Optimizing the specificity of nucleic acid hybridization

Journal name:
Nature Chemistry
Year published:
Published online


The specific hybridization of complementary sequences is an essential property of nucleic acids, enabling diverse biological and biotechnological reactions and functions. However, the specificity of nucleic acid hybridization is compromised for long strands, except near the melting temperature. Here, we analytically derived the thermodynamic properties of a hybridization probe that would enable near-optimal single-base discrimination and perform robustly across diverse temperature, salt and concentration conditions. We rationally designed ‘toehold exchange’ probes that approximate these properties, and comprehensively tested them against five different DNA targets and 55 spurious analogues with energetically representative single-base changes (replacements, deletions and insertions). These probes produced discrimination factors between 3 and 100+ (median, 26). Without retuning, our probes function robustly from 10 °C to 37 °C, from 1 mM Mg2+ to 47 mM Mg2+, and with nucleic acid concentrations from 1 nM to 5 µM. Experiments with RNA also showed effective single-base change discrimination.

At a glance


  1. Hybridization specificity of nucleic acids.
    Figure 1: Hybridization specificity of nucleic acids.

    a, Hybridization yield χ plotted against concentration-adjusted standard free energy ΔG′ = ΔG° + (Δn)RTln(c), where c is the concentration of the limiting species, and Δn = −1 for a standard bimolecular hybridization reaction. At room temperature, the binding of both the correct target (black dot) and the spurious target (red dot) are thermodynamically favourable and practically indistinguishable. In contrast, at the melting temperature, ΔG′ = −RTln((c/2)/(c/2)2)–RTln(c) = −0.41 kcal mol−1, the hybridization yield of the correct target is 50%, and much lower for the spurious target. b, In a hybridization-based assay or reaction, specificity is achieved when a spurious target that differs in sequence from the correct target by a single base (depicted as the red segment) does not hybridize significantly to the complement. c, The standard free energy difference (ΔΔG°) caused by a single-base change ranges from 1.83 to 6.57 kcal mol−1, and determines the upper bound on the discrimination factor: Qmax ≡ eΔΔG°/RT. (Graphic constructed using thermodynamic parameters by SantaLucia and Hicks22; see Supplementary Text S3 and Tables S1–S5 for detailed numerical values.) All 64 cases of single-base insertion were modelled to have identical ΔΔG°.

  2. Toehold exchange probes.
    Figure 2: Toehold exchange probes.

    a, The toehold exchange probe (PC) consists of a pre-hybridized complement strand C and a protector strand P. The probe can react with an intended target X to release P and the hybridized product XC. The probe is designed based on the sequence of the target so that the standard free energy (ΔG° = ΔG′) of the forward reaction is close to zero. In this example, seven new base pairs (green 5′ toehold of C) are formed, but seven existing base pairs are broken (blue 3′ toehold of C). For the sequences shown here, mathematical analysis predicts the hybridization yield χX = 0.34 at 25 °C, mimicking hybridization behaviour at close to the melting temperature of XC (Supplementary Text S4). b, Hybridization of a spurious target S with one base change is less thermodynamically favourable by +2.97 kcal mol−1, and is predicted to have χS = 0.0056. Thus, the discrimination factor Q is predicted to be 0.34/0.0056 = 61. For comparison, Qmax for ΔΔG° = 2.97 kcal mol−1 is 150 at 25 °C.

  3. Experimental demonstration of toehold exchange probes.
    Figure 3: Experimental demonstration of toehold exchange probes.

    a, The intended DNA target sequence and the experimental probe system. Outlined in red are the positions of the one-base changes in various spurious targets. P in the experimental system has a 30 nt poly-T tail at the 5′ end (purple) to help distinguish it from other species in gel experiments. b, Native PAGE results. PC was prepared with a 2:1 ratio of P to C, and annealed with a 1 µM concentration of PC. X or S was added to achieve final concentrations of 200 nM target (X or S), 100 nM PC and 100 nM P. Reactions proceeded at 25 °C for 1 h. Four different probes were tested (for example, the 7/5 probe has a 7 nt green toehold and a 5 nt blue toehold). Middle lanes show the reaction with different spurious targets S; ‘m’, ‘d’ and ‘i’ denote mismatch, deletion and insertion, respectively (for example, in m11C, the adenine at position 11 from the 5′ end was replaced by a cytosine; see Supplementary Table S7 for sequences). The rightmost lane shows the negative control (PC only). The P band is single-stranded and stains inconsistently with SybrGold. c, Hybridization yields χ inferred from b. χX are plotted as crosses, χS as filled circles. Lines connect χ values for the same target. d, Discrimination factors Q. e, Plot of hybridization yield χ versus reaction standard free energy ΔG°. Values of χ are plotted as crosses and filled circles against the reaction ΔG° calculated by NUPACK (Supplementary Text S6)31. The thick black trace shows the expected results from thermodynamic analysis (Supplementary Text S4). Adjusting all ΔG° by +1.5 kcal mol−1 (thin black trace) improves the agreement between model and data. f, 7/5 probe with a large excess of S (equal mixture of all 11 spurious targets). g, Fluorophore/quencher-labelled probe.

