Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Single-molecule visualization of DNA G-quadruplex formation in live cells

Subjects

Abstract

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: In vitro single-molecule fluorescence imaging of G4s.
Fig. 2: Single-molecule fluorescence imaging of G4s in living cells using the fluorescent probe SiR-PyPDS (1).
Fig. 3: G4s in living cells undergo dynamic folding and unfolding.
Fig. 4: The observation of G4s in live cells is altered by cell-cycle phase and transcription.

Similar content being viewed by others

Data availability

All data generated during this study are included in this published Article and its Supplementary Information.

References

  1. Sen, D. & Gilbert, W. Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature 334, 364–366 (1988).

    Article  CAS  Google Scholar 

  2. Hänsel-Hertsch, R., Di Antonio, M. & Balasubramanian, S. DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat. Rev. Mol. Cell Biol. 18, 279–284 (2017).

    Article  Google Scholar 

  3. Chambers et al. High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 33, 877–881 (2015).

    Article  Google Scholar 

  4. Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl Acad. Sci. USA 98, 8572–8577 (2001).

    Article  CAS  Google Scholar 

  5. Biffi, G., Tannahill, D., McCafferty, J. & Balasubramanian, S. Quantitative visualization of DNA G-quadruplex structures in human cells. Nat. Chem. 3, 182–186 (2013).

    Article  Google Scholar 

  6. Hänsel-Hertsch, R. et al. G-quadruplex structures mark human regulatory chromatin. Nat. Genet. 48, 1267–1272 (2016).

    Article  Google Scholar 

  7. Chen, X. C. et al. Tracking the dynamic folding and unfolding of RNA G-quadruplexes in live cells. Angew. Chem. Int. Ed. 57, 4702–4706 (2018).

    Article  CAS  Google Scholar 

  8. Laguerre, A. et al. Visualization of RNA G-quadruplexes in live cells. J. Am. Chem. Soc. 137, 8521–8525 (2015).

    Article  CAS  Google Scholar 

  9. Zhang, S. et al. Real-time monitoring of DNA G-quadruplexes in living cells with a small-molecule fluorescent probe. Nucleic Acids Res. 46, 7522–7532 (2018).

    Article  CAS  Google Scholar 

  10. Shivalingam, A. et al. The interactions between a small-molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy. Nat. Commun. 6, 8178 (2015).

    Article  CAS  Google Scholar 

  11. Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  Google Scholar 

  12. Rodriguez, R. et al. A novel small molecule that alters shelterin integrity and triggers a DNA-damage response at telomeres. J. Am. Chem. Soc. 130, 15758–15759 (2008).

    Article  CAS  Google Scholar 

  13. De Cian, A., Delemos, E., Mergny, J. L., Teulade-Fichou, M. P. & Monchaud, D. Highly efficient G-quadruplex recognition by bisquinolinium compounds. J. Am. Chem. Soc. 129, 1856–1857 (2007).

    Article  Google Scholar 

  14. Ying, L., Green, J. J., Li, H., Klenerman, D. & Balasubramanian, S. Studies on the structure and dynamics of the human telomeric G-quadruplex by single-molecule fluorescence resonance energy transfer. Proc. Natl Acad. Sci. USA 100, 14629–14634 (2003).

    Article  CAS  Google Scholar 

  15. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    Article  CAS  Google Scholar 

  16. Etheridge, T. J. et al. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy. Nucleic Acids Res. 42, e146 (2014).

    Article  Google Scholar 

  17. Guo, J. U. & Bartel, D. P. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria. Science 353, aaf537 (2016).

    Article  Google Scholar 

  18. Rodriguez, R. et al. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes. Nat. Chem. Biol. 8, 301–310 (2012).

    Article  CAS  Google Scholar 

  19. Chandradoss, S. D. et al. Surface passivation for single-molecule protein studies. J. Vis. Exp. 86, 50549 (2014).

    Google Scholar 

  20. Tinevez, J.-Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    Article  CAS  Google Scholar 

  21. Ponjavic, A. et al. Single-molecule light-sheet imaging of suspended T cells. Biophys. J. 114, 2200–2211 (2018).

    Article  CAS  Google Scholar 

  22. Ponjavic, A., Ye, Y., Laue, E., Lee, S. F. & Klenerman, D. Sensitive light-sheet microscopy in multiwell plates using an AFM cantilever. Biomed. Opt. Express 9, 5863–5880 (2018).

