title: Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.
abstract: Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
authors.forename: Vijay
authors.initials: V
authors.lastname: Singh
authors.forename: Shenliang
authors.initials: S
authors.lastname: Wang
authors.forename: Ke Min
authors.initials: KM
authors.lastname: Chan
authors.forename: Spencer A
authors.initials: SA
authors.lastname: Clark
authors.forename: Eric T
authors.initials: ET
authors.lastname: Kool
mesh_terms: D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000737:chemistry*; D007202:Indicators and Reagents; D050576:Membrane Fusion Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011506:Proteins / Q000737:chemistry* / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D050576:Membrane Fusion Proteins; D011506:Proteins; D004247:DNA; D006867:Hydrolases; C046137:haloalkane dehalogenase