In Vivo, Site-Specific, Covalent Conjugation of Quantum Dots to Proteins via Split-Intein Splicing

The ability to target proteins with nanostructures and/or nanodevices in vivo is important for understanding and controlling their biological function. Quantum dots (QDs) serve as an ideal model nanostructure due to their superior optical properties that permit visual confirmation of in vivo targeting and localization and due to their potential as a bio-imaging tool. Here, we describe the site-specific covalent conjugation of quantum dots to target proteins in vivo using an intein-based method. Experimental procedure includes the following steps: (1) fusion of Pleckstrin-homology (PH) domains with the N-terminus half of a split intein (I_N ); (2) conjugation of the C-terminal (I_C ) intein-derived peptide to streptavidin-coated QDs in vitro; and (3) in vivo expression of PH–I_N following microinjection of PH–I_N RNA and I_C –QDs into Xenopus embryos. Intein-splicing results in covalent conjugation of QDs with the C-terminus of the PH domain without interfering with protein localization or function. Such produced QD–PH conjugates could be monitored in real time within live embryos.