Jasenka Matulic-Adamic

Chemical modification of hammerhead ribozymes. Catalytic activity and nuclease resistance.

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 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 0.1 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.

Optimizing the Cell Efficacy of Synthetic Ribozymes SITE SELECTION AND CHEMICAL MODIFICATIONS OF RIBOZYMES TARGETING THE PROTO-ONCOGENE c-myb

Expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5′-end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2′-modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

Synthesis of 5-(β-D-ribofuranosyl)-pyridin-2-one: A “deletion-modiffied” analogue of uridine

Pyridin-2-one C-nucleoside2 was prepared using several different approaches. The most efficient pathway utilized the condensation of 2,3,5-tri-O-benzyl-d-ribono-1,4-lactone (4) and 2-(benzyloxy)-5-bromopyridine (5), followed by deoxygenation with Et3SiHBF3·Et2O and removal of the benzyl protecting groups.

Synthesis of 3-(β-D-ribofuranosyl)-2-fluoropyridine and 3-(β-D-ribofuranosyl)-pyridin-2-one

Abstract Pyridin-2-one C -nucleoside 13 was prepared in 7 steps from 2-fluoropyridine 1 and D-ribono-1,4-lactone 2 . The successful approach to β-ribofuranosides 12 and 13 consisted of the reductive opening of the furanose ring of hemiacetal 3 followed by intramolecular Mitsunobu cyclization.

Structure–function relationships in the hammerhead ribozyme probed by base rescue

We previously showed that the deleterious effects from introducing abasic nucleotides in the hammerhead ribozyme core can, in some instances, be relieved by exogenous addition of the ablated base and that the relative ability of different bases to rescue catalysis can be used to probe functional aspects of the ribozyme structure [Peracchi et al., Proc NatAcad Sci USA 93:11522]. Here we examine rescue at four additional positions, 3, 9, 12 and 13, to probe transition state interactions and to demonstrate the strengths and weaknesses of base rescue as a tool for structure-function studies. The results confirm functional roles for groups previously probed by mutagenesis, provide evidence that specific interactions observed in the ground-state X-ray structure are maintained in the transition state, and suggest formation in the transition state of other interactions that are absent in the ground state. In addition, the results suggest transition state roles for some groups that did not emerge as important in previous mutagenesis studies, presumably because base rescue has the ability to reveal interactions that are obscured by local structural redundancy in traditional mutagenesis. The base rescue results are complemented by comparing the effects of the abasic and phenyl nucleotide substitutions. The results together suggest that stacking of the bases at positions 9, 13 and 14 observed in the ground state is important for orienting other groups in the transition state. These findings add to our understanding of structure-function relationships in the hammerhead ribozyme and help delineate positions that may undergo rearrangements in the active hammerhead structure relative to the ground-state structure. Finally, the particularly efficient rescue by 2-methyladenine at position 13 relative to adenine and other bases suggests that natural base modifications may, in some instance, provide additional stability by taking advantage of hydrophobic interactions in folded RNAs.

Synthesis of nucleoside 5'-deoxy-5'-difluoromethylphosphonates

Abstract 1- O -Acetyl-2,3-di- O -benzoyl- d -ribofuranose 5-deoxy-5-difluoromethylphosphonate was synthesized in three steps from 1- O -methyl-2,3- O -isopropylidene-β- d -ribofuranose 5-deoxy-5-difluoromethylphosphonate. Condensation of this suitably derivatized sugar with silylated pyrimidines and purines afforded novel nucleoside 5′-deoxy-5′-difluoromethylphosphonates.

New Structural Motifs for Hammerhead Ribozymes. Catalytic Activity of Abasic Nucleotide Substituted Ribozymes

Abstract The synthesis of 1-deoxy-D-ribofuranose-3-(2-cyanoethyl N,N-diisopropylphosphoramidite) (6) from D-ribose and its incorporation into a hammerhead ribozyme is described.

An efficient synthesis of the ribozyme–folate conjugate

Abstract γ-Cysteamine modified folic acid was synthesized by reductive alkylation of N2-iBu-6-formylpterin with suitably protected cysteaminyl- l -glutamyl-p-aminobenzoic acid, followed by deprotection. Following activation with 2,2′-dipyridyl disulfide, this synthon was conjugated to the 5′-end of 5′-thiol modified ribozyme to afford target ribozyme–folate conjugate in a good yield.

Synthesis of 1-deoxy-1-C-(p-Aniline)-β-D-ribofuranose and its incorporation into hammerhead ribozymes

Abstract p-AnilineC-ribofuranoside 11 was prepared in 5 steps from 1-bromo-4-lithiobenzene (1) and D-ribono-1,4-lactone 2. A three step derivatization of 11 yielded 3′-O-phosphoramidite 14 which was incorporated into ribozymes using the solid phase phosphoramidite procedure.

Synthesis of pyridinone ribonucleoside 3′-O-phosphoramidites and their incorporation into oligoribonucleotides

Abstract Protected pyridin-2- and pyridin-4-one ribonucleosides 3 and 9 were synthesized using a one-pot reaction of silylated bases with 1-O- acetyl-tri -O- benzoyl-β- d -ribofuranose (2) in the presence of CF3SO3SiMe3. The nucleosides were converted in 4 steps into 3′-O-phosphoramidites 7 and 11 which were incorporated into hammerhead ribozyme substrates using solid-phase phosphoramidite chemistry.