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.
Subscribe to Journal
Get full journal access for 1 year
only $15.58 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Bath, J. & Turberfield, A. J. DNA nanomachines. Nature Nanotech. 2, 275–284 (2007).
Shih, W. Biomolecular self-assembly: Dynamic DNA. Nature Mater. 7, 98–100 (2008).
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).
Liu, J. & Lu, Y. A colorimetric lead biosensor using DNA enzyme-directed assembly of gold nanoparticles. J. Am. Chem. Soc. 125, 6642–6643 (2003).
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).
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).
Yin, P., Choi, H. M. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318–322 (2008).
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).
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).
Allan, V. J. & Schroer, T. A. Membrane motors. Curr. Opin. Cell Biol. 11, 476–482 (1999).
Griesbeck, O. Fluorescent proteins as sensors for cellular functions, Curr. Opin. Neurobiol. 14, 636–641 (2004).
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).
Gehring, K., Leroy, J.-L. & Guéron, M. A tetrameric DNA structure with protonated cytidine–cytidine base pairs. Nature 363, 561–565 (1993).
Mukherjee, S. Ghosh, R. N. & Maxfield, F. R. Endocytosis. Physiol. Rev. 77, 759–803 (1997).
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).
Stryer, L. & Haugland, R. P. Energy transfer: a spectroscopic ruler. Proc. Natl Acad. Sci. USA 58, 719–726 (1967).
Liu, D. & Balasubramanian, S. A proton-fuelled DNA nanomachine. Angew Chem. Int. Ed. 42, 5734–5736 (2003).
Liedl, T. & Simmel, F. C. Switching the conformation of a DNA molecule with a chemical oscillator. Nano Lett. 5, 1894–1898 (2005).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Simchowitz, L. & Cragoe, E. J. Jr Regulation of human neutrophil chemotaxis by intracellular pH. J. Biol. Chem. 261, 6492–6500 (1986).
Matsuyama, S. & Reed, J. C. Mitochondria-dependent apoptosis and cellular pH regulation. Cell Death Diff. 7, 1155–1165 (2000).
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).
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.
About this article
Cite this article
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
Journal of Cell Science (2020)
Gold Nanoparticles in Conjunction with Nucleic Acids as a Modern Molecular System for Cellular Delivery
Time-Resolved Activation of pH Sensing and Imaging in Vivo by a Remotely Controllable DNA Nanomachine
Nano Letters (2020)
Advanced Materials (2020)
Chemistry – A European Journal (2020)