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Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA

Abstract

The identification and differentiation of a large number of distinct molecular species with high temporal and spatial resolution is a major challenge in biomedical science. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinguishable fluorophores. Here, we used (deoxy)ribonucleic acid (DNA)-origami technology to construct submicrometre nanorods that act as fluorescent barcodes. We demonstrate that spatial control over the positioning of fluorophores on the surface of a stiff DNA nanorod can produce 216 distinct barcodes that can be decoded unambiguously using epifluorescence or total internal reflection fluorescence microscopy. Barcodes with higher spatial information density were demonstrated via the construction of super-resolution barcodes with features spaced by 40 nm. One species of the barcodes was used to tag yeast surface receptors, which suggests their potential applications as in situ imaging probes for diverse biomolecular and cellular entities in their native environments.

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Figure 1: Design of a barcode based on a DNA nanorod.
Figure 2: Fluorescent barcodes with single-labelled zones.
Figure 3: Fluorescent barcodes with dual-labelled zones.
Figure 4: Super-resolution fluorescent barcodes.
Figure 5: Fluorescent barcode with nonlinear geometry.
Figure 6: Tagging yeast cells with the GRG barcodes as in situ imaging probes.

References

  1. Fournier Bidoz, S. et al. Facile and rapid one-step mass preparation of quantum-dot barcodes. Angew. Chem. Int. Ed. 47, 5577–5581 (2008).

    CAS  Article  Google Scholar 

  2. Han, M., Gao, X., Su, J. Z. & Nie, S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nature Biotechnol. 19, 631–635 (2001).

    CAS  Article  Google Scholar 

  3. Li, Y., Cu, Y. T. H. & Luo, D. Multiplexed detection of pathogen DNA with DNA-based fluorescence nanobarcodes. Nature Biotechnol. 23, 885–889 (2005).

    CAS  Article  Google Scholar 

  4. Marcon, L. et al. ‘On-the-fly’ optical encoding of combinatorial peptide libraries for profiling of protease specificity. Mol. BioSyst. 6, 225–233 (2010).

    CAS  Article  Google Scholar 

  5. Xu, H. et al. Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay. Nucleic Acids Res. 31, e43 (2003).

    Article  Google Scholar 

  6. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    CAS  Article  Google Scholar 

  7. Lin, C., Liu, Y. & Yan, H. Self-assembled combinatorial encoding nanoarrays for multiplexed biosensing. Nano Lett. 7, 507–512 (2007).

    CAS  Article  Google Scholar 

  8. Levsky, J. M., Shenoy, S. M., Pezo, R. C. & Singer, R. H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    CAS  Article  Google Scholar 

  9. Braeckmans, K. et al. Encoding microcarriers by spatial selective photobleaching. Nature Mater. 2, 169–173 (2003).

    CAS  Article  Google Scholar 

  10. Dejneka, M. J. et al. Rare earth-doped glass microbarcodes. Proc. Natl Acad. Sci. USA 100, 389–393 (2003).

    CAS  Article  Google Scholar 

  11. Gudiksen, M. S., Lauhon, L. J., Wang, J., Smith, D. C. & Lieber, C. M. Growth of nanowire superlattice structures for nanoscale photonics and electronics. Nature 415, 617–620 (2002).

    CAS  Article  Google Scholar 

  12. Li, X. et al. Controlled fabrication of fluorescent barcode nanorods. ACS Nano 4, 4350–4360 (2010).

    CAS  Article  Google Scholar 

  13. Nicewarner-Pena, S. R. Submicrometer metallic barcodes. Science 294, 137–141 (2001).

    CAS  Article  Google Scholar 

  14. Pregibon, D. C., Toner, M. & Doyle, P. S. Multifunctional encoded particles for high-throughput biomolecule analysis. Science 315, 1393–1396 (2007).

