Molecular Evolution and Biomedical
Application of DNA and RNA Enzymes,
DNA and RNA Aptamers
My laboratory primarily focuses on the creation and in-depth
study of DNA and RNA molecules that have interesting
properties, such as the ability to function as enzymes (i.e., enzymes
that are made of DNA and RNA) or bind other molecules (DNA
and RNA aptamers). These special nucleic acid molecules may
hold great promise for a variety of applications, including, new
therapeutic agents to cure and control specific diseases.
Instead of relying on Mother Nature to supply us with DNA
or RNA molecules for our study, we create them in our own
hands using an evolution technology known as "In Vitro
Selection". It is a combinatorial approach that mimics Darwinian
evolution in a test tube. Typical experiments involve the following
(A) Construction of a random-sequence DNA pool. A
population containing up to 10E16 (10,000,000,000,000,000)
different single-stranded DNA or RNA molecules is generated
by automated DNA synthesis as well as enzymatic manipulations.
This extraordinarily diverse DNA or RNA pool (or library)
unquestionably contains molecules with special functions to
catalyze chemical reactions or to bind other molecules such as
(B) In vitro selection (see illustration). The above DNA
library is challenged to perform a specific task (such as catalysis
for a biological reaction) in carefully designed in vitro evolution
experiments. Strategies are implemented to allow the isolation
of capable molecules from incapable ones; the selected molecules
are amplified by PCR into a new generation for a new cycle of
selection-amplification. The cycle is repeated for a number of
times until the terminal population displays the desired properties.
(C) Forced evolution (see illustration). Usually, the above
selection experiments lead to the isolation of many different
molecules for any given task. Mutations are then introduced,
and the mutagenized molecules are forced to compete with each
other for the survival of the fittest under very demanding
conditions. The winning molecules will again be selected and
amplified, with more mutations being introduced. The process
will be repeated until the surviving population is very proficient
at completing the desired task.
(D) DNA cloning and sequencing. The identity of the
winning molecules in the terminal population will be revealed by
DNA cloning and sequencing techniques. Sequence comparison
studies will permit the identification of different classes of
the fittest functional DNA and RNA molecules for further studies.
(E) Biochemical analysis. A variety of chemical, biochemical,
and physical methods will be used to probe the structure and
function of the isolated molecules in order to understand the
molecular mechanisms for catalysis or binding (see an example).
(F) Biomedical and other applications. Thus created
special DNA or RNA molecules will then be fully assessed for
applications in biology, medicine and biotechnology. The potential
end uses of these functional nucleic acid sequences include
controlling the selective cellular functions, deactivating virus or
disease-prone genes, establishing novel drug-screening
Capping DNA with DNA. Li, Y., Liu, Y. & Breaker, R. R.
(2000). Biochemistry 39, 3106-3114. PubMed
Deoxyribozymes: New players in the ancient games of
biocatalysis. Li, Y. & Breaker, R. R. (1999). Curr.Opin.
Struct. Biol. 9, 315-323.PubMed
Phosphorylating DNA with DNA. Li, Y. & Breaker, R. R.
(1999). Proc. Natl. Acad. Sci. U. S. A. 96, 2746-2751.
Kinetics of RNA degradation by specific base catalysis
of transesterification involving the 2'-hydroxyl group.
Li, Y. & Breaker, R. R. (1999). J. Am. Chem. Soc.121,
DNA-enhanced peroxidase activity of DNA aptamer-
hemin complex. Travascio, P., Li, Y. & Sen, D. (1998).
Chem. Biol. 5, 505-517.PubMed
The modus operandi of a DNA enzyme: Enhancement
of substrate basicity. Li, Y. & Sen, D. (1998). Chem.
Biol. 5, 1-12. PubMed
Towards an efficient DNAzyme. Li, Y. & Sen, D. (1997).
Biochemistry 36, 5589-5599. PubMed
A catalytic DNA for porphyrin metallation. Li, Y. & Sen,
D. (1996). Nature Structural Biology 3, 743-747.
Recognition of anionic porphyrins by DNA aptamers.
Li, Y., Geyer, C. R. & Sen, D. (1996). Biochemistry 35,