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.