RRC ID 82249
Author Zhong C, Arai S, Okada Y.
Title Development of fluorescence lifetime biosensors for ATP, cAMP, citrate, and glucose using the mTurquoise2-based platform.
Journal Cell Rep Methods
Abstract 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 Ca2+ dynamics simultaneously in living cells by dual-color imaging. Our results complement recent studies, establishing mTQ2 as a valuable framework for developing FLIM biosensors.
Volume 4(11)
Pages 100902
Published 2024-11-18
DOI 10.1016/j.crmeth.2024.100902
PII S2667-2375(24)00292-3
PMID 39561716
MeSH Adenosine Triphosphate* / analysis Adenosine Triphosphate* / metabolism Biosensing Techniques* / methods Calcium / analysis Calcium / metabolism Citric Acid* / metabolism Cyclic AMP* / analysis Cyclic AMP* / metabolism Glucose* / analysis Glucose* / metabolism HEK293 Cells Humans Microscopy, Fluorescence / methods
Resource
DNA material pRSETA-qmTQ2-ATP (RDB20447) pcDNA3.1(-)-qmTQ2-ATP (RDB20448) pRSETA-qmTQ2-cAMP (RDB20449) pcDNA3.1(-)-qmTQ2-cAMP (RDB20450) pRSETA-qmTQ2-citrate (RDB20451) pRSETA-qmTQ2-glucose (RDB20452)
Human and Animal Cells COS-7(RCB0539)