title: Quantum dot targeting with lipoic acid ligase and HaloTag for single-molecule imaging on living cells.
abstract: We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 18-496, Cambridge, Massachusetts 02139, USA.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Liu
authors.forename: William S
authors.initials: WS
authors.lastname: Phipps
authors.forename: Ken H
authors.initials: KH
authors.lastname: Loh
authors.forename: Mark
authors.initials: M
authors.lastname: Howarth
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Ting
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D002470:Cell Survival; D003652:Decanoic Acids / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D010446:Peptide Fragments / Q000378:metabolism; D045663:Quantum Dots; D013379:Substrate Specificity; D008063:Thioctic Acid / Q000378:metabolism*
chemical_list: D003652:Decanoic Acids; D010446:Peptide Fragments; C031071:decanoic acid; D008063:Thioctic Acid; D006867:Hydrolases; C046137:haloalkane dehalogenase; D008025:Ligases