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

Journal name:
Nature Chemistry
Year published:
Published online


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.

At a glance


  1. Schematic representation of the double-stranded toehold exchange mechanism.
    Figure 1: Schematic representation of the double-stranded toehold exchange mechanism.

    a, The reaction starts with the hybridization of the initiation toeholds (orange and purple) to form a four-stranded complex C0. Next, the four-stranded complex undergoes a series of single-base reconfiguration events, known as branch migration27. The various states of branch migration (Ci) are roughly isoenergetic, and thus each branch-migration step is reversible and unbiased. When the branch migration reaches state Cn, at which the four-stranded complex is held together only by the dissociation toeholds (blue and green), these dissociation toeholds can dissociate spontaneously to release the two product molecules. b, A highly specific, conditionally fluorescent molecular probe based on four-stranded toehold exchange. The probe is functionalized at the balancing toeholds with a fluorophore and a quencher on separate strands; at the end of the reaction, the fluorophore is delocalized from the quencher and fluorescence increases. The lengths and sequences of the toeholds are designed so that the ΔGointended of the reaction between the probe and the intended target is roughly 0. The reaction between the probe and the SNP target results in two mismatch bubbles, and the reaction ΔGoSNP will be about 8 kcal mol−1. c, Plot of the analytic hybridization yield at equilibrium of the A + B ⇌ D + E reaction against the reaction ΔGo (assuming identical initial concentrations of A and B). Designing ΔGointended ≈ 0 ensures a balance of high specificity and high yield.

  2. Discrimination of SNPs by the dsDNA probe.
    Figure 2: Discrimination of SNPs by the dsDNA probe.

    a, Sequence of the intended target and the positions/identities of base-pair changes that lead to the 14 SNP targets. Circled 1, 2 and 3 denote the positions of the mismatch. Mismatches, insertions and deletions are shown in blue, red and green, respectively. ROX, carboxy-X-rhodamine fluorophore; RQ, Iowa Black Red Quencher. b, Hybridization yield as inferred from fluorescence kinetics (Supplementary Fig. S1 and Methods). The probe is present in solution initially, and the intended or SNP target is introduced at t ≈ 0. Experiments were run at 25 °C in 1 M Na+. The trace for the intended target is shown in black, and traces for SNP targets are shown in the colours described above (see Supplementary Fig. S2 for a zoom-in of SNP reactions). c, Reaction equilibration appears to be complete after four hours; to ensure equilibration, however, the reactions were allowed to proceed until t = 25 h. The hybridization yields at t = 25 h are taken to be the equilibrium values, and discrimination factors Q = χintended/χSNP are calculated for each SNP target. Observed Q values range between 17 and 99 (median = 43). Error bars show standard deviations calculated from three repetitions of each experiment.

  3. Concentration of SNP target needed to generate the same χ as a stoichiometric (relative to probe) amount of intended target.
    Figure 3: Concentration of SNP target needed to generate the same χ as a stoichiometric (relative to probe) amount of intended target.

    a, Sequences of intended and SNP targets used for the experiments shown in this figure. b, Hybridization yields of various concentrations of intended and ‘i8TA’ SNP targets. In all traces, the initial probe concentration [B]0 = 10 nM. c, Hybridization yields plotted against the stoichiometric ratio of the target. As with previous experiments, hybridization yield was inferred from the fluorescence value at t = 25 h. Experimentally determined values are shown as dots and the star, and solid lines show the analytic model prediction based on best-fit ΔGo values (Supplementary Text S1). All experiments other than the star data point were performed with 10 nM probe; the star data point reaction was performed with 2 nM probe to conserve reagents. R values are calculated based on best-fit models at 50% hybridization yield, and range between 260 and 12,000. Analysis shows and experiments verify that R ≈ Q2, with Q = χintended/χSNP being the discrimination factor.

