Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes

Qianzhu, H.; Butler, S. M.; Habel, E.; Abdelkader, E. H.; Paul, N.; Woolsey, R.; Zhou, X.; Martin, R. M.; Jolliffe, K. A.; Otting, G.; Huber, T.

doi:10.64898/2026.10.13.758698

bioRxiv · 10.64898/2026.10.13.758698

Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes

Qianzhu, H.·Butler, S. M.·Habel, E.·Abdelkader, E. H.·Paul, N.·Woolsey, R.·Zhou, X.·Martin, R. M.·Jolliffe, K. A.·Otting, G.·Huber, T.

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Abstract

Quantitative fluorescence lifetime imaging microscopy (FLIM) is limited by a lack of probes that combine single-residue specificity, minimal steric perturbation, visible-light excitation, and strong environment-dependent lifetime contrast. We introduce benzodiazole-based non-canonical amino acids (BD-ncAAs) as compact, genetically encoded sensors for site-specific FLIM. Using an engineered pyrrolysyl-tRNA synthetase pair, we incorporate three different BD-ncAAs into proteins in living cells. Site-specific incorporation of BD-ncAAs into a nanobody at different positions preserved native structure and antigen binding. The lead probe, cyano-substituted CBDS, exhibits visible-light excitation, large Stokes shifts, and pronounced environment-dependent fluorescence lifetimes spanning 1 to 24 ns. Under wash-free, live-cell FLIM conditions, CBDS quantitatively resolves localized structural microenvironments and transmembrane hydration gradients, while also distinguishing distinct states during protein secretion in eukaryotes. BD-ncAAs present genetically encoded, minimally perturbing reporters for tracking target proteins, detecting interactions with binding partners, and quantifying site-specific solvent accessibility in living cells by quantitative FLIM.

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Qianzhu, H., Butler, S. M., Habel, E., Abdelkader, E. H., Paul, N., Woolsey, R., Zhou, X., Martin, R. M., Jolliffe, K. A., Otting, G., Huber, T.. 2026-10-17. Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes. https://doi.org/10.64898/2026.10.13.758698

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