29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

Naphthalimide Derivatives: Tunable Nucleases and Proteases

Abstract-
Nucleases and proteases are classes of enzymes that cleave DNA and proteins, respectively, by various mechanisms. A novel series of chromophores derived from the naphthalimides (NI) has been synthesized and their ability to photoinitiate cleavage of DNA and proteins has been studied. Steady state photolysis of these compounds in the presence of pUC-19 supercoiled DNA was carried out using a 450W mercury lamp. Photocleavage products were separated and quantified by gel electrophoresis (2% agarose gel, post stained with 5ug/ml ethidium bromide). Apparent first order rate constants for depletion of supercoiled DNA were measured. The results of these experiments suggest that the presence of oxygen and halides inhibit the cleavage. Tuning redox potential of these compounds increased the efficiency of cleavage. Similar experiments were done with a synthetic 20bp DNA to investigate whether the damage was guanine specific or sequence neutral. Results of these experiments suggest that variation in concentration alters the type of cleavage. The mechanism of damage has been elucidated using laser flash photolysis. Using 355nm excitation, the first-order triplet-state decay at 480nm and the emergence of NI.- radical at 400nm were observed. From these investigations the reactive intermediates are observed in real time and the results are correlated with photo damage products. Preliminary mechanistic results from our protein damage experiments will also be presented. The results obtained in the studies of these chromophores have direct applications in genome sequencing, characterization of DNA protein interactions, photo-foot printing, and in photo-therapeutic applications.

Keywords: DNA photocleavage, Protein damage, Laser Flash Photolysis, Gel electrophoresis