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

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.