Energy Transfer Phenomenon with Nanoluciferase as Energy Donor

Abstract

Bioluminescence is a phenomenon that occurs where light is emitted from a chemical reaction that occurs in an organism.An enzyme called a luciferase assists in the catalysis of a reaction that releases the energy of broken bonds as light at a certain wavelength. Bioluminescence Resonance Energy Transfer (BRET) is a technique that utilizes a luciferase as an energy donor, whichemits energy from catalysis of a substrate. Fluorescence by Unbound Excitation from Luminescence (FUEL) deviates fromBRET slightly. FUEL involves an energy donor exciting an energy accepting particle; however, the acceptor and donor are unattached to each other. The luciferase being used as an energy donor is Nanoluciferase (Nluc)that emits light at 454 nm. Nluc beenmodified to expressa His-6 tag. The energy acceptors being used are Alexa 488, 555, and 647 and reemit light at the corresponding wavelength. Each of the Alexa dyes has been modified to include an anti-His antibody, causing attachment between Nluc and the respective Alexa dye.Energy transfer in the form of BRET did occur between Nluc and the Alexa 488 and minimally with Alexa 555. No energy transfer was noted with Alexa 647. Energy transfer was quantified using a fluorometer.