property of charge (electron hole) flow in DNA duplexes has been the subject of intensive study. RNA-DNA heteroduplexes have also been investigated; however, little information exists on the conductive properties of purely RNA duplexes. In investigating the relative conductive properties of a three molecule DNA-DNA duplex design, using piperidine and aniline to break strands at modified bases, we observed that duplexes with guanine-rich termini generated a large oxidative end-effect, which could serve as a highly sensitive reporter of charge flow through the duplexes. The end-effect was found faithfully to report attenuations in charge flow due to certain single-base mismatches within a duplex. Comparative charge flow experiments on DNA-DNA and RNA-RNA duplexes found large end-effects from both, suggesting that the A and B family of double helices conduct charge comparably. The sheer magnitude of the end-effect, and its high sensitivity to helical imperfections, suggest that it may be exploited as a sensitive reporter for DNA mismatches, as well as a versatile device for studying the structure, folding, and dynamics of complexly folded RNAs and DNAs.

Target-dependent on/off switch increases ribozyme fidelity.

Ribozymes, RNA molecules that catalyze the cleavage of RNA substrates, provide an interesting alternative to the RNA interference (RNAi) approach to gene inactivation, especially given the fact that RNAi seems to trigger an immunological response. Unfortunately, the limited substrate specificity of ribozymes is considered to be a significant hurdle in their development as molecular tools. Here, we report the molecular engineering of a ribozyme possessing a new biosensor module that switches the cleavage activity from 'off' (a 'safety lock') to 'on' solely in the presence of the appropriate RNA target substrate. Both proof-of-concept and the mechanism of action of this man-made riboswitch are demonstrated using hepatitis delta virus ribozymes that cleave RNA transcripts derived from the hepatitis B and C viruses. To our knowledge, this is the first report of a ribozyme bearing a target-dependent module that is activated by its RNA substrate, an arrangement which greatly diminishes non-specific effects. This new approach provides a highly specific and improved tool with significant potential for application in the fields of both functional genomics and gene therapy.

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 http://www.riboclub.org/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.

Development and comparison of procedures for the selection of delta ribozyme cleavage sites within the hepatitis B virus.

Delta ribozyme possesses several unique features related to the fact that it is the only catalytic RNA known to be naturally active in human cells. This makes it attractive as a therapeutic tool for the inactivation of clinically relevant RNAs. However, several hurdles must be overcome prior to the development of useful gene-inactivation systems based on delta ribozyme. We have developed three procedures for the selection of potential delta ribozyme target sites within the hepatitis B virus (HBV) pregenome: (i) the use of bioinformatic tools coupled to biochemical assays; (ii) RNase H hydrolysis with a pool of oligonucleotides; and (iii) cleavage assays with a pool of ribozymes. The results obtained with delta ribozyme show that these procedures are governed by several rules, some of which are different from those both for other catalytic RNAs and antisense oligonucleotides. Together, these procedures identified 12 sites in the HBV pregenome that can be cleaved by delta ribozymes, although with different efficiencies. Clearly, both target site accessibility and the ability to form an active ribozyme-substrate complex constitute interdependent factors that can best be addressed using a combinatorial library of either oligonucleotides or ribozymes.

Ribozyme-based gene-inactivation systems require a fine comprehension of their substrate specificities; the case of delta ribozyme.

The ability of ribozymes (i.e. RNA enzymes) to specifically recognize and subsequently catalyze the cleavage of an RNA substrate makes them attractive for the development of therapeutic tools for the inactivation of both viral RNAs and mRNAs associated with various diseases. Several applicable ribozyme models have been tested both in vitro and in a cellular environment, and have shown significant promise. However, several hurdles remain to be surpassed before we generate a useful gene-inactivation system based on a ribozyme. Among the most important requirements for further progress are a better understanding of the features that contribute to defining the substrate specificity for cleavage by a ribozyme, and the identification of the potential cleavage sites in a given target RNA. The goal of this review is to illustrate the importance of both of these factors at the RNA level in the development of any type of ribozyme based gene-therapy. This is achieved by reviewing the recent progress in both the structure-function relationships and the development of a gene-inactivation system of a model ribozyme, specifically delta ribozyme.

