29th Annual Meeting of the American Society of Photobiology

Downtown Marriot

Chicago, Il.

Developing Organic Photosensitizers to Probe Macromolecular Structure and Dynamics

Abstract-
Research in our laboratory uses complementary methods of transient laser spectroscopy and traditional bioanalytical methods to investigate photocleavage of nucleic acids and proteins. Specifically, unfuctionalized naphthalimide systems are employed to induce cleavage of the biological polymers by targeting the nucleotide bases in DNA and oxidizable aromatic side chains in proteins. Our current efforts include developing parallel systems that will induce site-specific (for sequencing applications) and non-specific (for footprinting applications) lesions in oligonucleotides following the absorption of a photon by the naphthalimide chromophore. We have elucidated three mechanisms by which the functionalized chromophores can initiate cleavage of oligonucleotides and peptides. These include: (i) Radical production via "self-quenching" reactions; (ii) production of reactive oxygen intermediates; and (iii) direct reaction with the macromolecule. Using laser flash photolysis, the excited states and reactive radicals have been identified. In conjunction with HPLC and electrophoretic product analysis, we have shown that the rate and mechanism of photoinitiated cleavage can be readily tuned using a single photosensitizer. For example, electron exchange between the electronically excited triplet state of the naphthalimide by ground-state chromophores results in highly reactive, non-specific cleavage agents. The utility of these systems as photofootprinting agents and site-specific endonucleases will be discussed. In parallel studies, we are preparing amino acid conjugates of the photosensitizers. Amino acids with charged or hydrophobic side chains will be used to facilitiate association with different protein residues. We have prepared tyrosine-functionalized 1,8-naphthalimide and are investigating their activity as "photoproteases." The work is aimed at designing of artificial "photoproteases," which is an area that remains virtually unexplored. In conjunction with developing chemical systems to carry out the reactions described above, we are integrating these systems into time-resolved assays, where macromolecular dynamics, following the initiation of a mixing or folding event, can be directly probed.

Keywords: photochemistry, electron transfer, naphthalimide, macromolecule