title: Design, synthesis, and application of the trimethoprim-based chemical tag for live-cell imaging.
abstract: Over the past decade, chemical tags have been developed to complement the use of fluorescent proteins in live-cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E. coli dihydrofolate reductase and the antibiotic trimethoprim and was subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live-cell imaging. Here we describe the general design, chemical synthesis, and application of TMP-tag for live-cell imaging. Alternate protocols for synthesizing and using the covalent and the fluorogenic TMP-tags are also included.
authors.affiliation: Department of Chemistry, Columbia University, New York, New York, USA.
authors.forename: Chaoran
authors.initials: C
authors.lastname: Jing
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
mesh_terms: D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry*; D002460:Cell Line; D015195:Drug Design; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D057054:Molecular Imaging / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014295:Trimethoprim / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim
keywords: chemical tag; fluorescence microscopy; live‐cell imaging; protein label