Genetically encoded dinitrobenzyl photostabilizer for enhancing the photostability of fluorescent proteins.

Authors

Guihua Li, Xia Wang, Jiben Xue, Jie Wang

Published in

RSC chemical biology. Sep 29, 2026. Epub Sep 29, 2026.

Abstract

Fluorescent proteins (FPs) are essential tools for biological imaging, but their application in long-term and super-resolution microscopy is often limited by photobleaching. Inspired by "self-healing" organic dyes, we report a genetically encoded intramolecular photostabilization strategy to improve the ability of fluorescent proteins to resist photobleaching. We designed a dinitrobenzene-based unnatural amino acid (UAA), 2-NBAK, to function as an efficient photostabilizer. Using an engineered orthogonal translation system, we performed a systematic spatial scan of 192 insertion sites across the mNeonGreen (mNG) β-barrel. Our screening revealed that optimal mNG-2-NBAK variants at specific outward-pointing side chains are proposed to quench the triplet state without disrupting the chromophore emission spectra. In vitro characterization and further live-cell confocal imaging demonstrated that the optimal variants exhibited more than a twofold extension of the fluorescence half-life under intense continuous irradiation. By transplanting the optimal mNG insertion sites into another FP, we established a proof-of-concept for enhancing green FP photostability, providing a genetically encoded framework adaptable to other spectral variants with energetically matched photostabilizers.

PMID:
42840676
Bibliographic data and abstract were imported from PubMed on 07 Oct 2026.

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