Structural basis for heterogeneous kinetics: Reengineering the hairpin ribozyme (original) (raw)
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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.
Substrate specificity and reaction kinetics of an X-motif ribozyme
RNA, 2003
The X-motif is an in vitro-selected ribozyme that catalyzes RNA cleavage by an internal phosphoester transfer reaction. This ribozyme class is distinguished by the fact that it emerged as the dominant clone among at least 12 different classes of ribozymes when in vitro selection was conducted to favor the isolation of high-speed catalysts. We have examined the structural and kinetic properties of the X-motif in order to provide a framework for its application as an RNA-cleaving agent and to explore how this ribozyme catalyzes phosphoester transfer with a predicted rate constant that is similar to those exhibited by the four natural self-cleaving ribozymes. The secondary structure of the X-motif includes four stem elements that form a central unpaired junction. In a bimolecular format, two of these base-paired arms define the substrate specificity of the ribozyme and can be changed to target different RNAs for cleavage. The requirements for nucleotide identity at the cleavage site are GD, where D = G, A, or U and cleavage occurs between the two nucleotides. The ribozyme has an absolute requirement for a divalent cation cofactor and exhibits kinetic behavior that is consistent with the obligate binding of at least two metal ions.
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.
Synthesis, deprotection, analysis and purification of RNA and ribosomes
Nucleic acids …, 1995
Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethyrthic-1 H-tetrazole (S-ethyltetrazole) as the activator. Base and Z-O-tbutyldimethylsllyl deprotection with methylamlne (MA) and anhydrous trtethylamlne/hydrogen fluoride In W-methylpyrrolidinone (TEAHF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of ollgoribonucleotlde purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods Improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.
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.
Nucleic Acids Research, 2002
A general approach is described for controlling the RNA-cleaving activity of nucleic acid enzymes (ribozymes and DNAzymes) via the use of oligonucleotide effectors (regulators). In contrast to the previously developed approaches of allosteric and facilitator-mediated regulation of such enzymes, this approach, called 'expansive' regulation, requires that the regulator bind simultaneously to both enzyme and substrate to form a branched three-way complex. Such three-way enzyme-substrate-regulator complexes are catalytically competent relative to the structurally unstable enzyme-substrate complexes. Using the 8-17 and bipartite DNAzymes and the hammerhead ribozyme as model systems, 20-to 30-fold rate enhancements were achieved in the presence of regulators of engineered variants of the above three enzymes, even under unoptimized conditions. Broadly, using this approach ribozyme and DNAzyme variants that are amenable to regulation by oligonucleotide effectors can be designed even in the absence of any knowledge of the folded structure of the relevant ribozyme or DNAzyme. Expansive regulation therefore represents a new and potentially useful technology for both the regulation of nucleic acid enzymes and the detection of specific RNA transcripts.
Total chemical synthesis of a ribozyme derived from a group I intron
Proceedings of the National Academy of Sciences, 1995
We describe the complete chemical synthesis of a ribozyme that catalyzes template-directed oligonucleotide ligation. The specific activity of the synthetic ribozyme is nearly identical to that of the same enzyme generated by in vitro transcription with T7 RNA polymerase. The ribozyme is derived from a group I intron and consists of three RNA fragments of 36, 43, and 59 nt that self-assemble to form a catalytically active complex. We have site-specifically substituted ribonucleotide analogs into this enzyme and have identified two 2'-hydroxyl groups that are required for full catalytic activity. In contrast, neither the 2'-hydroxyl nor the exocyclic amino group of the conserved guanosine in the guanosine binding site is necessary for catalysis. By allowing the ribozyme to be modified as easily as its substrates, this synthetic ribozyme system should be useful for testing specific hypotheses concerning ribozyme-substrate interactions and tertiary interactions within the ribozyme.
Kinetic Mechanism of the Hairpin Ribozyme
Journal of Biological Chemistry, 1997
To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozyme⅐ substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min ؊1) were typically 15-fold greater than cleavage rates (0.2 min ؊1), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (k on ؍ 4 ؋ 10 8 M ؊1 min ؊1), and the ribozyme⅐ substrate complex is very stable (K D < 25 pM; k off < 0.01 min ؊1). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.
Synthesis of 2′–modified nucleotides and their incorporation into hammerhead ribozymes
Nucleic acids …, 1995
Several 2'-modified ribonucleoside phosphoramidites have been prepared for structure-activity studies of the hammerhead ribozyme. The aim of these studies was to design and synthesize catalytically active and nuclease-resistant ribozymes. Synthetic schemes for stereoselective synthesis of the R isomer of 2'-deoxy-2'-Oallyl undine and cytidine phosphoramidites, based on the Keck allylation procedure, were developed. Protection of the 2'-amino group in 2'-deoxy-2'-aminouridine was optimized and a method for the convenient preparation of 5'--dimethoxytrityl-2'-deoxy-2'-phthalimidouridine 3'-0-(2-cyanoethyl-N,N-diisopropylphosphoramidite) was developed. During the attempted preparation of the 2'-Ot-butyIdimethylsilyl-3'-O-phosphoramidite of arabinouridine a reversed regioselectivity in the silylation reaction, compared with the published procedure, was observed, as well as the unexpected formation of the 2,2'-anhydronucleoside. A possible mechanism for this cyclization is proposed. The synthesis of 2'-deoxy-2'-methylene and 2'-deoxy-2'-difluoromethylene uridine phosphoramidites is described. Based on a '5-ribose' model for essential 2'-hydroxyls in the hammerhead ribozyme these 2'-modifled monomers were incorporated at positions U4 and/or U7 of the catalytic core. A number of these ribozymes had almost wild-type catalytic activity and improved stability in human serum, compared with an all-RNA molecule.