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Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins

Abstract

DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid–protein junction in the context of living cells. Here we report a covalent labelling reaction that 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 epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.

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Fig. 1: Chemistry of tagging proteins with PNA.
Fig. 2: Fluorescence microscopy characterization of PNA-tag-enabled fluorescent labelling of Cys-E3-EGFR-eGFP on live HEK293 cells.
Fig. 3: PNA-tag-enabled fluorescent labelling of Cys-P1-ETBR-GFPspark on HEK293 cells at different transient expression levels.
Fig. 4: PNA-tag-enabled multifluorophore labelling of Cys-P1-EGFR-eYFP on CHO cells.
Fig. 5: Reversible fluorescent labelling of PNA-tagged Cys-P1-EGFR-eYFP on serum-starved CHO cells and visualization of EGF-induced EGFR internalisation.

Data availability statement

Data supporting the results and conclusions are available within this paper and the Supplementary Information. Additional raw data are available at figshare, https://doi.org/10.6084/m9.figshare.c.5127728.

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Acknowledgements

We acknowledge financial support from the Deutsche Forschungsgemeinschaft (SPP 1623 and SFB 765). M.D.B. is a fellow of the International Max Planck Research School for Molecular Life Sciences (IMPRS-LS). P.W. is a member of the Graduate School Leipzig School of Natural Sciences—Building with Molecules and Nano-objects. We thank J. Lotze (University Leipzig), S. Korte and A. Herrmann (Humboldt University Berlin) for help with confocal laser scanning microscopy and K. Rurack (Bundesanstalt für Materialforschung und Prüfung, Berlin) for providing access to flow cytometry.

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G.C.G., K.G. and P.W. performed the experiments. G.C.G, K.G. and O.S. designed the experiments and analysed the data. M.D.B. and S.B. constructed the stable CHO cell lines. O.S. conceived the experiments. P.W. and A.G.B-S. designed experiments for labelling of ETBR. All authors discussed the results and contributed to the preparation and editing of the manuscript.

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Correspondence to Oliver Seitz.

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Extended data

Extended Data Fig. 1 Synthesis of thioester-linked K3 and P2 peptide-PNA conjugates via SPPS and strained cycloaddition.

a, Solid-phase synthesis affords K3 and P2 peptides containing thioester-linked azido hexanoic acid. b, After solid-phase assembly of PNA strands by using Fmoc/Bhoc-protected PNA monomers, PNA is cleaved by TFA treatment and submitted to an in-solution coupling with ALO (pictured), which provides the ALO-PNA conjugate. c, Strain-promoted azide-alkyne cycloaddition produces the thioester-linked PNA-peptide conjugates.

Extended Data Fig. 2 Time course experiment of transfer reaction between PNA11-K3 and Cys-E3.

a, UPLCTM traces of reaction time points. UPLC gradient 20-50 % in 4 min, detection at 260 nM. Experiment was repeated three times with similar results b) Structure of S-acylated side product PNA11-Cys(PNA11)-E3.

Extended Data Fig. 3 Analysis of PNA:DNA duplex stability by fluorescence microscope imaging of Cys-E3-hY2R tagged with PNA11 or PNA16 and hybridized with complementary FAM-DNA at 37 °C.

a, HEK293-Cys-E3-hY2R labelled with an 11mer duplex (PNA11-K3 tagging and FAM-DNA17 hybridisation). b, HEK293-Cys-E3-hY2R labelled with a 16mer duplex (PNA16-K3 tagging and FAM-DNA22 hybridisation). Stably transfected HEK293-Cys-E3-hY2R cells42,58 labelled with Hoechst33342 (shown in blue) were treated with PNA11-K3 or PNA16-K3 (100 nM) in buffer (HBSS with 0.1 mM TCEP, 20 mM HEPES, pH 7) for 4 min. After washing with basic buffer (200 mM NaHCO3 in DPBS, pH 8.4) for 1.5 min, complementary FAM-DNA (1 µM) containing 6mer overhangs (FAM-DNA17 or FAM-DNA22) was added for 5 min in 20 mM HEPES buffer. Cells were washed and microscopic studies were performed in OptiMEM at 37 °C. Hoechst33342 (λex:365, λem:420), 5/6-Carboxyfluorescein (FAM) (λex: 470/40, λem:525/50). Scale bar= 10 µm. See Supplementary 9.3 for full method and DNA sequences. Experiment was repeated 3 times independently with similar results.

Extended Data Fig. 4 Fluorescence microscopy analysis of Signal to Noise Ratio (SNR) of PNA15 labelled Cys-P1-EGFR-eYFP CHO cells stained with either one or five Cy7 fluorophores.

After PNA15 labelling, cells were incubated with 50 nM Complex I (1x Cy7: adaptor DNA-33mer with a single Cy7-15mer) or 50 nM Complex II (5x Cy7: adaptor DNA-105mer with five Cy7-15mers) in HBSS-BB before washing with HBSS-BB and imaging. From three independent experiments, 6-8 cells were analysed by line intensity profiles spanning a whole cell. For each line intensity profile, Cy7 or YFP signal was calculated as the max peak height at the membrane regions, and the noise calculated as the standard deviation of the signal from an empty background region. Dot plot is presented as the mean +/− SD with each point representing SNR for one cell (n = 20).

Extended Data Fig. 5 Spinning disk confocal microscopy analysis of PNA enabled reversible labelling of Cys-P1-EGFR-eYFP on CHO cells.

a, After staining of nuclei with Hoechst 33342, serum starved Cys-P1-EGFR-eYFP cells were treated with PNA15-P2 in HBSS for 4 minutes. Cells were then incubated with 50 nM Complex III (adaptor DNA-105mer + five Atto565-DNA-23mers) in HBSS-BB for 4 min. b, Stimulation with EGF (100 nM) for 15 mins. c, Toehold mediated strand displacement of Atto565-23mer DNA with 300 nM displacement DNA-23mer in presence of 100 nM EGF for 2 ×5 min in HBSS at 30 °C. d, Hybridisation with 100 nM Atto647N-DNA-15mer, 3 min. Excitation times: ATTO565: 200 ms YFP: 100 ms, Hoechst 33342:100 ms Atto647N: 300 ms. Diode lasers: Hoechst 33342) 405 nm; YFP) 488 nm; Atto565) 561 nm; Atto647N) 640 nm. Dichroic emission filters Hoechst 33342) λem = 460 ± 50 nm; YFP) λem 470 ± 24 nm; Atto565) λem 600 ± 50 nm. Atto647) λem = 700 ± 75 nm. Scale bar= 10μm. Experiments were repeated 3 times independently with similar results.

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Gavins, G.C., Gröger, K., Bartoschek, M.D. et al. Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins. Nat. Chem. 13, 15–23 (2021). https://doi.org/10.1038/s41557-020-00584-z

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