Engineering luminopsins with improved coupling efficiencies

Slaviero, A.; Gorantla, N.; Simkins, J. W.; Crespo, E. L.; Ikefuama, E. C.; Tree, M. O.; Prakash, M.; Bjorefeldt, A.; Barnett, L. M.; Lambert, G. G.; Lipscombe, D.; Moore, C. I.; Shaner, N. C.; Hochgeschwender, U.

doi:10.1101/2023.11.22.568342

bioRxiv · 10.1101/2023.11.22.568342

Engineering luminopsins with improved coupling efficiencies

Slaviero, A.·Gorantla, N.·Simkins, J. W.·Crespo, E. L.·Ikefuama, E. C.·Tree, M. O.·Prakash, M.·Bjorefeldt, A.·Barnett, L. M.·Lambert, G. G.·Lipscombe, D.·Moore, C. I.·Shaner, N. C.·Hochgeschwender, U.

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Abstract

SignificanceLuminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AimWe investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. ApproachWe quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. ResultsLuciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. ConclusionsWhole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Forster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

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Slaviero, A., Gorantla, N., Simkins, J. W., Crespo, E. L., Ikefuama, E. C., Tree, M. O., Prakash, M., Bjorefeldt, A., Barnett, L. M., Lambert, G. G., Lipscombe, D., Moore, C. I., Shaner, N. C., Hochgeschwender, U.. 2023-11-22. Engineering luminopsins with improved coupling efficiencies. https://doi.org/10.1101/2023.11.22.568342

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