    Article  CAS  Google Scholar 

  23. Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by programme grant funding from Cancer Research UK (C9681/A18618, to S.B.), core funding from Cancer Research UK (C14303/A17197, to S.B.), a Royal Society University Research Fellowship (UF120277, to S.F.L.), a Research Professorship (RP150066, D.K.), the EPSRC (EP/L027631/1, to D.K.) and a BBSRC David Phillips Fellowship (BB/R011605/1, to M.D.A.).

Author information

Authors and Affiliations

Authors

Contributions

M.D.A., A.P., D.K. and S.B. conceived and designed the experiments. M.D.A. performed the design, synthesis and biophysical characterization of G4 ligands. A.P. developed the microscope and protocols used for imaging. M.D.A., A.P. and R.T.R. performed in vitro imaging experiments. R.T.R. carried out surface preparation for in vitro experiments. M.D.A., A.R. and A.P. performed imaging experiments in cells. A.P. analysed imaging data. M.C., A.R. and X.Z. contributed to the synthesis and biophysical validation of the ligands. M.D.A., A.P., R.T.R., S.F.L., D.K. and S.B. contributed to the study design. J.S. assisted with DMS experiments. A.R. contributed to cellular staining and imaging. A.R. and L.-M.N. characterized the fluorescence properties of the G4 ligands. M.D.A., A.P., D.K. and S.B. interpreted the results and co-wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to David Klenerman or Shankar Balasubramanian.

Ethics declarations

Competing interests

S.B. is a founder and shareholder of Cambridge Epigenetix Ltd.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 SiR-PyPDS analogues synthesized.

SiR-PyPDS analogues (1, 2, 3 and 4) with different chemical linkers between the PyPDS and the SiR scaffold synthesised in this study. TSTU was used as amide coupling reagent in the synthesis of all 3 analogues.

Extended Data Fig. 2 Single-step photobleaching confirms detection of individual probes.

a, 25 pM SiR-PyPDS binding to MYC in vitro. The red square indicates a single binding event. b, Intensity traces from three binding events in (a), showing probes undergoing single-step photobleaching. The insets show time lapses for each molecule. Similar single-step photobleaching could be consistently observed in all single-molecule video acquisitions.

Extended Data Fig. 3 FRET between SiR-PyPDS and Alexa Fluor 488-labelled MYC confirms direct binding to G4s.

a, Emission spectrum of 488-MYC-G4 at 1µM and SiR-PyPDS at various stoichiometric ratios. As the probe concentration increases, donor emission drops and acceptor emission increases, indicating FRET. b, In vitro G4 FRET experiment. 250 pM of SiR-PyPDS (shown in red with acceptor excitation) interacting with Alexa Fluor 488-labelled MYC-G4 (with ~1% surface coverage). The green channel shows acceptor emission under donor excitation. FRET between MYC and SiR-PyPDS is highlighted with white arrows. c, 10 nM SiR-PyPDS interacting with 488-MYC-G4 (0.001% surface coverage). Temporal intensity traces of donor (green) and acceptor (red) emission under donor excitation. Anti-correlated intensity fluctuation upon acceptor photobleaching indicates single-molecule FRET between PyPDS and MYC. d, Example time lapse of acceptor (top, red) and donor (bottom, green) emission from (c). Experiments a-d were performed as 3 independent replicates all providing similar results.

Extended Data Fig. 4 Single-molecule imaging with SiR-PyPDS can be used to quantify MYC-G4 prevalence in vitro.

a, Number of detected binding events increases with probe concentrations. b, MYC fluorescence showing that the concentration of MYC on the surface can be controlled by mixing with a competing biotinylated oligomer. c, Number of detected events increases with G4 concentration. Sample images for each condition is shown beneath each plot. Error bars indicate mean ± sd. n=12 measurements taken from 2 independent replicates.

Extended Data Fig. 5 Induction of G4-folding by increasing concentrations of SiR-PyPDS measured with dually labelled FRET oligos.

a-c, Fluorescence emission spectra under Cy3 excitation for each G4 sequence. Experiments a-c were performed as 3 independent replicates all providing similar results.

Extended Data Fig. 6 The effect on SiR-PyPDS binding on unfolding kinetics of G4 DNA sequences in vitro.

G4 two-phase unfolding kinetics were measured by introducing 10 µM of respective complimentary DNA oligonucleotide at t=0 to trap the unfolded G4 oligonucleotide state. Data presented here are of best fit of a two-phase association model. Error indicates the standard error of the fit. n=1 measurement for each condition. Each experiment has been repeated 3 times providing consistent results.

