Ribozymes

doi:10.1016/S0959-440X(94)90099-X

Current Opinion in Structural Biology

Volume 4, Issue 3, June 1994, Pages 322-330

https://doi.org/10.1016/S0959-440X(94)90099-X Get rights and content

Abstract

In the past year there has been increasing interest in the six naturally occurring ribozyme systems. The main emphasis has been on approaches to increase our understanding of the three-dimensional active conformation and the details of the reaction mechanism of these structures. There is continued interest in the application of the hammerhead system in vivo to control viral replication, especially in animal cells.

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Construction of new ribozymes requiring short regulator oligonucleiotides as a cofactor
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A hairpin loop and an oligonucleotide bound to the loop form one-half of the pseudoknot structure. We have designed an allosteric hammerhead ribozyme, which is activated by the introduction of this motif by using a short complementary oligonucleotide as a cofactor. Stem II of the hammerhead ribozyme was substituted with a non-self-complementary loop sequence (loop II) to abolish the cleavage activity. The new ribozyme had almost no cleavage activity of the target RNA. However, it exhibited the cleavage activity in the presence of a cofactor oligoribonucleotide, which is complementary to loop II of the ribozyme. The activity is assumed to be derived from the formation of a pseudo-stem structure between the cofactor oligonucleotide and loop II. The structure including the loop may be similar to the pseudo-half-knot structure. The activation efficiencies of the cofactor oligonucleotides were decreased as the lengths of the oligonucleotides increased, and the ribozyme with a longer loop II was more active than that with a short loop II. Oligoribonucleotides with 3′-dangling purine bases served as efficient cofactors of the ribozyme, and a 2′-O-methyloligonucleotide enhanced the cleavage activity of the ribozyme most efficiently, by as much as about 750-fold as compared with that in the absence of the oligonucleotide. Cofactor oligonucleotides with a cytidine base at the 3′-end also activated a ribozyme with the G10.1·G11.1 mutation, which eliminates the cleavage activity in the wild-type. The binding sites of the oligonucleotide were identified by photo-crosslinking experiments and were found to be the predicted sites in the loop. This is the first report of a design aimed at positively controlling the activity of ribozymes by employing a structural motif. This method can be applied to control the activities of other functional RNAs with hairpin loops.
Ribozymes: The characteristics and properties of catalytic RNAs
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Ribozymes, or catalytic RNAs, were discovered a little more than 15 years ago. They are found in the organelles of plants and lower eukaryotes, in amphibians, in prokaryotes, in bacteriophages, and in viroids and satellite viruses that infect plants. An example is also known of a ribozyme in hepatitis delta virus, a serious human pathogen. Additional ribozymes are bound to be found in the future, and it is tempting to regard the RNA component(s) of various ribonucleoprotein complexes as the catalytic engine, while the proteins serve as mere scaffolding – an unheard-of notion 15 years ago! In nature, ribozymes are involved in the processing of RNA precursors. However, all the characterized ribozymes have been converted, with some clever engineering, into RNA enzymes that can cleave or modify targeted RNAs (or even DNAs) without becoming altered themselves. While their success in vitro is unquestioned, ribozymes are increasingly used in vivo as valuable tools for studying and regulating gene expression. This review is intended as a brief introduction to the characteristics of the different identified ribozymes and their properties.
Cellular delivery of hammerhead ribozymes conjugated to a transferrin receptor antibody
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Chemically-modified hammerhead ribozymes are sequence-specific RNA enzymes that can cleave target mRNA. These molecules have potential application as biological tools to understand gene expression and as therapeutic agents for the selective down-regulation of genes implicated in disease. However, as a result of their polyanionic character and relatively large molecular weights, ribozyme delivery to target cells is relatively inefficient. Using nuclease resistant 2′-O-methyl-modified ribozymes targeting the c-erbB1 oncogene, we have evaluated the potential use of human monoclonal transferrin-receptor antibody (TRA)-ribozyme conjugates for the improved delivery of ribozymes to A431 tumour cells. A 37-mer ribozyme derivatized with a free thiol-group at the 5′-end and bearing an internal [³²P]-radiolabel was conjugated to either TRA or a non-specific IgG antibody using the heterobifunctional crosslinker, succinimidyl 4-(maleimido methyl)cyclohexane-1-carboxylate (SMCC). Up to six molecules of the ribozyme could be conjugated to one molecule of antibody. Cellular uptake studies in cultured human epidermoid A431 carcinoma cells showed that approximately a three-fold increase in cellular association could be obtained with the TRA-ribozyme conjugate compared to the free ribozyme. Cellular association of the conjugate was temperature-dependent and was inhibited by competition with excess free transferrin receptor antibody implying that conjugate uptake was consistent with the transferrin receptor-mediated endocytosis pathway. Treatment of cells with monensin further enhanced TRA-ribozyme conjugate cell association indicating that ribozyme delivery of conjugates may be further improved by strategies that modulate vesicular trafficking in cells.
Inhibition of influenza A virus reproduction by a ribozyme targeted against PB1 mRNA
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A ribozyme gene directed at a specific cleavage of mRNA coding for PB1 protein, a component of RNA-dependent RNA-polymerase of influenza A virus, was constructed. The avian adenovirus CELO virus-associated RNA (VA RNA CELO) promoter and human cytomegalovirus (CMV) promoter were used for the permanent expression of the ribozyme in cell lines. The cells were infected with influenza A virus strains A/Singapore/1/57 and A/WSN/33, and the suppression of the virus reproduction and virus-specific protein synthesis was measured. The maximal level of the inhibition of virus reproduction as compared to the reproduction in non-transformed cells was 93.5%. Defective recombinant adenoviruses were constructed carrying the genes of functional and non-functional ribozymes under the control of human cytomegalovirus (CMV) promoter. The reproduction of A/WSN/33 virus in CV-1 cells preinfected with recombinant adenoviruses was shown to be suppressed.

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Ribozymes

Abstract

J Mol Biol

Nucleic Acids Res

Virology

FASEB J

J Biol Chem

Nature

J Mol Biol

Nucleic Acids Res

J Mol Biol

Self-Splicing of Group I Introns

Annu Rev Biochem

Recent Studies of Ribonuclease P

FASEB J

Ribozymes

Crit Rev Plant Sci

Small Catalytic RNAs

Annu Rev Biochem

Structural Conventions for Group I Introns

Nucleic Acids Res

Defining the Inside and Outside of a Catalytic RNA Molecule

Science

Important 2′-Hydroxyl Groups within the Core of a Group I Intron

Biochemistry

Guanosine Binding to the Tetrahymena Ribozyme: Thermodynamic Coupling with Oligonucleotide Binding

Dynamics of Ribozyme Binding of Substrate Revealed by Fluorescence-Detected Stopped-Flow Methods

Science

Metal Ion Catalysis in the Tetrahymena Ribozyme Reaction

Nature

A Highly Sensitive Probe for Guanine N7 in Folded Structures of RNA: Application to tRNAPhe and Tetrahymena Group I Intron

Biochemistry

Modelling of the Three-Dimensional Architecture of Group I Catalytic Introns Based on Comparative Sequence Analysis

J Mol Biol

Movement of the Guide Sequence During RNA Catalysis by a Group I Ribozyme

Science

Novel RNA Polymerization Reaction Catalyzed by a Group I Ribozyme

The EMBO Jour

A Highly Sensitive Probe for Guanine N7 in Folded Structures of RNA: Application to tRNA^Phe and Tetrahymena Group I Intron