    CAS  Article  Google Scholar 

  15. Geiss, G. K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotechnol. 26, 317–325 (2008).

    CAS  Article  Google Scholar 

  16. Xiao, M. et al. Direct determination of haplotypes from single DNA molecules. Nature Methods 6, 199–201 (2009).

    CAS  Article  Google Scholar 

  17. Toomre, D. & Bewersdorf, J. A new wave of cellular imaging. Annu. Rev. Cell Dev. Biol. 26, 285–314 (2010).

    CAS  Article  Google Scholar 

  18. Seeman, N. C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).

    CAS  Article  Google Scholar 

  19. Aldaye, F. A., Palmer, A. L. & Sleiman, H. F. Assembling materials with DNA as the guide. Science 321, 1795–1799 (2008).

    CAS  Article  Google Scholar 

  20. Lin, C., Liu, Y. & Yan, H. Designer DNA nanoarchitectures. Biochemistry 48, 1663–1674 (2009).

    CAS  Article  Google Scholar 

  21. Nangreave, J., Han, D., Liu, Y. & Yan, H. DNA origami: a history and current perspective. Curr. Opin. Chem. Biol. 14, 608–615 (2010).

    CAS  Article  Google Scholar 

  22. Shih, W. M. & Lin, C. Knitting complex weaves with DNA origami. Curr. Opin. Struct. Biol. 20, 276–282 (2010).

    CAS  Article  Google Scholar 

  23. Tørring, T., Voigt, N. V., Nangreave, J., Yan, H. & Gothelf, K. V. DNA origami: a quantum leap for self-assembly of complex structures. Chem. Soc. Rev. 40, 5636–5646 (2011).

    Article  Google Scholar 

  24. Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    CAS  Article  Google Scholar 

  25. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

    CAS  Article  Google Scholar 

  26. Dietz, H., Douglas, S. M. & Shih, W. M. Folding DNA into twisted and curved nanoscale shapes. Science 325, 725–730 (2009).

    CAS  Article  Google Scholar 

  27. Andersen, E. S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73–76 (2009).

    CAS  Article  Google Scholar 

  28. Han, D., Pal, S., Liu, Y. & Yan, H. Folding and cutting DNA into reconfigurable topological nanostructures. Nature Nanotechnol. 5, 712–717 (2010).

    CAS  Article  Google Scholar 

  29. Liedl, T., Högberg, B., Tytell, J., Ingber, D. E. & Shih, W. M. Self-assembly of three-dimensional prestressed tensegrity structures from DNA. Nature Nanotechnol. 5, 520–524 (2010).

    CAS  Article  Google Scholar 

  30. Han, D. et al. DNA origami with complex curvatures in three-dimensional space. Science 332, 342–346 (2011).

    CAS  Article  Google Scholar 

  31. 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).

    CAS  Article  Google Scholar 

  32. Steinhauer, C., Jungmann, R., Sobey, T. L., Simmel, F. C. & Tinnefeld, P. DNA origami as a nanoscopic ruler for super-resolution microscopy. Angew. Chem. Int. Ed. 48, 8870–8873 (2009).

    CAS  Article  Google Scholar 

  33. Lund, K. et al. Molecular robots guided by prescriptive landscapes. Nature 465, 206–210 (2010).

    CAS  Article  Google Scholar 

  34. Pal, S., Deng, Z., Ding, B., Yan, H. & Liu, Y. DNA-origami-directed self-assembly of discrete silver-nanoparticle architectures. Angew. Chem. Int. Ed. 49, 2700–2704 (2010).

    CAS  Article  Google Scholar 

  35. Bui, H. et al. Programmable periodicity of quantum dot arrays with DNA origami nanotubes. Nano Lett. 10, 3367–3372 (2010).

    CAS  Article  Google Scholar 

  36. Douglas, S. M., Chou, J. J. & Shih, W. M. DNA–nanotube-induced alignment of membrane proteins for NMR structure determination. Proc. Natl Acad. Sci. USA 104, 6644–6648 (2007).

