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A DNA nanomachine that maps spatial and temporal pH changes inside living cells

Abstract

DNA nanomachines are synthetic assemblies that switch between defined molecular conformations upon stimulation by external triggers. Previously, the performance of DNA devices has been limited to in vitro applications. Here we report the construction of a DNA nanomachine called the I-switch, which is triggered by protons and functions as a pH sensor based on fluorescence resonance energy transfer (FRET) inside living cells. It is an efficient reporter of pH from pH 5.5 to 6.8, with a high dynamic range between pH 5.8 and 7. To demonstrate its ability to function inside living cells we use the I-switch to map spatial and temporal pH changes associated with endosome maturation. The performance of our DNA nanodevices inside living systems illustrates the potential of DNA scaffolds responsive to more complex triggers in sensing, diagnostics and targeted therapies in living systems.

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Figure 1: In vitro characterization of the I-switch.
Figure 2: I-switch internalization and function within endosomes of Drosophila haemocytes.
Figure 3: Spatial and temporal mapping of pH changes during endocytosis using the I-switch in living cells.
Figure 4: Protein tagging and pH mapping using the I-switch.

References

  1. Bath, J. & Turberfield, A. J. DNA nanomachines. Nature Nanotech. 2, 275–284 (2007).

    Article  CAS  Google Scholar 

  2. Shih, W. Biomolecular self-assembly: Dynamic DNA. Nature Mater. 7, 98–100 (2008).

    Article  CAS  Google Scholar 

  3. Yurke, B., Turberfield, A. J., Mills, A. P. Jr, Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605–608 (2000).

    Article  CAS  Google Scholar 

  4. Liu, J. & Lu, Y. A colorimetric lead biosensor using DNA enzyme-directed assembly of gold nanoparticles. J. Am. Chem. Soc. 125, 6642–6643 (2003).

    Article  CAS  Google Scholar 

  5. Alberti, P. & Mergny, J.-L. DNA duplex–quadruplex exchange as the basis for a nanomolecular machine. Proc. Natl Acad. Sci. USA. 100, 1569–1573 (2003).

    Article  CAS  Google Scholar 

  6. Benenson, Y., Gil, B., Ben-Dor, U., Adar, R. & Shapiro, E. An autonomous molecular computer for logical control of gene expression. Nature 429, 423–428 (2004).

    Article  CAS  Google Scholar 

  7. Yin, P., Choi, H. M. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318–322 (2008).

    Article  CAS  Google Scholar 

  8. Beyer, S. & Simmel, F. C. A modular DNA signal translator for the controlled release of a protein by an aptamer. Nucleic Acids Res. 34, 1581–1587 (2006).

    Article  CAS  Google Scholar 

  9. Mao, C., Sun, W., Shen, Z. & Seeman, N. C. A nanomechanical device based on the B-Z transition of DNA. Nature 397, 144–146 (1999).

    Article  CAS  Google Scholar 

  10. Allan, V. J. & Schroer, T. A. Membrane motors. Curr. Opin. Cell Biol. 11, 476–482 (1999).

    Article  CAS  Google Scholar 

  11. Griesbeck, O. Fluorescent proteins as sensors for cellular functions, Curr. Opin. Neurobiol. 14, 636–641 (2004).

    Article  CAS  Google Scholar 

  12. Ai1, H. W., Hazelwood, K. L., Davidson, M. W. & Campbell, R. E. Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nature Methods 5, 401–403 (2008).

    Article  CAS  Google Scholar 

  13. Gehring, K., Leroy, J.-L. & Guéron, M. A tetrameric DNA structure with protonated cytidine–cytidine base pairs. Nature 363, 561–565 (1993).

    Article  CAS  Google Scholar 

  14. Mukherjee, S. Ghosh, R. N. & Maxfield, F. R. Endocytosis. Physiol. Rev. 77, 759–803 (1997).

    Article  CAS  Google Scholar 

  15. Montesano, R., Roth, J., Robert, A. & Orci, L. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature 296, 651–653 (1982).

    Article  CAS  Google Scholar 

  16. Stryer, L. & Haugland, R. P. Energy transfer: a spectroscopic ruler. Proc. Natl Acad. Sci. USA 58, 719–726 (1967).

    Article  CAS  Google Scholar 

  17. Liu, D. & Balasubramanian, S. A proton-fuelled DNA nanomachine. Angew Chem. Int. Ed. 42, 5734–5736 (2003).

    Article  CAS  Google Scholar 

  18. Liedl, T. & Simmel, F. C. Switching the conformation of a DNA molecule with a chemical oscillator. Nano Lett. 5, 1894–1898 (2005).

