Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes
- 1Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
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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BibTeX
@misc{qianzhu2026quantitative,
title = {{Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes}},
author = {H. Qianzhu and S. M. Butler and E. Habel and E. H. Abdelkader and N. Paul and R. Woolsey and X. Zhou and R. M. Martin and K. A. Jolliffe and G. Otting and T. Huber},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.13.758698},
url = {https://www.biorxiv.org/content/10.64898/2026.10.13.758698v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Quantitative fluorescence lifetime imaging of protein microenvironments by genetically encoded compact benzodiazole probes
AU - H. Qianzhu
AU - S. M. Butler
AU - E. Habel
AU - E. H. Abdelkader
AU - N. Paul
AU - R. Woolsey
AU - X. Zhou
AU - R. M. Martin
AU - K. A. Jolliffe
AU - G. Otting
AU - T. Huber
PY - 2026
DA - 2026/10/13
PB - bioRxiv
DO - 10.64898/2026.10.13.758698
UR - https://www.biorxiv.org/content/10.64898/2026.10.13.758698v1
AB - 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.
ER -