Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins

Abstract

DNA nanotechnology is an emerging field, which promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress in the area is the ability to create the nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction, which installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled coil peptide tag. Once installed the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness, and achieving reversible labelling by way of toehold mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled coil systems, with EGFR and ETBR, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of EGFR on CHO cells.

Strategies for Site‐Specific Labeling of Receptor Proteins on the Surfaces of Living Cells by Using Genetically Encoded Peptide Tags

“…[82] SpyTag/SpyCatcher [83] AHIVMVDAYKPTK 5 μM SpyCatcher-Alexa Fluor 555 in PBS, supplemented with 5 mM Mg 2 + (reaction time: 15 min at 25°C). [83] Heterodimerization of coiled-coil peptides [84][85][86][87][88][89][90][91] see Table 2 Fluorescein arsenical helix binder (FIAsH) [92,93] CCXXCC (Tetracysteine motif) Extracellular labeling: incubation with 5 mM MES and 0.5 mM TCEP (for 20-30 min) prior to addition of FlAsH reactant (2-5 μM). [92] Ni 2 + loaded NTA [94,95] His 6 or His 10 15 nM of dye-conjugated, Ni 2 + -loaded NTA for noncovalent labeling in < 15 min.…”

“…CoilR LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETR [91] MiniE LEIEAAFLERENTALETRVAELRQRVQRLRNE After a blocking step with BSA, labeling was performed with 100 nM peptide probe (reaction time: 30 min at 4°C). [91] MiniR LEIRVAFLRQRNTALRTEVAELEQEVQRLENR P1/P2 system [89,139] P1-tag EIQALEEENAQLEQENAALEEEIAQLEY Covalent cargo transfer is carried out on in < 5 min using 100 nM peptide probe at room temperature. [89] See also Figure 10.…”

“…[91] MiniR LEIRVAFLRQRNTALRTEVAELEQEVQRLENR P1/P2 system [89,139] P1-tag EIQALEEENAQLEQENAALEEEIAQLEY Covalent cargo transfer is carried out on in < 5 min using 100 nM peptide probe at room temperature. [89] See also Figure 10.…”

“…Recently, Gavins, et al reported on a novel coiledcoil labeling system which enables the erasable imaging of GPCRs and RTKs (Figure 10A). [89] The method relies on the 28 aa P1 and P2 coiled-coil peptides introduced by Jerala. [139] An Nterminal Cys-P1-tag was fused to the human EGFR, and the endothelin B receptor (ET B R), a rhodopsin-like GPCR and labeled upon incubation with a "complementary" peptide probe P2.…”

“…Internalized receptors are visualized with higher signal-to-noise ratios upon toeholdmediated strand displacement (lower row). Microscopy data adapted from Gavins, et al [89] Simultaneous binding of multiple Ni-NTA units to multiple imidazole groups along a His 6 -tag boosts the binding affinity as shown for different NTAs binding to oligo-His 6 -tags: mono-NTA (K D~1 3 μM) < bis-NTA (K D~6 8 nM) < tris-NTA (K D~2 .1 nM). [154] The graded affinity of tris-NTA probes for His 6 -and His 10 -tags enabled imaging of intracellular His 10 -tagged POIs by complexation with His 6 -tagged cell penetrating peptides.…”