Carolyn Gonzalez

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.

Remarkable enhancement of binding affinity of Heterocycle-modified DNA to DNA and RNA. Synthesis, characterization and biophysical evaluation of N2-imidazolylpropylguanine and N2-imidazolylpropyl-2-aminoadenine modified oligonucleotides

Abstract Oligonucleotides containing novel N2-Imidazolylpropylguanine and N2-Imidazolylpropyl-2-aminoadenine moieties were synthesized and studied for their hybridization and biophysical properties. Interestingly, these heterocycle modified oligonucleotides showed a remarkable enhancement of heteroduplex binding affinity when hybridized to complementary DNA.

Highly efficient synthesis of 2′-O-amino nucleosides and their incorporation in hammerhead ribozymes

Abstract 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 2′- O -amino adenosine into position A9 of a hammerhead ribozyme resulted in a slight improvement of catalytic rates.

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.

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.

SYNTHESIS AND INCORPORATION OF 2'-AMINO ACID CONJUGATED NUCLEOTIDES INTO RIBOZYMES

Abstract 2′-Amino-2′-deoxyuridine ( 1 ) was conjugated with N -Fmoc protected L-amino acids. 5′- O -Dimethoxytritylation followed by 3′- O -phosphitylation afforded 3′- O -phosphoramidites 4a-d . These monomers were incorporated into ribozymes using the solid phase phosphoramidite method. The catalytic activity of these ribozymes is reported.

2′-O-Methyl Thiomethyl Modifications in Hammerhead Ribozymes

Abstract The synthesis of all four phosphoramidites of 2′-O-methylthiomethyl ribonucleosides and their incorporation into hammerhead ribozymes and influence on nuclease stability and catalytic activity is described.