Base-caged Adenosine Triphosphate as a Model System for Photoactivatable Small Interfering RNA
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