Cytoplasmic Delivery and Selective, Multicomponent Labeling with Oligoarginine-Linked Protein Tags

• Published in: Bioconjugate chemistry 2015-03, Vol.26 (3), p.460-465
• Main Authors: Zou, Xiaoyan, Rajendran, Megha, Magda, Darren, Miller, Lawrence W
• Format: Article
• Language: English
• Subjects: Amino acids; Animals; Arginine - chemistry; Arginine - metabolism; Biochemistry; Cell culture; Cytoplasm; Cytoplasm - metabolism; Dogs; Drug Delivery Systems - methods; HeLa Cells; Humans; Ligands; Madin Darby Canine Kidney Cells; Membranes; Mice; NIH 3T3 Cells; Oligopeptides - chemistry; Oligopeptides - metabolism; Protein Transport - physiology; Proteins; Staining and Labeling - methods
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc500550z

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 Tb3+ 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 (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.