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Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA

Nature Chemistry volume 5, pages 782–789 (2013)Cite this article

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Abstract

Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to a 12,000-fold excess of a target that contains a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base-pair subsequence of the Escherichia coli rpoB gene. That our probes are constructed from multiple oligonucleotides circumvents synthesis limitations and enables long continuous DNA sequences to be probed.

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Figure 1: Schematic representation of the double-stranded toehold exchange mechanism.
Figure 2: Discrimination of SNPs by the dsDNA probe.
Figure 3: Concentration of SNP target needed to generate the same χ as a stoichiometric (relative to probe) amount of intended target.
Figure 4: Characterization of the background, temperature, salinity and time robustness of the probe.
Figure 5: Detection of SNPs in E. coli-derived samples.

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References

  1. Gunderson, K. L., Steemers, F. J., Lee, G., Mendoza, L. G. & Chee, M. S. A genome-wide scalable SNP genotyping assay using microarray technology. Nature Biotechnol. 37, 549–554 (2005).

    CAS  Google Scholar 

  2. Kim, S. & Misra A. SNP genotyping: technologies and biomedical applications. Annu. Rev. Biomed. Eng. 9, 289–320 (2007).

    CAS  PubMed  Google Scholar 

  3. Arnold, C. et al. Single-nucleotide polymorphism-based differentiation and drug resistance detection in Mycobacterium tuberculosis from isolates or directly from sputum. Clin. Microbiol. Infect. 11, 122–130 (2005).

    CAS  PubMed  Google Scholar 

  4. Bang, H. et al. Improved rapid molecular diagnosis of multidrug-resistant tuberculosis using a new reverse hybridization assay, REBA MTB-MDR. J. Med. Microbiol. 60, 1447–1454 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470 (1995).

    CAS  Google Scholar 

  6. Saiki, R. K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491 (1988).

    CAS  PubMed  Google Scholar 

  7. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 1728–1732 (2005).

    CAS  PubMed  Google Scholar 

  8. Landegren, U., Kaiser, R., Sanders, J. & Hood, L. A ligase-mediated gene detection technique. Science 241, 1077–1080 (1988).

    CAS  PubMed  Google Scholar 

  9. Tong, A. K., Li, Z., Jones, G. S., Russo, J. J. & Ju, J. Combinatorial fluorescence energy transfer tags for multiplex biological assays. Nature Biotechnol. 19, 756–759 (2001).

    CAS  Google Scholar 

  10. Botstein, D., White, R. L., Skolnick, M. & Davis, R. W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314 (1980).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hall, J. G. et al. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc. Natl Acad. Sci. USA 97, 8272–8277 (2000).

    CAS  PubMed  Google Scholar 

  12. Xu, Y., Karalkar, N. B., & Kool, E. T. Nonenzymatic autoligation in direct three-color detection of RNA and DNA point mutations. Nature Biotechnol. 19, 148–152 (2001).

    CAS  Google Scholar 

  13. Grossmann, T. N. & Seitz, O. Nucleic acid templated reactions: consequences of probe reactivity and readout strategy for amplified signaling and sequence selectivity. Chem. Eur. J. 15, 6723–6730 (2009).

    CAS  PubMed  Google Scholar 

  14. Singh, S. K., Koshkin, A. A., Wengel, J. & Nielsen, P. LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition. Chem. Commun. 4, 455–456 (1998).

    Google Scholar 

  15. Egholm, M., Buchardt, O., Nielsen, P. E. & Berg, R. H. Peptide nucleic acids (PNA). Oligonucleotide analogs with an achiral peptide backbone. J. Am. Chem. Soc. 114, 1895–1897 (1992).

    CAS  Google Scholar 

  16. Simeonov, A. & Nikiforov, T. T. Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. Nucleic Acids Res. 30, e91 (2002).

    PubMed  PubMed Central  Google Scholar 

  17. Komiyama, M. et al. PNA for one-base differentiating protection of DNA from nuclease and its use for SNPs detection. J. Am. Chem. Soc. 125, 3758–3762 (2003).

    CAS  PubMed  Google Scholar 

  18. Tyagi, S. & Kramer, F. R. Molecular beacons: probes that upon hybridization. Nature Biotechnol. 14, 303–308 (1996).

    CAS  Google Scholar 

  19. Zhang, D. Y., Chen, S. X. & Yin, P. Optimizing the specificity of nucleic acid hybridization. Nature Chem. 4, 208–214 (2012).

    CAS  Google Scholar 

  20. Guo, Z., Liu, Q. & Smith, L. M. Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization. Nature Biotechnol. 15, 331–335 (1997).

    CAS  Google Scholar 

  21. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).

    CAS  PubMed  Google Scholar 

  22. Xiao, Y. et al. Fluorescence detection of single-nucleotide polymorphisms with a single, self-complementary, triple-stem DNA probe. Angew. Chem Int. Ed. 48, 4354–4358 (2009).

    CAS  Google Scholar 

  23. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nature Methods 6, 331–338 (2009).

    CAS  PubMed  Google Scholar 

  24. Manganelli, R., Tyagi, S. & Smith, I. Real-time PCR using molecular beacons. Methods Mol. Med. 54, 295–310 (2001).

    CAS  PubMed  Google Scholar 

  25. Severinov, K., Soushko, M., Goldfarb, A. & Nikiforov, V. Rifampicin region revisited. J. Biol. Chem. 268, 14820–14825 (1993).

    CAS  PubMed  Google Scholar 

  26. Telenti, A. et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341, 648–650 (1993).

    Google Scholar 

  27. Thompson, B. J., Camien, M. N. & Warner, R. C. Kinetics of branch migration in double-stranded DNA. Proc. Natl Acad. Sci. 73, 2299–2303 (1976).