  4. Characterization of the background, temperature, salinity and time robustness of the probe.
    Figure 4: Characterization of the background, temperature, salinity and time robustness of the probe.

    ac, The probe operates robustly to discriminate SNPs in the presence of high concentrations of 50 nucleotide polynucleotide strands (a), in different salinity buffers (b) and at different temperatures (c) (see also Supplementary Figs S5 and S6). d, The discrimination factor Q approaches its final value after about ten minutes of reaction, and maintains a high discrimination indefinitely. The initial rise and bumpiness in Q can be attributed to fluorescence-signal instability directly after the addition of target to solution (Supplementary Fig. S2).

  5. Detection of SNPs in E. coli-derived samples.
    Figure 5: Detection of SNPs in E. coli-derived samples.

    a, Rifampicin resistance is typically conferred by mutations in one of two regions in the rpoB gene, nucleotides 1,531–1,599 and 1,684–1,728, which correspond to amino acid residues 511–533 and 562–576, respectively. Here, we generated three distinct probes, one to test one region, one to test the other, and one to test both simultaneously (see Supplementary Fig. S17 for sequences of probes and targets). b, DNA from ten rifampicin-resistant colonies was extracted and individually amplified by colony PCR. Subsequently, unbalanced PCR using an excess of one primer with an overhang was used to generate the initiation toeholds. These DNA samples were allowed to react with our fluorescent probes, which were constructed by annealing four separate oligonucleotides, and possessed non-overlapping nicks that do not interfere with probe function. c, The left side of each column shows the approximate position of the mutations, as determined by sequencing. The right side of each column shows the fluorescence response of the rpoB subsequences to the two fluorescent probes. A mutation in the green (blue) region would result in no increase in the fluorescence for Probe 1 (Probe 2), as shown in the green (blue) trace. The experimental results agree with the sequencing results in all experiments. The fluorescence data shown in the experimental panels on the left represent the behaviour over three hours of the reaction; the right panels show ten hours of reaction (see Supplementary Figs S11–S15 for zoomed-in view of data).