    Article  CAS  Google Scholar 

  19. Ohkuma, S. & Poole, B. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl Acad. Sci. USA 75, 3327–3331 (1978).

    Article  CAS  Google Scholar 

  20. Overly, C. C., Lee, K. D., Berthiaumet, E. & Hollenbeck, P. J. Quantitative measurement of intraorganelle pH in the endosomal–lysosomal pathway in neurons by using ratiometric imaging with pyranine. Proc. Natl Acad. Sci. USA 92, 3156–3160 (1995).

    Article  CAS  Google Scholar 

  21. Guha, A., Sriram, V., Krishnan, K. S. & Mayor, S. Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes. J. Cell Sci. 116, 3373–3386 (2003).

    Article  CAS  Google Scholar 

  22. Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins Nature 394, 192–195 (1998).

    Article  CAS  Google Scholar 

  23. Yamashiro, D. J. & Maxfield, F. R. Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells. J. Cell Biol. 105, 2723–2733 (1987).

    Article  CAS  Google Scholar 

  24. Sipe, D. M. & Murphy, R. F. High-resolution kinetics of transferrin acidification in BALB/3T3 cells exposed to pH 6 followed by temperature sensitive alkalinization during recycling. Proc. Natl Acad. Sci. USA 84, 7119–7123 (1987).

    Article  CAS  Google Scholar 

  25. Thomas, J. A., Buschbaum, R. N., Zimniak, A. & Racker, E. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18, 2210–2218 (1979).

    Article  CAS  Google Scholar 

  26. Murphy, R. F., Powers, S. & Cantor, C. R. Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J. Cell Biol. 98, 1757–1762 (1984).

    Article  CAS  Google Scholar 

  27. Roberta, L. G. & Acosta, D. Ratiometric measurement of intracellular pH of cultured cells with BCECF in a fluorescence multi-well plate reader. In Vitro Cell. Devel. Biol. Animal 33, 256–260 (1997).

    Article  Google Scholar 

  28. Koo, M. K., Oh, C. H., Holme, A. L. & Pervaiz, S. Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry. Cytometry A 71A, 87–93 (2007).

    Article  CAS  Google Scholar 

  29. Disbrow, G. L., Hanover, J. A. & Schlegel, R. Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH. J. Virol. 79, 5839–5846 (2005).

    Article  CAS  Google Scholar 

  30. Downey, G. P. et al. Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcγRIIA Receptors J. Biol. Chem. 274, 28436–28444 (1999).

    Article  CAS  Google Scholar 

  31. Simchowitz, L. & Cragoe, E. J. Jr Regulation of human neutrophil chemotaxis by intracellular pH. J. Biol. Chem. 261, 6492–6500 (1986).

    CAS  Google Scholar 

  32. Matsuyama, S. & Reed, J. C. Mitochondria-dependent apoptosis and cellular pH regulation. Cell Death Diff. 7, 1155–1165 (2000).

    Article  CAS  Google Scholar 

  33. Altan, N., Chen, Y., Schindler, M. & Simon, S. M. Defective acidification in human breast tumor cells and implications for chemotherapy. J. Exp. Med. 187, 1583–1598 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

The author would like to thank E. Westhof, V. Malhotra, V. Rodrigues, G.V. Shivashankar and A. Sarin for critical input, M. Gonzalez-Gaitan for the Rab-5–GFP flies, V. Rangaraju for technical assistance and the CIFF facility at NCBS. S.M. and S.M.G. thank the CSIR for Fellowships. This work was funded by the Nano Science and Technology Initiative, DST, Government of India, and the Innovative Young Biotechnologist Award, DBT (Government of India) to Y.K.

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S.M., S.M. and Y.K. conceived, designed and analysed the experiments. S.M. and S.M.G. performed the experiments. S.M. and Y.K. wrote the paper. D.G. performed time-resolved experiments. G.D.G. contributed the SR+ cell line.

Corresponding author

Correspondence to Yamuna Krishnan.

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Modi, S., M. G., S., Goswami, D. et al. A DNA nanomachine that maps spatial and temporal pH changes inside living cells. Nature Nanotech 4, 325–330 (2009). https://doi.org/10.1038/nnano.2009.83

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