The new world of ribozymes (original) (raw)
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Ribozymes: recent advances in the development of RNA tools
FEMS Microbiology Reviews, 2003
The discovery 20 years ago that some RNA molecules, called ribozymes, are able to catalyze chemical reactions was a breakthrough in biology. Over the last two decades numerous natural RNA motifs endowed with catalytic activity have been described. They all fit within a few well-defined types that respond to a specific RNA structure. The prototype catalytic domain of each one has been engineered to generate trans-acting ribozymes that catalyze the site-specific cleavage of other RNA molecules. On the 20th anniversary of ribozyme discovery we briefly summarize the main features of the different natural catalytic RNAs. We also describe progress towards developing strategies to ensure an efficient ribozyme-based technology, dedicating special attention to the ones aimed to achieve a new generation of therapeutic agents.
Ribozymes: the characteristics and properties of catalytic RNAs
FEMS Microbiology Reviews, 1999
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. ß Contents 0168-6445 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 -6 4 4 5 ( 9 9 ) 0 0 0 0 7 -8 * Tel.
Thirty-five years of research into ribozymes and nucleic acid catalysis: where do we stand today?
F1000Research, 2016
Since the discovery of the first catalytic RNA in 1981, the field of ribozyme research has developed from the discovery of catalytic RNA motifs in nature and the elucidation of their structures and catalytic mechanisms, into a field of engineering and design towards application in diagnostics, molecular biology and medicine. Owing to the development of powerful protocols for selection of nucleic acid catalysts with a desired functionality from random libraries, the spectrum of nucleic acid supported reactions has greatly enlarged, and importantly, ribozymes have been accompanied by DNAzymes. Current areas of research are the engineering of allosteric ribozymes for artificial regulation of gene expression, the design of ribozymes and DNAzymes for medicinal and environmental diagnostics, and the demonstration of RNA world relevant ribozyme activities. In addition, new catalytic motifs or novel genomic locations of known motifs continue to be discovered in all branches of life by the h...
Modulating RNA structure and catalysis: lessons from small cleaving ribozymes
Cellular and Molecular Life Sciences, 2009
RNA is a key molecule in life, and comprehending its structure/function relationships is a crucial step towards a more complete understanding of molecular biology. Even though most of the information required for their correct folding is contained in their primary sequences, we are as yet unable to accurately predict both the folding pathways and active tertiary structures of RNA species. Ribozymes are interesting molecules to study when addressing these questions because any modifications in their structures are often reflected in their catalytic properties. The recent progress in the study of the structures, the folding pathways and the modulation of the small ribozymes derived from natural, self-cleaving, RNA motifs have significantly contributed to today's knowledge in the field.
Nucleic Acids Research, 2001
Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small mol. catalysis to the design of macromol., biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phenanthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of mol. design principles from small-mol. cleavage to macromol. cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.
Chemically Modified Hammerhead Ribozymes with Improved Catalytic Rates
Biochemistry, 1996
A site-specific chemical modification strategy has been employed to elucidate structurefunction relationships at the only phylogenetically nonconserved position within the core of the hammerhead ribozyme (N7). Four different base substitutions at position 7 resulted in increased catalytic rates. A pyridin-4-one base substitution increased the rate of the chemical step up to 12-fold. These results are the first examples of chemical modifications within a catalytic RNA that enhance the rate of the chemical step. Four base substitutions resulted in decreased catalytic rates. The results do not correlate with proposed hydrogen bond interactions (Pley et al., 1994; Scott et al., 1995). This study demonstrates the utility of using unnatural nucleotide analogssrather than mutagenesis with the four standard nucleotides alonesto elucidate structure-function relationships of small RNAs.
RNA synthesis by in vitro selected ribozymes for recreating an RNA world
Life (Basel, Switzerland), 2015
The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme's possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory.
Ribozyme Catalysis Revisited: Is Water Involved?
Molecular Cell, 2007
Enzymatic catalysis by RNA was discovered 25 years ago, yet mechanistic insights are emerging only slowly. Thought to be metalloenzymes at first, some ribozymes proved more versatile than anticipated when shown to utilize their own functional groups for catalysis. Recent evidence suggests that some may also judiciously place structural water molecules to shuttle protons in acid-base catalyzed reactions.
Identification and characterization of a short 2'-3' bond-forming ribozyme
RNA, 2008
A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 29-39 phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.
Chemical Modification of Hammerhead Ribozymes
Journal of biological …, 1995
A systematic study of selectively modified, 36-mer hammerhead ribozymes has resulted in the identification of a generic, catalytically active and nuclease stable ribozyme motif containing 5 ribose residues, 29 -30 2-O-Me nucleotides, 1-2 other 2-modified nucleotides at positions U4 and U7, and a 3-3-linked nucleotide "cap." Eight 2-modified uridine residues were introduced at positions U4 and/or U7. From the resulting set of ribozymes, several have almost wild-type catalytic activity and significantly improved stability. Specifically, ribozymes containing 2-NH 2 substitutions at U4 and U7, or 2-C-allyl substitutions at U4, retain most of their catalytic activity when compared to the all-RNA parent. Their serum half-lives were 5-8 h in a variety of biological fluids, including human serum, while the all-RNA parent ribozyme exhibits a stability half-life of only ϳ0.1 min. The addition of a 3-3-linked nucleotide "cap" (inverted T) did not affect catalysis but increased the serum half-lives of these two ribozymes to >260 h at nanomolar concentrations. This represents an overall increase in stability/activity of 53,000 -80,000-fold compared to the all-RNA parent ribozyme.