Unusual subcellular confinement of the fragile X mental retardation protein (FMRP) in circulating human platelets: complete polyribosome dissociation.

FMRP, a RNA-binding protein, was shown in association with polyribosomes in every cell types studied so far, suggesting a ubiquitous role as a translational regulator. Platelets are known for their limited protein synthesis potential. However, current investigations put forward that RNA metabolism is more developed than previously thought. Unexpectedly, our results provide evidence that FMRP, in platelets, is not constitutively associated with heavy particles, such as polyribosomes, and possesses a sedimentation coefficient of less than 10S contrasting with values of 150 to 500S as reported in other cell types. In summary, this report brings to light platelets as a simple human biological system to delineate novel FMRP functions as well as strengthening our comprehension of the pathophysiology of the fragile X syndrome which results from the absence of FMRP.

Silencing of amyloid precursor protein expression using a new engineered delta ribozyme.

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-ß peptides (Aß) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40-42 residue peptide (Aß(40-42)) derived from amyloid protein precursor (APP). Most likely, reducing Aß levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aß accumulation in the cells, we have selectively chosen to target the primary step in the Aß cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aß levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD.

Functional characterization of the SOFA delta ribozyme.

Molecular engineering has led to the development of a novel target-dependent riboswitch that increases deltaribozyme fidelity. This delta ribozyme possesses a specific on/off adapter (SOFA) that switches the cleavage activity from off (a "safety lock") to on solely in the presence of the desired RNA substrate. In this report, we investigate the influence of both the structure and the sequence of each domain of the SOFA module. Analysis of the cleavage activity, using a large collection of substrates and SOFA-ribozyme mutants, together with RNase H probing provided several insights into the nature of the sequence and the optimal design of each domain of the SOFA module. For example, we determined that (1) the optimal size of the blocker sequence, which keeps the ribozyme off in the absence of the substrate, is 4 nucleotides (nt); (2) a single nucleotide difference between the substrate and the biosensor domain, which is responsible for the initial binding of the substrate that subsequently switches the SOFA-ribozyme on, is sufficient to cause non-recognition of the appropriate substrate; (3) the stabilizer, which joins the 5' and 3' ends of the SOFA-ribozyme, plays only a structural role; and (4) the optimal spacer sequence, which serves to separate the binding regions of the biosensor and catalytic domain of the ribozyme on the substrate, is from 1 to 5 nt long. Together, these data should facilitate the design of more efficient SOFA-ribozymes with significant potential for many applications in gene-inactivation systems.

Silencing of SPC2 expression using an engineered delta ribozyme in the mouse betaTC-3 endocrine cell line.

Endoproteolytic processing is carried out by subtilase-like pro-protein convertases in mammalian cells. In order to understand the distinct roles of a member of this family (SPC2), gene silencing in cultured cells is an ideal approach. Previous studies showed limited success in either the degree of inhibition obtained or the stability of the cell lines. Here we demonstrate the high potential of delta ribozyme as a post-transcriptional gene silencing tool in cultured cells. We used an expression vector based on the RNA polymerase III promoter to establish betaTC-3 stable cell lines expressing the chimeric tRNA(Val)-delta ribozyme transcript targeting SPC2 mRNA. Northern and Western blot hybridizations showed a specific reduction of SPC2 mRNA and protein. Validation of processing effects was tested by measuring the levels of dynorphin A-(1-8), which are present in betaTC-3 cells as a result of the unique cleavage of dynorphin A-(1-17) by SPC2. Moreover, a differential proteomic analysis confirmed these results and allowed identification of secretogranin II as a potential substrate of SPC2. The development of efficient, specific, and durable silencing tools, such as described in the present work, will be of great importance in elucidating the functions of the subtilase-like pro-protein convertases in regard to peptide processing and derived cellular events.