    CAS  PubMed  Google Scholar 

  28. Panyutin, I. G. & Hsieh, P. The kinetics of spontaneous DNA branch migration. Proc. Natl Acad. Sci. 91, 2021–2025 (1994).

    CAS  PubMed  Google Scholar 

  29. Panyutin, I. G. & Hsieh, P. Formation of a single base mismatch impedes spontaneous DNA branch migration. J. Mol. Biol. 230, 413–424 (1993).

    CAS  PubMed  Google Scholar 

  30. Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand displacement reactions. Nature Chem. 3, 103–114 (2011).

    CAS  Google Scholar 

  31. Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 1585–1588 (2006).

    CAS  PubMed  Google Scholar 

  32. Zhang, D. Y., Turberfield, A. J., Yurke, B. & Winfree, E. Engineering entropy-driven reactions and networks catalyzed by DNA. Science 318, 1121–1125 (2007).

    CAS  PubMed  Google Scholar 

  33. Soloveichik, D., Seelig, G. & Winfree, E. DNA as a universal substrate for chemical kinetics. Proc. Natl Acad. Sci. 107, 5393–5398 (2010).

    CAS  PubMed  Google Scholar 

  34. Zhang, D. Y. & Winfree, E. Robustness and modularity properties of a non-covalent DNA catalytic reaction. Nucleic Acids Res. 38, 4182–4197 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Qian, L. & Winfree, E. Scaling up digital circuit computation with DNA strand displacement cascades. Science 332, 1196–1201 (2011).

    CAS  PubMed  Google Scholar 

  36. Nandagopal, N. & Elowitz M. B. Synthetic biology: integrated gene circuits. Science 333, 1244–1248 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Purnick, P. E. M. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nature Rev. Mol. Cell Biol. 10, 410–422 (2009).

    CAS  Google Scholar 

  38. Bunka, D. H. J., Platonova, O. & Stockley, P. G. Development of aptamer therapeutics. Curr. Opin. Pharmacol. 10, 557–562 (2010).

    CAS  PubMed  Google Scholar 

  39. SantaLucia, J. & Hicks, D. The thermodynamics of DNA structural motifs. Annu. Rev. Biochem. 33, 415–440 (2004).

    CAS  Google Scholar 

  40. Marras, S. A., Kramer, F. R. & Tyagi, S. Efficiencies of resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30, e122 (2002).

    PubMed  PubMed Central  Google Scholar 

  41. Biswas, I., Yamamoto, A. & Hsieh, P. Branch migration through DNA sequence heterology. J. Mol. Biol. 279, 795–806 (1998).

    CAS  PubMed  Google Scholar 

  42. Lishanski, A. Screening for single-nucleotide polymorphisms using branch migration inhibition in PCR-amplified DNA. Clin. Chem. 46, 1464–1470 (2000).

    CAS  PubMed  Google Scholar 

  43. Yang, Q. et al. Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring. Genome Res. 13, 1754–1764 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Liu, Y. P., Behr, M. A., Small, P. M. & Kurn, N. Genotypic determination of Mycobacterium tuberculosis antibiotic resistance using a novel mutation detection method, the branch migration inhibition M. tuberculosis antibiotic resistance test. J. Clin. Microbiol. 38, 3656–3662 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. McNerney, R. & Daley, P. Towards a point-of-care test for active tuberculosis: obstacles and opportunities. Nature Rev. Microbiol. 9, 204–213 (2011).

    CAS  Google Scholar 

  46. Niemz, A., Ferguson, T. M. & Boyle, D. S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29, 240–250 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Piatek, A. S. et al. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nature Biotechnol. 16, 359–363 (1998).

    CAS  Google Scholar 

  48. Boehme, C. C. et al. Rapid molecular detection of tuberculosis and rifampin resistance. N. Engl. J. Med. 363, 1005–1015 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Browning, S. R. & Browning, B. L. Haplotype phasing: existing methods and new developments. Nature Rev. Genetics 12, 703–714 (2011).

    CAS  Google Scholar 

  50. Seelig, G., Yurke, B. & Winfree, E. Catalyzed relaxation of a metastable DNA fuel. J. Am. Chem. Soc. 128, 12211–12220 (2006).

    CAS  PubMed  Google Scholar 

  51. Gao, Y., Wolf, L. K. & Georgiadis, R. M. Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison. Nucleic Acids Res. 34, 3370–3377 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Zhang, J., Finney, R. P., Clifford, R. J., Derr, L. K. & Buetow, K. H. Genomics 85, 297308 (2005).

    Google Scholar 

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Acknowledgements

The authors thank Eric Klavins for insightful discussion and helpful manuscript preparation suggestions. This work was funded by National Institutes of Health Award 1K99EB015331 to D.Y.Z., by National Science Foundation CAREER Award 0954566 to G.S. and by a Defense Advanced Research Projects Agency Young Faculty Award to G.S.

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Authors and Affiliations

  1. Department of Electrical Engineering, University of Washington, Seattle, 98195, Washington, USA

    Sherry Xi Chen

  2. Department of Bioengineering, Rice University, Houston, 77005, Texas, USA

    David Yu Zhang

  3. Department of Electrical Engineering, Department of Computer Science and Engineering, University of Washington, Seattle, 98195, Washington, USA

    Georg Seelig

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  1. Sherry Xi Chen
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Contributions

S.X.C., D.Y.Z. and G.S. conceived the project and designed the experiments. S.X.C. conducted the experiments. S.X.C., D.Y.Z. and G.S. analysed the data and co-wrote the paper.

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Correspondence to David Yu Zhang or Georg Seelig.

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The authors declare no competing financial interests.

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Chen, S., Zhang, D. & Seelig, G. Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA. Nature Chem 5, 782–789 (2013). https://doi.org/10.1038/nchem.1713

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