  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, 549554 (2005).
  2. Kim, S. & Misra A. SNP genotyping: technologies and biomedical applications. Annu. Rev. Biomed. Eng. 9, 289320 (2007).
  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, 122130 (2005).
  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, 14471454 (2011).
  5. Schena, M., Shalon, D., Davis, R. W. & Brown, P. O. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467470 (1995).
  6. Saiki, R. K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487491 (1988).
  7. Shendure, J. et al. Accurate multiplex polony sequencing of an evolved bacterial genome. Science 309, 17281732 (2005).
  8. Landegren, U., Kaiser, R., Sanders, J. & Hood, L. A ligase-mediated gene detection technique. Science 241, 10771080 (1988).
  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, 756759 (2001).
  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).
  11. Hall, J. G. et al. Sensitive detection of DNA polymorphisms by the serial invasive signal amplification reaction. Proc. Natl Acad. Sci. USA 97, 82728277 (2000).
  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, 148152 (2001).
  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, 67236730 (2009).
  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, 455456 (1998).
  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, 18951897 (1992).
  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).
  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, 37583762 (2003).
  18. Tyagi, S. & Kramer, F. R. Molecular beacons: probes that upon hybridization. Nature Biotechnol. 14, 303308 (1996).
  19. Zhang, D. Y., Chen, S. X. & Yin, P. Optimizing the specificity of nucleic acid hybridization. Nature Chem. 4, 208214 (2012).
  20. Guo, Z., Liu, Q. & Smith, L. M. Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization. Nature Biotechnol. 15, 331335 (1997).
  21. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 1730317314 (2009).
  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, 43544358 (2009).
  23. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nature Methods 6, 331338 (2009).
  24. Manganelli, R., Tyagi, S. & Smith, I. Real-time PCR using molecular beacons. Methods Mol. Med. 54, 295310 (2001).
  25. Severinov, K., Soushko, M., Goldfarb, A. & Nikiforov, V. Rifampicin region revisited. J. Biol. Chem. 268, 1482014825 (1993).
  26. Telenti, A. et al. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet 341, 648650 (1993).
  27. Thompson, B. J., Camien, M. N. & Warner, R. C. Kinetics of branch migration in double-stranded DNA. Proc. Natl Acad. Sci. 73, 22992303 (1976).
  28. Panyutin, I. G. & Hsieh, P. The kinetics of spontaneous DNA branch migration. Proc. Natl Acad. Sci. 91, 20212025 (1994).
  29. Panyutin, I. G. & Hsieh, P. Formation of a single base mismatch impedes spontaneous DNA branch migration. J. Mol. Biol. 230, 413424 (1993).
  30. Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand displacement reactions. Nature Chem. 3, 103114 (2011).
  31. Seelig, G., Soloveichik, D., Zhang, D. Y. & Winfree, E. Enzyme-free nucleic acid logic circuits. Science 314, 15851588 (2006).
  32. Zhang, D. Y., Turberfield, A. J., Yurke, B. & Winfree, E. Engineering entropy-driven reactions and networks catalyzed by DNA. Science 318, 11211125 (2007).
  33. Soloveichik, D., Seelig, G. & Winfree, E. DNA as a universal substrate for chemical kinetics. Proc. Natl Acad. Sci. 107, 53935398 (2010).
  34. Zhang, D. Y. & Winfree, E. Robustness and modularity properties of a non-covalent DNA catalytic reaction. Nucleic Acids Res. 38, 41824197 (2010).
  35. Qian, L. & Winfree, E. Scaling up digital circuit computation with DNA strand displacement cascades. Science 332, 11961201 (2011).
  36. Nandagopal, N. & Elowitz M. B. Synthetic biology: integrated gene circuits. Science 333, 12441248 (2011).
  37. Purnick, P. E. M. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nature Rev. Mol. Cell Biol. 10, 410422 (2009).
  38. Bunka, D. H. J., Platonova, O. & Stockley, P. G. Development of aptamer therapeutics. Curr. Opin. Pharmacol. 10, 557562 (2010).
  39. SantaLucia, J. & Hicks, D. The thermodynamics of DNA structural motifs. Annu. Rev. Biochem. 33, 415440 (2004).
  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).
  41. Biswas, I., Yamamoto, A. & Hsieh, P. Branch migration through DNA sequence heterology. J. Mol. Biol. 279, 795806 (1998).
  42. Lishanski, A. Screening for single-nucleotide polymorphisms using branch migration inhibition in PCR-amplified DNA. Clin. Chem. 46, 14641470 (2000).
  43. Yang, Q. et al. Allele-specific Holliday junction formation: a new mechanism of allelic discrimination for SNP scoring. Genome Res. 13, 17541764 (2003).
  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, 36563662 (2000).
  45. McNerney, R. & Daley, P. Towards a point-of-care test for active tuberculosis: obstacles and opportunities. Nature Rev. Microbiol. 9, 204213 (2011).
  46. Niemz, A., Ferguson, T. M. & Boyle, D. S. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29, 240250 (2011).
  47. Piatek, A. S. et al. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nature Biotechnol. 16, 359363 (1998).
  48. Boehme, C. C. et al. Rapid molecular detection of tuberculosis and rifampin resistance. N. Engl. J. Med. 363, 10051015 (2010).
  49. Browning, S. R. & Browning, B. L. Haplotype phasing: existing methods and new developments. Nature Rev. Genetics 12, 703714 (2011).
  50. Seelig, G., Yurke, B. & Winfree, E. Catalyzed relaxation of a metastable DNA fuel. J. Am. Chem. Soc. 128, 1221112220 (2006).
  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, 33703377 (2006).
  52. Zhang, J., Finney, R. P., Clifford, R. J., Derr, L. K. & Buetow, K. H. Genomics 85, 297308 (2005).

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Author information


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

    • Sherry Xi Chen
  2. Department of Bioengineering, Rice University, Houston, Texas 77005, USA

    • David Yu Zhang
  3. Department of Electrical Engineering, Department of Computer Science and Engineering, University of Washington, Seattle, Washington 98195, USA

    • Georg Seelig


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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