    CAS  Article  Google Scholar 

  37. Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    CAS  Article  Google Scholar 

  38. Huang, B., Babcock, H. & Zhuang, X. Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047–1058 (2010).

    CAS  Article  Google Scholar 

  39. Vogelsang, J. et al. Make them blink: probes for super-resolution microscopy. ChemPhysChem. 11, 2475–2490 (2010).

    CAS  Article  Google Scholar 

  40. Walter, N. G., Huang, C. Y., Manzo, A. J. & Sobhy, M. A. Do-it-yourself guide: how to use the modern single-molecule toolkit. Nature Methods 5, 475–489 (2008).

    CAS  Article  Google Scholar 

  41. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  42. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–795 (2006).

    CAS  Article  Google Scholar 

  43. Yildiz, A. et al. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

    CAS  Article  Google Scholar 

  44. Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nature Methods 8, 499–508 (2011).

    CAS  Article  Google Scholar 

  45. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    CAS  Article  Google Scholar 

  46. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnol. 21, 86–89 (2003).

    CAS  Article  Google Scholar 

  47. Klein, T. et al. Live-cell dSTORM with SNAP-tag fusion proteins. Nature Methods 8, 7–9 (2011).

    CAS  Article  Google Scholar 

  48. Cunin, F. et al. Biomolecular screening with encoded porous-silicon photonic crystals. Nature Mater. 1, 39–41 (2002).

    CAS  Article  Google Scholar 

  49. Matesanz-Isabel, J. et al. New B-cell CD molecules. Immunol. Lett. 134, 104–112 (2011).

    CAS  Article  Google Scholar 

  50. Maecker, H. T., McCoy, J. P. & Nussenblatt, R. Standardizing immunophenotyping for the Human Immunology Project. Nature Rev. Immunol. 12, 191–200 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank C. Steinhauer and S. P. Laurien for help with super-resolution microscopy software development, the Harvard Center for Biological Imaging, as well as the Nikon Imaging Center at Harvard Medical School, for the use of their microscopes and S. M. Douglas for providing the transmission electron microscopy images used in Supplementary Fig. S5. This work is supported by a National Institutes of Health (NIH) Director's New Innovator Award (1DP2OD007292), a National Science Foundation Faculty Early Career Development Award (CCF1054898), an Office of Naval Research Young Investigator Program Award (N000141110914), an Office of Naval Research grant (N000141010827) and a Wyss Institute for Biologically Engineering Faculty Startup Fund to P.Y., and an NIH Director's New Innovator Award (1DP2OD004641) and a Wyss Institute for Biologically Inspired Engineering Faculty Award to W.M.S. C. Li, D.L. and G.M.C. acknowledge support from the National Human Genome Research Institute, Centers of Excellence in Genomic Science. R.J. acknowledges support from the Alexander von Humboldt-Foundation through a Feodor Lynen fellowship.

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Authors

Contributions

C. Lin conceived the project, designed and conducted the majority of the experiments, analysed the data and prepared the majority of the manuscript. R.J. conceived the super-resolution barcode study, designed and conducted experiments for this study, analysed the data and prepared the manuscript. A.M.L. wrote the MATLAB script for the automated barcode deciphering and prepared the manuscript. C. Li wrote the C script for the barcode geometry characterization. D.L. (with C. Lin) performed the yeast-tagging experiment. G.M.C. championed multiplexed in situ, supervised C. Li and D.L., and critiqued the data and the manuscript. W.M.S. conceived the project, discussed the results and prepared the manuscript. P.Y. conceived, designed and supervised the study, interpreted the data and prepared the manuscript. All authors reviewed and approved the manuscript.

Corresponding authors

Correspondence to William M. Shih or Peng Yin.

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Supplementary information

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Supplementary information (PDF 20281 kb)

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High-resolution Fig. S3 (PDF 8483 kb)

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High-resolution Fig. S7 (PDF 5239 kb)

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High-resolution Fig. S8 (PDF 11430 kb)

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High-resolution Fig. S9 (PDF 5958 kb)

Supplementary information

High-resolution Fig. S12 (PDF 5111 kb)

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Lin, C., Jungmann, R., Leifer, A. et al. Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. Nature Chem 4, 832–839 (2012). https://doi.org/10.1038/nchem.1451

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