Biotin methyl ester enhances cargo release in RUSH system and enables rapid biotinylation with TurboID.

Biotin-streptavidin technology is a key tool in biotechnology. The engineering of streptavidin or streptavidin-like molecules and organic synthesis of biotin derivatives offer a variety of applications to meet the needs of researchers. Here, we report that owing to its masked charged carboxyl group, biotin methyl ester (BME) improves cell penetration and allows the retention using selective hooks (RUSH) system to initiate cargo release promptly and uniformly in all cells. In addition, because of the rapid hydrolysis of BME to biotin inside cells, BME enabled faster biotinylation than biotin, by the promiscuous biotin ligase TurboID. These properties allow BME to act as a biotin prodrug, enhance the RUSH system and TurboID, and highlight the potential of biotin-streptavidin technology for numerous applications in living cells. Additionally, BME may be a good biotin supplement for patients with multivitamin-responsive inherited metabolic disorders.