Mechanism and application of thiol–disulfide redox biosensors with a fluorescence-lifetime readout DOI Creative Commons
Paul C. Rosen,

Andrew Glaser,

Juan Ramón Martínez‐François

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(19)

Published: May 6, 2025

Genetically encoded biosensors with changes in fluorescence lifetime (as opposed to intensity) can quantify small molecules complex contexts, even vivo. However, lifetime-readout sensors are poorly understood at a molecular level, complicating their development. Although there many that have fluorescence-intensity changes, currently only few fluorescence-lifetime changes. Here, we optimized two for thiol–disulfide redox (RoTq-Off and RoTq-On) opposite response oxidation. Using biophysical approaches, showed the high-lifetime states of these lock chromophore more firmly place than low-lifetime do. Two-photon imaging RoTq-On fused glutaredoxin (Grx1) enabled robust, straightforward monitoring cytosolic glutathione state acute mouse brain slices. The motional mechanism described here is probably common may inform design other sensors; Grx1-RoTq-On fusion sensor will be useful studying physiology.

Language: Английский

Mechanism and application of thiol–disulfide redox biosensors with a fluorescence-lifetime readout DOI Creative Commons
Paul C. Rosen,

Andrew Glaser,

Juan Ramón Martínez‐François

et al.

Proceedings of the National Academy of Sciences, Journal Year: 2025, Volume and Issue: 122(19)

Published: May 6, 2025

Genetically encoded biosensors with changes in fluorescence lifetime (as opposed to intensity) can quantify small molecules complex contexts, even vivo. However, lifetime-readout sensors are poorly understood at a molecular level, complicating their development. Although there many that have fluorescence-intensity changes, currently only few fluorescence-lifetime changes. Here, we optimized two for thiol–disulfide redox (RoTq-Off and RoTq-On) opposite response oxidation. Using biophysical approaches, showed the high-lifetime states of these lock chromophore more firmly place than low-lifetime do. Two-photon imaging RoTq-On fused glutaredoxin (Grx1) enabled robust, straightforward monitoring cytosolic glutathione state acute mouse brain slices. The motional mechanism described here is probably common may inform design other sensors; Grx1-RoTq-On fusion sensor will be useful studying physiology.

Language: Английский

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