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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Efficient DNA fluorescence labeling via base excision trapping
Nature Communications Open Access 26 August 2022
Can DyeCycling break the photobleaching limit in single-molecule FRET?
Nano Research Open Access 13 May 2022
Hydrolysis of 5-methylfuran-2-yl to 2,5-dioxopentanyl allows for stable bio-orthogonal proximity-induced ligation
Communications Chemistry Open Access 22 October 2021
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
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.
Rizzuto, R., Brini, M., Pizzo, P., Murgia, M. & Pozzan, T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr. Biol. 5, 635–642 (1995).
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).
Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).
Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).
Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).
Lotze, J., Reinhardt, U., Seitz, O. & Beck-Sickinger, A. G. Peptide-tags for site-specific protein labelling in vitro and in vivo. Mol. Biosyst. 12, 1731–1745 (2016).
Seeman, N. C. & Sleiman, H. F. DNA nanotechnology. Nat. Rev. Mater. 3, 17068 (2018).
Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).
Duose, D. Y. et al. Configuring robust DNA strand displacement reactions for in situ molecular analyses. Nucleic Acids Res. 40, 3289–3298 (2012).
Jungmann, R. et al. Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett. 10, 4756–4761 (2010).
Diezmann, F. & Seitz, O. DNA-guided display of proteins and protein ligands for the interrogation of biology. Chem. Soc. Rev. 40, 5789–5801 (2011).
Peri-Naor, R., Ilani, T., Motiei, L. & Margulies, D. Protein–protein communication and enzyme activation mediated by a synthetic chemical transducer. J. Am. Chem. Soc. 137, 9507–9510 (2015).
Schade, M. et al. Remote control of lipophilic nucleic acids domain partitioning by DNA hybridization and enzymatic cleavage. J. Am. Chem. Soc. 134, 20490–20497 (2012).
Röglin, L., Ahmadian, M. R. & Seitz, O. DNA-controlled reversible switching of peptide conformation and bioactivity. Angew. Chem. Int. Ed. 46, 2704–2707 (2007).
Freeman, R. et al. Instructing cells with programmable peptide DNA hybrids. Nat. Comm. 8, 15982 (2017).
Ueki, R., Atsuta, S., Ueki, A. & Sando, S. Nongenetic reprogramming of the ligand specificity of growth factor receptors by bispecific DNA aptamers. J. Am. Chem. Soc. 139, 6554–6557 (2017).
Leung, K., Chakraborty, K., Saminathan, A. & Krishnan, Y. A DNA nanomachine chemically resolves lysosomes in live cells. Nat. Nanotechnol. 14, 176–183 (2019).
Janssen, B. M. G., Van Rosmalen, M., Van Beek, L. & Merkx, M. Antibody activation using DNA-based logic gates. Angew. Chem. Int. Ed. 54, 2530–2533 (2015).
Qian, L., Winfree, E. & Bruck, J. Neural network computation with DNA strand displacement cascades. Nature 475, 368–372 (2011).
Elbaz, J. et al. DNA computing circuits using libraries of DNAzyme subunits. Nat. Nanotechnol. 5, 417–422 (2010).
Hemphill, J. & Deiters, A. DNA computation in mammalian cells: microRNA logic operations. J. Am. Chem. Soc. 135, 10512–10518 (2013).
You, M. et al. DNA ‘nano-claw’: logic-based autonomous cancer targeting and therapy. J. Am. Chem. Soc. 136, 1256–1259 (2014).
Egholm, M. et al. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules. Nature 365, 566–568 (1993).
Demidov, V. V. et al. Stability of peptide nucleic acids in human serum and cellular extracts. Biochem. Pharmacol. 48, 1310–1313 (1994).
Söderberg, O. et al. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3, 995–1000 (2006).
Meyer, R., Giselbrecht, S., Rapp, B. E., Hirtz, M. & Niemeyer, C. M. Advances in DNA-directed immobilization. Curr. Opin. Chem. Biol. 18, 8–15 (2014).
Trads, J. B., Tørring, T. & Gothelf, K. V. Site-selective conjugation of native proteins with DNA. Acc. Chem. Res. 50, 1367–1374 (2017).
Kazane, S. A. et al. Self-assembled antibody multimers through peptide nucleic acid conjugation. J. Am. Chem. Soc. 135, 340–346 (2013).
Dickgiesser, S. et al. Self-assembled hybrid aptamer–Fc conjugates for targeted delivery: a modular chemoenzymatic approach. ACS Chem. Biol. 10, 2158–2165 (2015).
Leonidova, A. et al. In vivo demonstration of an active tumor pretargeting approach with peptide nucleic acid bioconjugates as complementary system. Chem. Sci. 6, 5601–5616 (2015).
Mahal, L. K., Yarema, K. J. & Bertozzi, C. R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).
Kayser, H. et al. Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-d-hexosamines as precursors. J. Biol. Chem. 267, 16934–16938 (1992).
Chandra, R. A., Douglas, E. S., Mathies, R. A., Bertozzi, C. R. & Francis, M. B. Programmable cell adhesion encoded by DNA hybridization. Angew. Chem., Int. Ed. 45, 896–901 (2006).
Shi, P. et al. Poylvalent display of biomolecules on live cells. Angew. Chem. Int. Ed. 130, 6916–6920 (2018).
Saccà, B. et al. Orthogonal protein decoration of DNA origami. Angew. Chem. Int. Ed. 49, 9378–9383 (2010).
