Development of fluorescence lifetime biosensors for ATP, cAMP, citrate, and glucose using the mTurquoise2-based platform.

Cell Rep Methods

Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research (BDR), Suita, Osaka 565-0874, Japan; Department of Cell Biology, Graduate School of Medicine, the University of Tokyo, Hongo, Tokyo 113-0033, Japan; Department of Physics, Graduate School of Science, the University of Tokyo, Hongo, Tokyo 113-0033, Japan; Universal Biology Institute (UBI), the University of Tokyo, Hongo, Tokyo 113-0033, Japan; Internatinonal Research Center for Neurointelligence (WPI-IRCN), the University of Tokyo, Hongo, Tokyo 113-0033, Japan. Electronic address:

Published: November 2024

Single-fluorescent protein (FP)-based FLIM (fluorescence lifetime imaging microscopy) biosensors can visualize intracellular processes quantitatively. They require a single wavelength for detection, which facilitates multi-color imaging. However, their development has been limited by the absence of a general design framework and complex screening processes. In this study, we engineered FLIM biosensors for ATP (adenosine triphosphate), cAMP (cyclic adenosine monophosphate), citrate, and glucose by inserting each sensing domain into mTurquoise2 (mTQ2) between Tyr-145 and Phe-146 using peptide linkers. Fluorescence intensity-based screening yielded FLIM biosensors with a 0.5 to 1.0 ns dynamic range upon analyte binding, demonstrating that the mTQ2(1-145)-GT-X-EF-mTQ2(146-238) backbone is a versatile platform for FLIM biosensors, allowing for simple intensity-based screening while providing dual-functional biosensors for both FLIM and intensity-based imaging. As a proof of concept, we monitored cAMP and Ca dynamics simultaneously in living cells by dual-color imaging. Our results complement recent studies, establishing mTQ2 as a valuable framework for developing FLIM biosensors.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11705765PMC
http://dx.doi.org/10.1016/j.crmeth.2024.100902DOI Listing

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