title: Cytoplasmic delivery and selective, multicomponent labeling with oligoarginine-linked protein tags.
abstract: Strategies that leverage bio-orthogonal interactions between small molecule ligands and genetically encoded amino acid sequences can be used to attach high-performance fluorophores to proteins in living cells. However, a major limitation of chemical protein labeling is that cells' plasma membranes are impermeable to many useful probes and biolabels. Here, we show that conjugation to nonaarginine, a cell penetrating peptide (CPP), enables passive cytoplasmic delivery of otherwise membrane-impermeant, small molecule protein labels. Heterodimers consisting of a luminescent Tb(3+) complex, Lumi4, linked to benzyl guanine, benzyl cytosine, and trimethoprim were conjugated to the peptide CysArg9 with a reducible disulfide linker. When added to culture medium, the peptide conjugates rapidly (<30 min) enter the cytoplasm and diffuse freely throughout cells. The benzyl guanine, benzyl cytosine, and trimethoprim derivatives bind selectively to fusion proteins tagged with SNAP-Tag, CLIP-Tag, and Escherichia coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogues in the same cell, and this labeling can be detected using two-color, time-gated Förster resonance energy transfer (FRET) microscopy. Finally, we present quantitative data showing that cytoplasmic uptake of nonaarginine-conjugated probes occurs in multiple cell types (MDCK, HeLa, NIH 3T3), most cells in a culture (>75%) are loaded with probe, and the cellular probe concentration can be controlled by varying incubation conditions. CPP-mediated delivery of Lumi4-linked protein labels will greatly increase the utility of lanthanide-based FRET microscopy. Moreover, our results strongly suggest that this approach can be adapted to deliver a wide variety of protein-targeted fluorophores or other functional probes that were previously unavailable for intracellular imaging studies.
authors: †Department of Chemistry University of Illinois at Chicago 845 West Taylor Street Chicago, Illinois 60607, United States.
authors: Xiaoyan
authors: X
authors: Zou
authors: †Department of Chemistry University of Illinois at Chicago 845 West Taylor Street Chicago, Illinois 60607, United States.
authors: Megha
authors: M
authors: Rajendran
authors: ‡Lumiphore, Inc., 4677 Meade Street Richmond, California 94804, United States.
authors: Darren
authors: D
authors: Magda
authors: †Department of Chemistry University of Illinois at Chicago 845 West Taylor Street Chicago, Illinois 60607, United States.
authors: Lawrence W
authors: LW
authors: Miller
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004285:Dogs; D016503:Drug Delivery Systems / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D041681:NIH 3T3 Cells; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D009842:Oligopeptides; D001120:Arginine