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 bio-labels. 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 (<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 E. coli dihydrofolate reductase (eDHFR), respectively. Furthermore, eDHFR and SNAP-Tag fusions can be labeled with Lumi4 analogs 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.

Brightly Luminescent and Kinetically Inert Lanthanide Bioprobes Based on Linear and Preorganized Chelators

“…Anionic probes that diffuse freely throughout cells can be conjugated to cell penetrating peptides and targeting ligands to enable passive cytoplasmic delivery and specific biomolecular or subcellular targeting, as we have previously demonstrated with Lumi4(Tb). 6, 39 …”

Strategies for the enhanced intracellular delivery of nanomaterials

“…Typically, this organelle is targeted when infectious and autoimmune diseases and familial hypercholesterolemia occur [8]. There are several approaches available that allow the delivery of nanomaterials into the cytoplasm, such as CPPs [175] and cationic nanomaterials [176]. Liposomes with fusogenic properties [177] or that are pH sensitive have also been studied as drug carriers [178], given that they have the ability to destabilize endosomal membranes and release drugs into the cytoplasm [9].…”

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

“…2 A) (36). When added to culture medium, TMP-Lumi4-R 9 directly enters the cytoplasm of living cells and binds selectively to overexpressed E. coli dihydrofolate reductase (eDHFR) fusion proteins with high affinity (~1 nM K D ) (36)(37)(38). Time-gated images were acquired on an epifluorescence microscope equipped with an ICCD camera as the gated detector and an LED emitting at 365 nm as the pulsed light source (28).…”