Intracellular bioconjugation of targeted proteins with semiconductor quantum dots.
We demonstrate controlled in vivo bioconjugation of a targeted intracellular protein to semiconductor quantum dots (QDs). Metal-affinity driven coordination of oligohistidine-appended proteins for chelated divalent cations was exploited to facilitate this interaction. Monomeric mCherry red fluorescent protein recombinantly engineered to express an N-terminal hexahistidine sequence was expressed from a eukaryotic plasmid vector following transfection into COS-1 cells. QDs solubilized with a carboxylated polymeric ligand and pretreated with Ni(2+) were then microinjected into the mCherry-expressing COS-1 cells. Förster resonance energy transfer (FRET) between the central QD donors and mCherry acceptors specifically coordinated to their surface was utilized to probe and confirm intracellular conjugate formation. We unexpectedly found that mCherry attachment to the QDs also substantially improves its resistance to photobleaching. This proof-of-concept, highlighting targeted intracellular bioconjugation to QDs, suggests that many cytoplasmic proteins expressing the ubiquitous hexahistidine affinity handle can be specifically attached to QDs in vivo. This approach can facilitate long-term monitoring of their spatio-temporal activity or, alternatively, allow engineering and in situ assembly of designer chimeric QD-fluorescent protein sensors.
Boeneman
Kelly
K
Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, 4555 Overlook Avenue, S.W., Washington, DC 20375, USA.
Delehanty
James B
JB
Susumu
Kimihiro
K
Stewart
Michael H
MH
Medintz
Igor L
IL
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006639:Histidine / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D045663:Quantum Dots; D011994:Recombinant Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012666:Semiconductors; D014162:Transfection; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D006639:Histidine; D008164:Luminescent Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C471213:His-His-His-His-His-His