title: Optimized fluorescent trimethoprim derivatives for in vivo protein labeling.
abstract: The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.
authors: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors: Nathaniel T
authors: NT
authors: Calloway
authors: Michael
authors: M
authors: Choob
authors: Ana
authors: A
authors: Sanz
authors: Michael P
authors: MP
authors: Sheetz
authors: Lawrence W
authors: LW
authors: Miller
authors: Virginia W
authors: VW
authors: Cornish
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D005453:Fluorescence; D008853:Microscopy; D015335:Molecular Probes; D011506:Proteins / Q000737:chemistry*; D014295:Trimethoprim / Q000737:chemistry*
chemical_list: D017931:DNA Primers; D015335:Molecular Probes; D011506:Proteins; D014295:Trimethoprim