High Affinity scFv−Hapten Pair as a Tool for Quantum Dot Labeling and Tracking of Single Proteins in Live Cells (original) (raw)
We describe a general approach to label cell surface proteins using quantum dots (QD) for singlemolecule tracking. QDs coated with small-hapten modified peptides are targeted to cell surface fusion proteins containing the corresponding single-chain fragment antibody (scFv). The approach is illustrated with the small hapten fluorescein (FL) and a high-affinity anti-FL scFv fused to two different proteins in yeast and murine neuronal cell line N2a. Advances in fluorescence microscopy have allowed imaging of single molecules in live cells. 1,2 However, organic dyes and fluorescent proteins (FPs) are prone to rapid photobleaching and fluorescence saturation, limiting the temporal resolution of single-molecule observations. As an alternative fluorophore, quantum dots (QDs) provide brighter and more stable fluorophores that are comparable in size to FPs. 3 However, since QDs are extrinsic probes (not genetically encoded), methods for targeting QDs to proteins of interest are needed. Furthermore, to allow for multicolor imaging, orthogonal targeting approaches must be developed to label different proteins with different color QDs. Antibody-QD conjugates represent one approach for protein labeling. 4 However, the large size of the antibody-QD probe may interfere with protein function or localization. Solutions developed to functionalize proteins with fluorophores could be (and in some cases have been) used with QDs. 1,5,6 We propose a new labeling strategy that keeps the generality and built-in orthogonality of antibodies but results in much smaller probe sizes. The approach is based on high-affinity single-chain variable fragment antibodies (scFv) against small molecules (or haptens). 7 An scFv and the corresponding hapten constitute affinity partners that can be separately attached to a target protein and QD. We illustrate this approach with the 4M5.3 scFv developed against fluorescein by the Wittrup group. 8 This affinity pair is characterized by a dissociation constant of 48 fM in Low Salt Buffer. 8 We call the resulting QDs Fluoresceinfunctionalized pc-QDs or FL-pc-QDs. Here, we fully characterize this system, demonstrate
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