Project Summary The proposed research objective is to acquire an advanced gel imaging system to enhance the ongoing research on studying the structure-function relationship between triplex DNA and a class of newly discovered flavonoid-based triplex-specific binding ligands. Knowledge of ligand-mediated triplex formation can be used to design potent antigene enhancers in the antigene strategy for disease treatment. This gel imaging system will be used in three sets of experiments. 1) A plasmid DNA containing four restriction endonuclease DraI cleavage sites will be constructed. One of the DraI cleavage sites resides in a triplex-forming region. The ligand-mediated triplex formation will protect this cleavage site from DraI digestion. The cleaved DNA fragments can be separated by agarose gel electrophoresis and observed on the new gel imaging system via fluorescence. Based on the DNA fragments obtained from DraI digestion, we can determine the efficiency of ligand-mediated triplex formation on inhibition of enzymatic activities. High-quality gel images will be obtained. 2) Fluorescent probe labeled triplex-forming DNA oligonucleotides will be synthesized. The purine strand of duplex DNA is labeled with a green probe, and the triplex-forming oligonucleotide is labeled with a red probe. The formed DNA will be separated using polyacrylamide gel electrophoresis. The ligand-mediated triplex formation will result in different color schemes representing the formation of duplex and triplex DNA (duplex DNA: a green band; triplex DNA: a yellow band; no duplex or triplex DNA formation: separated red and green bands). This color scheme can be readily observed using multiplex detection on the new gel imaging system. 3) A plasmid DNA containing a red fluorescent protein (mcherry) will be constructed. The upstream of the mcherry sequence contains a triplex-forming site. The ligand- mediated triplex formation will reduce the synthesis of red fluorescent protein in live cells (E. coli). The fluorescence in live cells (in vivo) can be visualized using the plated-based imaging on the new gel imaging system. The acquisition of an advanced gel imaging system allows us to conduct high-quality gel imaging experiments proposed in the parent award with 24/7 access and to expand our research capability to perform multiplex imaging and in vivo imaging. In addition, it introduces state-of-the-art imaging technology to our undergraduate and graduate students to enhance their experiential learning experience at Pacific and improve the research infrastructure at our undergraduate-centered institution.