Taylor, M. J., Husain, K., Gartner, Z. J., Mayor, S. & Vale, R. D. A DNA-based T cell receptor reveals a role for receptor clustering in ligand discrimination. Cell 169, 108–119.e20 (2017).
Lovendahl, K. N., Hayward, A. N. & Gordon, W. R. Sequence-directed covalent protein–DNA linkages in a single step using HUH-tags. J. Am. Chem. Soc. 139, 7030–7035 (2017).
Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).
Spagnuolo, C. C., Vermeij, R. J. & Jares-Erijman, E. A. Improved photostable FRET-competent biarsenical−tetracysteine probes based on fluorinated fluoresceins. J. Am. Chem. Soc. 128, 12040–12041 (2006).
Baalmann, M., Best, M. & Wombacher, R. in Noncanonical Amino Acids: Methods and Protocols (ed Lemke, E. A.) 365–387 (Springer, 2018).
Chen, I., Howarth, M., Lin, W. & Ting, A. Y. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat. Methods 2, 99–104 (2005).
Reinhardt, U., Lotze, J., Morl, K., Beck-Sickinger, A. G. & Seitz, O. Rapid covalent fluorescence labeling of membrane proteins on live cells via coiled-coil templated acyl transfer. Bioconjugate Chem. 26, 2106–2117 (2015).
Reinhardt, U. et al. Peptide-templated acyl transfer: a chemical method for the labeling of membrane proteins on live cells. Angew. Chem. Int. Ed. 53, 10237–10241 (2014).
Litowski, J. R. & Hodges, R. S. Designing heterodimeric two-stranded α-helical coiled-coils. Effects of hydrophobicity and α-helical propensity on protein folding, stability, and specificity. J. Biol. Chem. 277, 37272–37279 (2002).
Yano, Y. et al. Coiled-coil tag—probe system for quick labeling of membrane receptors in living cells. ACS Chem. Biol. 3, 341–345 (2008).
Dawson, P. E., Muir, T. W., Clark-Lewis, I. & Kent, S. B. H. Synthesis of proteins by native chemical ligation. Science 266, 776–779 (1994).
Chang, P. V. et al. Copper-free click chemistry in living animals. Proc. Natl Acad. Sci. USA 107, 1821–1826 (2010).
Rohde, H., Schmalisch, J., Harpaz, Z., Diezmann, F. & Seitz, O. Ascorbate as an alternative to thiol additives in native chemical ligation. ChemBioChem 12, 1396–1400 (2011).
Haase, C. & Seitz, O. Internal cysteine accelerates thioester-based peptide ligation. Eur. J. Org. Chem. 2009, 2096–2101 (2009).
Roskoski, R. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol. Res. 79, 34–74 (2014).
Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 103, 211–225 (2000).
Yamaguchi, T., Murata, Y., Fujiyoshi, Y. & Doi, T. Regulated interaction of endothelin B receptor with caveolin-1. Eur. J. Biochem. 270, 1816–1827 (2003).
Mazzuca, M. Q. & Khalil, R. A. Vascular endothelin receptor type B: structure, function and dysregulation in vascular disease. Biochem. Pharmacol. 84, 147–162 (2012).
Gradišar, H. & Jerala, R. De novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers. J. Pept. Sci. 17, 100–106 (2011).
Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).
Yurke, B., Turberfield, A. J., Mills, A. P., Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605–608 (2000).
Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand-displacement reactions. Nat. Chem. 3, 103 (2011).
Lotze, J. et al. Time-resolved tracking of separately internalized neuropeptide Y2 receptors by two-color pulse-chase. ACS Chem. Biol. 13, 618–627 (2018).
Olson, X., Kotani, S., Yurke, B., Graugnard, E. & Hughes, W. L. Kinetics of DNA strand displacement systems with locked nucleic acids. J. Phys. Chem. B 121, 2594–2602 (2017).
Schweller, R. M. et al. Multiplexed in situ immunofluorescence using dynamic DNA complexes. Angew. Chem. Int. Ed. 51, 9292–9296 (2012).
Bandlow, V. et al. Spatial screening of hemagglutinin on influenza A virus particles: sialyl-LacNAc displays on DNA and PEG scaffolds reveal the requirements for bivalency enhanced interactions with weak monovalent binders. J. Am. Chem. Soc. 139, 16389–16397 (2017).
Liang, S. I. et al. Phosphorylated EGFR dimers are not sufficient to activate ras. Cell Rep 22, 2593–2600 (2018).
Thomas, F., Boyle, A. L., Burton, A. J. & Woolfson, D. N. A set of de novo designed parallel heterodimeric coiled coils with quantified dissociation constants in the micromolar to sub-nanomolar regime. J. Am. Chem. Soc. 135, 5161–5166 (2013).
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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
Supplementary discussion, figures and tables.
Rights and permissions
About this article
Cite this article
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
This article is cited by
Spatiotemporal and global profiling of DNA–protein interactions enables discovery of low-affinity transcription factors
Nature Chemistry (2023)
Nanomechanical assay for ultrasensitive and rapid detection of SARS-CoV-2 based on peptide nucleic acid
Nano Research (2023)
Efficient DNA fluorescence labeling via base excision trapping
Nature Communications (2022)
Can DyeCycling break the photobleaching limit in single-molecule FRET?
Nano Research (2022)
Fishing for nucleic acid with a coiled hook
Nature Chemistry (2021)