1-Deazaadenosine: synthesis and activity of base-modified hammerhead ribozymes (original) (raw)
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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.
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.
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.
Highly efficient synthesis of 2′-O-amino nucleosides and their incorporation in hammerhead ribozymes
Tetrahedron Letters, 1998
2'-O-Amino nucleosides were prepared from arabino nucleosides by triflate displacement with N-hydroxy phthalimide. The corresponding phosphoramidites suitable for automated oligonucleotide synthesis, were also prepared. Incorporation of 2'-O-amino uridine into position U7 or T-O-amino adenosine into position A9 of a hammerhead ribozyme resulted in a slight improvement of catalytic rates.
Nucleic Acids Research, 1992
The catalytic function of the hairpin ribozyme has been investigated by modification-interference analysis of both ribozyme and substrate, using ribonucleoside phosphorothioates. Thiophosphate substitutions in two ribozyme domains were examined by using a novel and highly efficient two-piece ribozyme assembled from two Independently synthesized oligoribonucleotides. The catalytic proficiency of the two-piece construct (KM = 48 nM, kcat = 2.3 min~1) is nearly identical to that of the one-piece ribozyme. The two-piece ribozyme is essentially unaffected by substitution with thiophosphates 5' to all guanosines, cytidines, and uridlnes. In contrast, incorporation of multiple adenosine phosphorothioates in the 5' domain of the ribozyme decreases ribozyme activity by a factor of 25. Modification-interference experiments using ribozymes partially substituted with adenosine phosphorothioate suggest that thiophosphates 5' to A 7 , Ag and A 10 interfere with cleavage to a greater extent than substitutions at other sites within the molecule, but the effect is modest. Within the substrate, phosphorothioate substitution does not directly interfere with cleavage, rather, increasing thiophosphate content decreases the stability of the rlbozyme-substrate complex. We describe the construction of a hairpin ribozyme containing dinucleotide extensions at its 5' and 3' ends. Full substitution of this molecule with G and C phosphorothioates results in a ribozyme with greatly enhanced stability against cellular ribonucleases without significant loss of catalytic efficiency.
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.