Biomedical subjects

R Tritz

Publications and source records attributed to R Tritz.

8 recordsLinked to original sources

PubMed1997

Intracellular application of hairpin ribozyme genes against hepatitis B virus.

HBV, a partially double-stranded DNA virus, replicates through a pregenomic RNA (pgRNA) intermediate, which provides a therapeutic opportunity for a novel antiviral gene therapy based on ribozyme RNA cleavage. Three hairpin ribozymes (Rzs) were designed which have the potential to disrupt HBV replication by targeting the pgRNA as well as specific mRNAs encoding the HBV surface antigen (HBsAg), the polymerase and the X protein. The ability of each ribozyme to cleave approximately 0.3 kb HBV subgenomic RNA fragments was tested in vitro. Two of the three Rzs tested (BR1 and BR3) were capable of cleaving their respective RNA substrates, while their catalytically disabled mutated counterpart Rzs were not. Structural modifications were performed on these two Rzs, with the goal of increasing catalytic efficiency both in vitro and in cells. To determine the Rz activities in liver cells, the cDNAs for each of the anti-HBV Rzs (and their catalytically disabled negative controls) were cloned into retroviral vectors. Unmodified ribozymes co-expressed with HBV in human liver Huh7 cells reduced the level of viral particle production by up to 66% based on the endogenous polymerase assay, while the structurally modified ribozymes inhibited HBV production up to 83%. These encouraging results indicate the feasibility of ribozyme-mediated gene therapy for the treatment of HBV infections.

Gene Expression↗

PubMed1996

A potential therapeutic application of hairpin ribozymes: in vitro and in vivo studies of gene therapy for hepatitis C virus infection.

Two effective ribozymes (CR2 and CR4) that target HCV RNA 5' UTR and capsid gene regions were generated. Ribozyme cleavage was demonstrated in vitro, which can be enhanced by facilitator RNA molecules. In tissue culture cells, these two ribozymes can inhibit the expression of a cotransfected reporter gene containing HCV RNA target sequences. Furthermore, transduction of human hepatoma cells, HepG2, with retroviral vectors carrying CR2 or CR4 ribozymes enabled the cells to resist the infection by retroviral particles containing HCV target sequences. These results represent the first positive step towards the application of hairpin ribozymes in gene therapy for the treatment of HCV infection.

Animals↗

PubMed1995

Detection of cleavage products from an in vivo transcribed cis hairpin ribozyme in turnips using the CaMV plant virus.

In order to examine ribozyme (Rz) activity in vivo, we have adapted a virus to deliver Rz to plants. DNA fragments that code for both active and mutant cis-hairpin Rz were cloned into the double-stranded DNA plant virus, cauliflower mosaic virus (CaMV). These Rz constructs successfully infected Brassica campestris rapa (turnip). The plants that were infected with the active-Rz construct showed, on average, a one-week delay in the appearance of viral symptoms, when compared to the mutant-Rz control. Since CaMV replicates through reverse transcription of a full-length RNA intermediate, Rz cloned into the CaMV DNA should be transcribed within this viral RNA. If these Rz constructs cleave, the amount of intact virus RNA should be reduced, resulting in attenuated viral symptoms. In addition, lysate RNase protection assays showed fragments corresponding to the sizes of both the 5' and 3' cis cleavage products in the active Rz tissue. No cleavage products were observed from plant tissue infected with the mutant Rz. Both the attenuated systemic viral symptoms and the cleavage products from the protection assay strongly support in vivo transcription and cleavage of this hairpin Rz. This is the first report of an in vivo transcribed Rz showing cleaved products by direct RNA analysis (non-PCR) in plants or animals.

Base Sequence↗

PubMed1994

Mutagenesis of the hairpin ribozyme.

Extensive in vitro mutagenesis studies have been performed on the hairpin ribozyme and substrate in an effort to refine the overall secondary structure of the molecule and provide further insight into what elements are essential for activity. A secondary structure consisting of four helices and five loop regions remains the basic model as originally proposed. Two helices, helix 1 and 2, form between the substrate and ribozyme while helices 3 and 4 are within the ribozyme itself. Our results suggest that helices 3 and 4 are smaller than previously proposed, consisting of four base pairs and three base pairs respectively. Helix 4 can be extended without loss of activity and loop 3 at the closed end of the hairpin model can be varied in sequence with retention of activity. There is an unpaired nucleotide between helices 2 and 3 consisting of a single A base, suggesting the opportunity for flexibility within the tertiary structure at this point. Comparisons are made between the new data and previously published mutagenesis and phylogenetic data. Substrate targeting rules require base pairing between helices 1 and 2 with cleavage (*) occurring in a preferred 5'(g/c/u)n*guc3' sequence of the substrate.

Base Composition↗

PubMed1992

A method for generating transcripts with defined 5' and 3' termini by autolytic processing.

Plasmids containing both the hammerhead and hairpin ribozyme autocatalytic cassettes were constructed for the purpose of generating RNA transcripts with specific termini at both the 5' and 3' ends. Following transcription, the RNA encoded by these cassettes was capable of intramolecular cleavage. This resulted in the generation of a processed RNA, which was located between the two cassettes, with specifically engineered 5' and 3' ends. The two different ribozymes were selected for their efficient intramolecular cleavage ability and to reduce the possibility of DNA recombination that could occur if identical cassettes were used. An application of this technique was the generation of a processed RNA which was itself a ribozyme, with specific 5' and 3' termini. The ribozyme generated was a hairpin ribozyme specific for a sequence in the gene encoding hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoA reductase). The processed ribozyme was fully catalytically active against an RNA substrate sequence of HMGCoA reductase.

Autolysis↗

PubMed1990

'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA.

We have identified the catalytic domain within the sequence of the negative strand of the satellite RNA of tobacco ringspot virus. Minimum energy RNA folding calculations predict a two dimensional model with four major helical regions which are supported by mutagenesis experiments. This model for the catalytic complex consists of a 50 base catalytic RNA and a 14 base substrate RNA folded together in a type of hairpin two dimensional structure. Part of the recognition region between the catalyst and substrate is two helices of 6 bases and 4 bases respectively. Catalytic activity remains when the bases in these two helices are changed but base pairing is maintained. Thus an appropriately engineered 'hairpin' catalyst is capable of cleaving heterologous RNA.

Base Sequence↗

PubMed1989

RNA catalytic properties of the minimum (-)sTRSV sequence.

We have identified an RNA catalytic domain within the sequence of the 359 base long negative-strand satellite RNA of tobacco ringspot virus. The catalytic domain contains two minimal sequences of satellite RNA, a 50-base catalytic RNA sequence, and a 14-base substrate RNA sequence. The catalytic complex of catalytic RNA/substrate RNA represents a structure not previously found in any RNA catalytic reaction described to date. The reaction is truly catalytic since the catalytic RNA has multiple substrate cleavage events and is not consumed during the course of the reaction. A linear relationship is seen between reaction rate and catalytic RNA concentration. The reaction has a Km of 0.03 microM, a kcat of 2.1/min, a temperature optimum of near 37 degrees C, and an energy of activation of 19 kcal/mol.

Base Sequence↗

PubMed1988

Leucine-tRNA ligase complexes.

The methodologies described in this chapter allow the reproducible preparation of native high-molecular-weight synthetase complexes of leuRL. These complexes have the ability to preferentially utilize extracellular leucine immediately upon transport and are likely the forms of the enzyme most important in the utilization of leucine for protein synthesis.

Amino Acyl-tRNA Synthetases↗