News and Publications
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
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
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
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.
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,
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,
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.