Extended Data Fig. 7 Single-step photobleaching confirms detection of individual probes in cells.

a, 20 nM SiR-PyPDS binding to targets in a living cell. The red square indicates a single binding event. b, Intensity traces from three binding events in A, showing probes undergoing single-step photobleaching. The insets show time lapses for each molecule. Similar single-step photobleaching could be observed in all single-molecule video acquisitions.

Extended Data Fig. 8 SiR-PyPDS mainly accumulates in lysosomes.

Representative confocal and HILO microscopy images obtained in the presence of SiR-PyPDS (1 µM in confocal and 40 nM in HILO) and LysoTracker Green (50 nM), confirming co-localisation of extra-nuclear staining with lysosomes. Experiments have been repeated 3 times providing similar results.

Extended Data Fig. 9 Total nuclear accumulation of SiR-PyPDS and SiR-iPyPDS in U2OS cells.

Total fluorescence intensity measured inside the nuclei of >300 U2OS cells after incubation with 10μM SiR-PyPDS or SiR-iPyPDS by standard confocal microscopy at 633 nm. Each point on the graph represents the total fluorescence of SiR measured at 633 nm per nuclei, data are plotted as the mean of >300 nuclei measured in 3 independent replicates. Total fluorescence measurement revealed comparable ability of the two molecules to accumulate in the nuclei. Error bars indicate mean ± sd.

Extended Data Fig. 10 Cellular displacement experiments of SiR-PyPDS with the established G4-ligands PDS and PhenDC3.

Displacement of SiR-PyPDS in cells by competition with 10 µM of unlabelled G4-ligands PDS and PhenDC3. Cells were pre-incubated 30 minutes with PDS or PhenDC3 at 10 μM prior standard single-molecule imaging with SiR-PyPDS. Each point on the graph depicts the number of long-lived SiR-PyPDS event measured in independent replicates. Data are plotted as the mean of 3 or more independent replicates. Error bars indicate mean ± sd. * P < 0.05, two-sided Mann–Whitney U-test. n = 5,3 and 3 measurements taken from 3 independent replicates for no displacement, PDS displacement and PhenDC3 displacement respectively.

Supplementary information

Supplementary Information

Supplementary Methods and Figs. 1–5.

Reporting Summary

Supplementary Video 1

The fluorescent G4-ligand SiR-PyPDS specifically binds G4-MYC but not single-stranded DNA (mut). The control isomer SiR-iPyPDS shows significantly reduced binding to G4-MYC. Videos for final analysis have been collected from three or more independent replicates.

Supplementary Video 2

G4 binding by SiR-PyPDS can be inhibited by addition of 10 μM PhenDC3, demonstrating that observed binding events are G4 specific. Videos for final analysis have been collected from three or more independent replicates.

Supplementary Video 3

Long-lived binding events can be observed in living cells by SiR-PyPDS but not by the control isomer SiR-iPyPDS, suggesting that observed events are G4 specific. Videos for final analysis have been collected from three or more independent replicates.

Supplementary Video 4

When cells are treated with unlabelled PDS or PhenDC3, SiR-PyPDS ceases to bind to G4s in cells. White dashed circles indicate the nucleus. Videos for final analysis have been collected from three or more independent replicates.

Supplementary Video 5

Time-lapse imaging enables determination of the residence time of SiR-PyPDS binding to G4s. Videos for final analysis have been collected from three or more independent replicates.

Supplementary Video 6

When cells are treated with DMS, increased treatment durations result in a reduction in observed G4-binding events, as G4s become trapped in an unfolded state and can therefore no longer be bound by SiR-PyPDS. Videos for final analysis have been collected from three or more independent replicates.

Supplementary Video 7

G4 binding events can readily be observed during replication (S) and transcription (G1/S) cell cycle phases. In the quiescent state (G0/G1) minimal G4s can be observed. Minimal binding is also seen if replication and transcription are inhibited chemically (arrest). Videos for final analysis have been collected from three or more independent replicates.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Di Antonio, M., Ponjavic, A., Radzevičius, A. et al. Single-molecule visualization of DNA G-quadruplex formation in live cells. Nat. Chem. 12, 832–837 (2020). https://doi.org/10.1038/s41557-020-0506-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-020-0506-4

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing