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The Skaggs Institute For Chemical Biology
Scientific Report 1999-2000


Catalytic Nucleic Acids for Treating the Molecular Basis of Disease


G.F. Joyce, S.W. Santoro, J. Nowakowski, H. Kühne, R. Kumar, S.E. Jones

During the past decade, antisense technology has emerged as a promising approach for the treatment of cancer and inflammatory diseases. The antisense strategy uses short oligodeoxynucleotides that bind to 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

We developed DNA enzymes that can be directed to cleave almost any targeted RNA under cellular conditions. Our most versatile DNA enzyme is the "10-23" motif, which contains a catalytic domain of 15 nucleotides flanked by substrate-recognition domains of 7-12 nucleotides each. 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.

When the DNA enzyme is used to target RNAs in either cultured cells or whole organisms, the enzyme must be stabilized against degradation by nucleases. We accomplish this stabilization by introducing chemical modifications at both the 5´ and the 3´ ends of the molecule. Another challenge is to choose an accessible cleavage site within the target RNA. One approach is to target the start codon of a messenger RNA, which tends to be an accessible site. Another approach, which is more tedious but nearly always successful, is to screen a large number of potential target sites in order to find ones that are cleaved efficiently.

During the past 2 years, more than a dozen laboratories have used the 10-23 DNA enzyme to inactivate disease-related target RNAs. One particularly striking example is the work of Khachigian and colleagues at the University of New South Wales in Sydney, Australia, in which the DNA enzyme was instilled into the carotid artery of rats after balloon angioplasty; treatment with the enzyme inhibited vascular restenosis, which often occurs after balloon-induced injury. Another group at the Institute for Cancer Research in Oslo, Norway, used the DNA enzyme to inhibit expression of protein kinase Cα in various tumor cell lines. By inhibiting expression of this protein, the DNA enzyme caused the cancer cells to undergo apoptotic cell death, mediated by proteins that would otherwise be inhibited by protein kinase Cα.

Structure of a Nucleic Acid 4-Way Junction

We embarked on an effort to obtain a high-resolution crystal structure of the 10-23 DNA enzyme. In collaboration with D. Stout, The Scripps Research Institute, we obtained diffraction-quality crystals of the enzyme-substrate complex and of several heavy-atom derivatives. This work led to the determination of a crystal structure at 3-Å resolution. Unfortunately, the complex underwent rearrangement in the crystal so that it was no longer in a catalytically relevant conformation. Interestingly, however, it rearranged to give a 4-way junction structure that has important biological implications. Four-way junctions are a common feature of nucleic acid structure. They form the basis of the Holliday junction, which is the key intermediate of DNA recombination.

Unlike a true Holliday junction, which contains 4 strands of DNA, our structure contains 2 strands of DNA and 2 strands of RNA. Our structure agrees closely, however, with extensive data in the literature on the likely structure of the Holliday junction in solution. Six months after our work was published, the structure of an all-DNA 4-way junction appeared. This all-DNA structure is similar to our structure, other than subtle differences attributable to the presence of DNA in all 4 arms.

More recently, we obtained a second crystal structure of a 4-way junction, containing the same nucleotides as before but in a conformation that is rotated by 135° relative to the first conformation (Fig. 1). A comparison of the 2 structures provides insight into "crossover isomerization," in which 2 strands of a 4-way junction are exchanged, resulting in genetic recombination.

The large rotation of the helical axes is accommodated primarily by changes in the torsion angles of the sugar-phosphate backbone. The first conformation, which has an angle of +55° between the helical axes, maximizes base stacking at the expense of electrostatic repulsion between phosphate groups. The second conformation, which has an angle of -80°, minimizes electrostatic clashes at the expense of reduced base stacking. Both conformations are stabilized by metal ions bound at specific sites unique to each conformer. Taken together, these counterbalancing influences explain how strand exchange can be accomplished.

Publications

Jaeger, L., Wright, M.C., Joyce, G.F. A complex ligase ribozyme evolved in vitro from a group I ribozyme domain. Proc. Natl. Acad. Sci. U. S. A. 96:14712, 1999.

Joyce, G.F. The counterforce. Curr. Biol. 9:R500, 1999.

Joyce, G.F. Employing DNAzymes to target bcr-abl mRNA in chronic myelogenous leukemia. Blood Cells Mol. Dis. 26:60, 2000.

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, 1999, p. 21.

Nowakowski, J., Shim, P.J., Joyce, G.F., Stout, C.D. Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides. Acta Crystallogr. Biol. Crystallogr. D 55:1885, 1999.

Nowakowski, J., Shim, P.J., Stout, C.D., Joyce, G.F. Alternative conformations of a nucleic acid four-way junction. J. Mol. Biol. 300:93, 2000.

Ordoukhanian, P., Joyce, G.F. A molecular description of the evolution of resistance. Chem. Biol. 6:881, 1999.

Rogers, J., Joyce, G.F. A ribozyme that lacks cytidine. Nature 402:323, 1999.

Santoro, S.W., Joyce, G.F., Sakthivel, K., Gramatikova, S., Barbas, C.F. RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc. 122:2433, 2000.

Sheppard, T.L., Ordoukhanian, P., Joyce, G.F. A DNA enzyme with N-glycosylase activity. Proc. Natl. Acad. Sci. U. S. A. 97:7802, 2000.

Sheppard, T.L., Wong, C.-H., Joyce, G.F. Nucleoglycoconjugates: Design and synthesis of a new class of DNA-carbohydrate conjugates. Angew. Chem. Int. Ed., in press.

 

 







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