Fluorescent properties and photostability of streptavidin‐conjugated StayGOLD protein for DNA labeling

Abstract Here, we report a strepatavidin(SA)‐conjugated StayGOLD fluorescent protein as a DNA labeling agent. We investigate the photophysical properties and photostability of the streptavidin‐coupled StayGOLD fluorescent protein compared to SA‐mNeonGreen. Fluorescent proteins, particularly StayGOLD variants, are recognized for their enhanced stability and brightness, making them suitable for prolonged imaging. Using SA‐StayGOLD in DNA labeling, we observed significant improvements in fluorescence intensity and reduced photobleaching relative to SA‐mNeonGreen. Photophysical analyses suggest that StayGOLD's stability arises from specific structural features, including interactions of protein residues and water molecules with the chromophore. Structural comparisons revealed differing chromophore environments between SA‐StayGOLD and SA‐mNeonGreen, with arginine (R86) in StayGOLD appearing as a particularly relevant factor for its photostability. These findings confirm SA‐StayGOLD as a superior tool for single‐molecule DNA imaging, where high fluorescence intensity and photostability are essential for data quality.