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The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999


Catalytic Nucleic Acids for Treating the Molecular Basis of Disease


G.F. Joyce, S.W. Santoro, J. Nowakowski, T.L. Sheppard, R. Kumar, S.E. Jones

During the past decade, antisense technology has emerged as a promising approach for the treatment of cancer and inflammatory and viral diseases. The antisense strategy uses short oligonucleotides that bind particular cellular RNAs, leading to inactivation of the RNAs. The discovery and subsequent development of catalytic nucleic acids enhanced antisense technology by providing agents that both recognize and inactivate a target RNA. We used in vitro evolution techniques to develop novel nucleic acid enzymes that cleave RNA or DNA targets in a sequence-specific manner. We are investigating the structure and mechanism of these enzymes and are applying the enzymes to the inactivation of disease-related genes in living cells.

Rna-Cleaving Dna Enzymes

Several years ago, we used in vitro selection to develop the first example of a DNA enzyme. Since then, we have produced additional examples, including DNA enzymes that can be directed to cleave almost any targeted RNA under cellular conditions. Compared with synthetic RNA enzymes, DNA enzymes are easier to prepare and are more stable in biological tissues. Our most powerful and versatile DNA enzyme is the "10-23" motif, which contains a catalytic domain of 15 nucleotides flanked by substrate-recognition domains of 7­10 nucleotides each (Fig. 1). The catalytic efficiency of this enzyme is the highest of any known nucleic acid enzyme, limited only by the rate of enzyme-substrate association. The enzyme binds its RNA substrate through Watson-Crick pairing, enabling us to target different RNAs simply by altering the sequence of the recognition domains.

We introduced chemical modifications at the 5´ and 3´ ends of the 10-23 enzyme to increase its stability in biological tissues. During the past year, several laboratories showed that stabilized forms of the enzyme can be used to inactivate specific target messenger RNAs in cells. Most notably, D. Snyder and colleagues at the City of Hope Medical Center in Duarte, California, and K. Taira and colleagues at the Institute of Applied Biochemistry in Tsukuba, Japan, showed that the enzyme inhibited bcr-abl oncogene expression in bone marrow cells obtained from patients with leukemia. This inhibition resulted in apoptotic cell death of the leukemic cells but not the normal cells.

In collaboration with C. Barbas, The Scripps Research Institute, we developed an RNA-cleaving DNA enzyme that contains 3 essential imidazole-functionalized deoxyuridine residues in place of thymidine. The in vitro selection scheme was the same as that used to obtain the 10-23 enzyme, but in this case, the modified residues, synthesized previously by Barbas and colleagues, were used to provide added functionality similar to that of the amino acid histidine. The resulting modified DNA enzyme, the "16.2-11" motif, can be made to cleave RNA substrates that contain the sequence AUG (Fig. 1). The enzyme has a multiple turnover rate of greater than 1/min in the presence of micromolar concentrations of zinc.

Structural Studies

We have tried to obtain a high-resolution crystal structure of the 10-23 DNA enzyme. No DNA enzyme structure has ever been determined. In collaboration with D. Stout, The Scripps Research Institute, we obtained diffraction-quality crystals of the enzyme-substrate complex and of 6 corresponding heavy-atom derivatives. Data were collected at the Stanford Synchrotron Radiation Laboratory, and the structure of the complex was solved at 3.0-Å resolution.

The structure obtained in the crystal was not the same as that of the molecules in solution. Instead, an 82-nucleotide complex was formed, consisting of 2 strands of DNA and 2 strands of RNA. Interestingly, this complex has the structure of a 4-way junction, analogous to the structure of the Holliday junction that occurs during genetic recombination. Our structure is the first reported of an all-nucleic-acid 4-way junction, revealing a specifically positioned metal ion that stabilizes the sharp turn of the DNA backbone at the junction. Efforts continue to obtain the structure of the 10-23 DNA enzyme in a catalytically relevant conformation, which would guide further development of the enzyme as a therapeutic agent.

Publications

Joyce, G.F. Reactions catalyzed by RNA and DNA enzymes. In: The RNA World, 2nd ed. Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, p. 687.

Joyce, G.F., Orgel, L.E. Prospects for understanding the origin of the RNA world. In: The RNA World, 2nd ed. Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999, p. 49.

Kumar, R.M., Joyce, G.F. Developing ribozymes for therapeutic application through in vitro evolution. In: Intracellular Ribozyme Technology: Protocols and Applications. Rossi, J.J., Couture, L. (Eds.). Horizon Scientific Press, Norfolk, England, in press.

Nowakowski, J., Shim, P.J., Prasad, G.S., Stout, C.D., Joyce, G.F. Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme. Nat. Struct. Biol. 6:151, 1999.

Santoro, S.W., Joyce, G.F. Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37:13330, 1998.

 

 







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