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This paper is restricted to users on the Connecticut College campus until May 17, 2023.


In previous research, bioluminescence proteins have been used for in vivo bioluminescence deep tissue imaging. A familiar organism that produces bioluminescent light, is a firefly. Firefly luciferase normally produces yellow-green light (λmax ~ 560 nm) with LH2. There are several reasons for improving bioluminescent light to emit in the near-infrared window (nIR) between 650 nm and 900 nm. The main reasons for improving nIR detectability include that the light transmits more efficiently through mammalian tissues due to less light scattering, nIR light is also less likely to be absorbed by hemoglobin, and water. Overall this provides better resolution for deep tissue imaging.

This thesis project included using directed evolution techniques to improve the Branchini Lab’s best luciferase enzyme with its complementary substrate analog (PLR3 with OH-QLH2) for potential use in in vivo bioluminescence imaging applications. Directed evolution methodology was used to optimize the PLR3 enzyme and specifically target where the substrate binds in the N2-region. The objective was to increase the specific activity of the enzyme/substrate pair and minimize the loss of nIR emission by targeting the N2-region.

All specific activity improvements over the original template, PLR3, came from a GeneMorph® library screen, which produced PLR3/C47 and PLR3/C47T. In HEK293T live cells C47 with OH-QLH2 had a 1.5-fold increase in bioluminescence, and C47T with OH-QLH2 had a 1.9-fold increase in bioluminescence compared to PLR3 with OH-QLH2. The increase in bioluminescence from PLR3/C47 and PLR3/C47T in the live HEK293T cells demonstrates that the directed evolution methods used optimize the PLR3 enzyme for potential in vivo bioluminescence imaging uses was successful.

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