Master of Science in Biological and Agricultural Engineering (MSBAE)
Biological and Agricultural Engineering
Photocaged adenosine triphosphate (ATP) is one of the earliest examples of exerting spatial-temporal control over the activity of a substrate. The activity of ATP is blocked until near-ultraviolet light exposure photocleaves the cage moiety. Caged ATP has been used for a myriad of applications including kinetic studies of ATP-dependent enzymes. Traditional caging of ATP occurs at the gamma-phosphate, which has been found to competitively inhibit several enzymatic systems. It was hypothesized that blocking access to the adenosine N6 position via cage molecule would prevent the initial enzyme-substrate binding event from occurring prior to photolysis, effectively minimizing competitive inhibition. Utilizing a convertible nucleoside analog of ATP, this work synthesized, purified, and characterized a form of caged ATP which, by attaching the cage molecule to the nucleobase, did not inhibit the enzymatic activity of luciferase in vitro. Characterization was accomplished via UV/Vis spectroscopy, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and mass spectrometry (MS). Base-caged ATP was evaluated in a firefly luciferase enzymatic assay to determine the degree of bioactivity in the caged and photoactivated states and compared to the results of native (uncaged) ATP and gamma-NPE-caged ATP. Photolysis was conducted via 308 nm light from a transilluminator. Base-caged ATP did not inhibit the enzymatic system and the convertible nucleoside synthesis approach offers significant advantages over other caging techniques.
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Hendrix, Amanda Yoesting, "Base-caged Adenosine Triphosphate as a Model System for Photoactivatable Small Interfering RNA" (2012). LSU Master's Theses. 527.
Monroe, W. Todd