Modified Hammerhead Ribozymes as Potential Therapeutics (original) (raw)
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Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability
BMC chemical biology, 2004
BACKGROUND: Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues. RESULTS: A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and t...
Synthetic Hammerhead Ribozymes as Therapeutic Tools to Control Disease Genes
Current Gene Therapy, 2005
Ribozymes are RNA molecules that have the ability to catalyse the cleavage and formation of covalent bonds in RNA strands at specific sites. The "hammerhead" motif, approximately 30-nucleotide long, is the smallest endonucleolytic cis-acting ribozyme structure found in natural circular RNAs of some plant viroids. Hammerhead ribozymes became appealing when it was shown that it is possible to produce transacting ribozymes directed against RNA sequences of interest. Since then, gene-tailored ribozymes have been designed, produced and given to cells to knock down the expression of specific genes. At present, this technology has advanced so much that many hammerhead ribozymes are being used in clinical trials. With this work we would provide some guidelines to design efficient transacting hammerhead ribozymes as well as review the recent results obtained with them as gene therapy tools.
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, 1992
Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of transacting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the nonenzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop 11, has a sixfold greater kc, value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop 11 (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.
Can hammerhead ribozymes be efficient tools to inactivate gene function
Nucleic Acids Research, 1994
In order to improve hammerhead ribozyme efficiency and specificity, we have analyzed, both in vitro and in vivo, the activity of a series of ribozyme/substrate combinations that have the same target sequence but differ in the length of the ribozyme/substrate duplex or in their structure, i.e., the total length of the RNA. In vitro, we have found that optimal kcatlKm (at 370C) is obtained when the ribozyme/substrate duplex has a length of 12 bases, which according to the base composition represents a calculated free energy of binding of -16 kcal/mol. We discuss the importance of this value for ribozyme specificity and present strategies that may improve it. Increasing the length of the duplex from 14 to 17 bases (from -19 to -26 kcal/mol) produces a reduced ribozyme activity which is probably due to a slower rate of product dissociation. In addition, inclusion of either the substrate or the ribozyme in a long transcript produces a reduction (10 fold) of the kcat/Km, probably because of a different accessibility of the target sequence. In vivo, the activity of the trans-acting ribozyme was extremely low and detected in only one case: with a ribozyme/substrate duplex length of 13 bases and with both ribozyme and substrate embedded in short RNAs expressed at a very high level. The similarity of the results obtained in vitro and in vivo indicates that it is possible to use an in vitro system to optimize ribozymes which are to be used in vivo. Satisfactory results were obtained in vivo only with cisacting ribozymes. Altogether these results suggest that the ribozyme/substrate hybridization step is the limiting step in vivo and therefore it is not clear if ribozymes represent an improvement over antisense RNAs.
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.
Ribozymes as therapeutic tools for genetic disease
Human Molecular Genetics, 1998
The discovery that RNA can act as a biological catalyst, as well as a genetic molecule, indicated that there was a time when biological reactions were catalysed in the absence of protein-based enzymes. It also provided the platform to develop those catalytic RNA molecules, called ribozymes, as trans-acting tools for RNA manipulation. Viral diseases or diseases due to genetic lesions could be targeted therapeutically through ribozymes, provided that the sequence of the genetic information involved in the disease is known. The hammerhead ribozyme, one of the smallest ribozymes identified, is able to induce site-specific cleavage of RNA, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Its ability to down-regulate gene expression through RNA cleavage makes the hammerhead ribozyme a candidate for genetic therapy. This could be particularly useful for dominant genetic diseases by down-regulating the expression of mutant alleles. The group I intron ribozyme, on the other hand, is capable of site-specific RNA trans-splicing. It can be engineered to replace part of an RNA with sequence attached to its 3′ end. Such application may have importance in the repair of mutant mRNA molecules giving rise to genetic diseases. However, to achieve successful ribozyme-mediated RNA-directed therapy, several parameters including ribozyme stability, activity and efficient delivery must be considered. Ribozymes are promising genetic therapy agents and should, in the future, play an important role in designing strategies for the therapy of genetic diseases.