Project Summary
The proposed research objectives are to study the structure-function relationship between
triplex DNA and a class of newly discovered flavonoid-based triplex-specific binding ligands and use
the obtained knowledge to design and develop more potent triplex binding ligands as antigene
enhancers in the antigene strategy for disease treatment. This proposal can be categorized into three
portions, structural modification (synthesis), determination of ligand-DNA interactions by biophysical
methods, and study of inhibition of enzymatic activities. The synthesis project includes changing the
linker length, removing the functional groups, and extending the aromatic surface. Each structural
modification is intended to address a specific inquiry on the structure-function relationship. The
synthesis procedures involve basic organic techniques. The synthesized molecules will then be used to
study their interactions with several DNA structures by various biophysical methods. The stabilization
of triplex and duplex DNA with ligands under various conditions (pH, ionic strength) will be
determined using thermal denaturation monitored by UC. The conformation changes of the DNA and
binding stoichiometry will be measured using circular dichroism. Selective binding to DNAs with
specific conformations will be determined using competition dialysis in a relatively high throughput
fashion. The transition enthalpy of the ligand-triplex DNA complex will be directly measured using
differential scanning calorimetry (DSC). The thermodynamics parameters, including enthalpy,
entropy, free energy, and binding site size, will be determined using isothermal titration calorimetry
(ITC). Notably, results from biophysical studies will be used for cross-inspection to ensure the accuracy
of the conclusions. After completion of the biophysical studies, ligands with the best binding strength
and specificity will be chosen for biochemical reactions. A plasmid DNA containing a triplex-forming
site will be used. In this plasmid DNA, four restriction endonuclease DraI cleavage sites are present in
the plasmid, one of which residues exactly at the junction of the triplex helix site. The formation of
triplex DNA should protect the DraI cleavage site, leading to the disappearance and appearance of
products with specific lengths, which can be readily detected by the agarose gel electrophoresis. The
results from the proposed work will be the first effort in the scientific community to understand these
newly discovered triplex-specific binding ligands. The nature of the research is suitable for training
undergraduate and graduate researchers at an undergraduate-focused institution.
