biostudies-literatureUnknownIlievski FEuropean Research CouncilSingle-molecule tracking (SMT) is a powerful tool for real-time studies of protein interactions in living cells. Dye-labelled SNAP-tag and HaloTag self-labelling proteins have simplified SMT significantly, due to their superior photophysical properties compared to fluorescent proteins. However, due to their size, fusion of these tags to a protein of interest often results in loss of protein function. We introduce FLORENCE - a universal labelling method for SMT, based on genetic code expansion (GCE). We overcome significant caveats related to re-coded strains, vectors, and dyes and report successful tracking of site-specifically intracellularly labelled proteins in genomically re-coded <i>E. coli.</i> Our findings establish a robust <i>in vivo</i> protein-labelling strategy, expanding the cRSC chemical biologyhttps://www.ebi.ac.uk/biostudies/studies/S-EPMC12684252biostudies-literatureOptimization of the genetic code expansion technology for intracellular labelling and single-molecule tracking of proteins in genomically re-coded &lt;i&gt;E. coli&lt;/i&gt;.PMC12684252947747Borg AIlievski FBrandis GJohansson MWikstrom LVolkov ILfalseOptimization of the genetic code expansion technology for intracellular labelling and single-molecule tracking of proteins in genomically re-coded &lt;i&gt;E. coli&lt;/i&gt;.Single-molecule tracking (SMT) is a powerful tool for real-time studies of protein interactions in living cells. Dye-labelled SNAP-tag and HaloTag self-labelling proteins have simplified SMT significantly, due to their superior photophysical properties compared to fluorescent proteins. However, due to their size, fusion of these tags to a protein of interest often results in loss of protein function. We introduce FLORENCE - a universal labelling method for SMT, based on genetic code expansion (GCE). We overcome significant caveats related to re-coded strains, vectors, and dyes and report successful tracking of site-specifically intracellularly labelled proteins in genomically re-coded <i>E. coli.</i> Our findings establish a robust <i>in vivo</i> protein-labelling strategy, expanding the c2025-01-01T00:00:00Z2025 Nov2026-06-14T03:23:40.233Z2026-06-14T03:09:20.748ZS-EPMC126842524136847610.1039/d5cb00221d