  4. Results for additional DNA and RNA targets.
    Figure 4: Results for additional DNA and RNA targets.

    a, Sequences of four additional DNA targets (see Supplementary Tables S6–S8 for sequences of protectors P and spurious targets S). b, Hybridization yield versus adjusted ΔG°. The same ΔG° adjustments (inset, in kcal mol−1) were applied to all reactions within a set. Systems in Fig. 3 are referred to as X1. (See Supplementary Figs S8–S11 for detailed results.) c, Distribution of observed Q for all 7/5 probes (55 data points in total). Owing to the limitations in quantifying gel band intensities, it was not possible to consistently measure Q values above 100 (see Methods). Median observed Q was 26. d, Comparison of observed Q and Q predicted based on ΔΔG° values (Supplementary Text S6). e, RNA target and probe. The target sequence is the RNA analogue of the X2 DNA system shown in a, and is identical to the human let7g microRNA. f, Native PAGE results. PC was prepared with a 2:1 ratio of P to C and annealed at [PC] = 3 µM. X or S was added to achieve final concentrations of 2 µM X or S, 1 µM PC and 1 µM P.

  5. Performance of the 7/5 probe for the X1 target at different conditions.
    Figure 5: Performance of the 7/5 probe for the X1 target at different conditions.

    a, PAGE results. As in Fig. 3, the upper band is the unreacted PC complex, the middle band is excess P and the bottom band is the product XC or SC band. b, High discrimination factors were observed for all conditions tested (see Supplementary Figs S12–S14 for more details and results quantitation), with median Q = 34 across conditions. The histogram includes data from Fig. 3b, where the X1 7/5 probe was operated at standard conditions of 25 °C, 11.5 mM Mg2+ and 100 nM PC.


  1. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215233 (2009).
  2. Saiki, R. K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487491 (1988).
  3. 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).
  4. 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 Genet. 37, 549554 (2005).
  5. Koltai, H. & Weingarten-Baror, C. Specificity of DNA microarray hybridization: characterization, effectors, and approaches for data correction. Nucleic Acids Res. 36, 23952405 (2008).
  6. DeLong, E. F., Wickham, G. S. & Pace, N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 243, 13601363 (1989).
  7. Amann, R. I., Krumholz, L. & Stahl, D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172, 762770 (1990).
  8. Seeman, N. C. Nanomaterials based on DNA. Annu. Rev. Biochem. 79, 6587 (2010).
  9. Rothemund, P. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297302 (2006).
  10. Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414418 (2009).
  11. Aldaye, F. A., Palmer, A. L. & Sleiman, H. F. Assembling materials with DNA as the guide. Science 321, 17951799 (2008).
  12. Yin, P., Choi, H. M. T., Calvert, C. R. & Pierce, N. A. Programming biomolecular self-assembly pathways. Nature 451, 318322 (2008).
  13. Zhang, D. Y. & Seelig, G. Dynamic DNA nanotechnology using strand displacement reactions. Nature Chem. 3, 103113 (2011).
  14. Tyagi, S. & Kramer, F. R. Molecular beacons: probes that fluoresce upon hybridization. Nature Biotechnol. 14, 303308 (1996).
  15. Tyagi, S., Bratu, D. P. & Kramer, F. R. Multicolor molecular beacons for allele discrimination. Nature Biotechnol. 16, 4953 (1998).
  16. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nature Methods 6, 331338 (2009).
  17. Bonnet, G., Tyagi, S., Libchaber, A. & Kramer, F. R. Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Natl Acad. Sci. USA 96, 61716176 (1999).
  18. Tsourkas, A., Behlke, M. A., Rose, S. D. & Bao, G. Hybridization kinetics and thermodynamics of molecular beacons. Nucleic Acids Res. 31, 13191330 (2003).
  19. 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).
  20. Kolpashchikov, D. M. A binary DNA probe for highly specific nucleic acid recognition. J. Am. Chem. Soc. 128, 1062510628 (2006).
  21. Dave N. & Liu, J. Fast molecular beacon hybridization in organic solvents with improved target specificity. J. Phys. Chem. B 114, 1569415699 (2010).
  22. SantaLucia, J. & Hicks, D. The thermodynamics of DNA structural motifs. Ann. Rev. Biophys. Biomol. Struct. 33, 415440 (2004).
  23. Peyret, N. Prediction of Nucleic Acid Hybridization: Parameters and Algorithms. Doctoral thesis, Wayne State University (2000).
  24. Tan, Z. J. & Chen, S. J. Nucleic acid helix stability: effects of salt concentration, cation valence and size, and chain length. Biophys. J. 90, 11751190 (2006).
  25. Yurke, B., Turberfield, A. J., Mills, A. P., Simmel, F. C. & Neumann, J. L. A DNA-fuelled molecular machine made of DNA. Nature 406, 605608 (2000).
  26. Zhang, D. Y. & Winfree, E. Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 1730317314 (2009).
  27. Zhang, D. Y., Turberfield, A. J., Yurke, B. & Winfree, E. Engineering entropy-driven reactions and networks catalyzed by DNA. Science 318, 11211125 (2007).
  28. He, G., Rapireddy, S., Bahal, R., Sahu, B. & Ly, D. H. Strand invasion of extended, mixed-sequence B-DNA by γPNAs. J. Am. Chem. Soc. 131, 1208812090 (2009).
  29. Petersen, M. & Wengel, J. LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 21, 7481 (2003).
  30. Bommarito, S., Peyret, N. & SantaLucia, J. Thermodynamic parameters for DNA sequences with dangling ends. Nucleic Acids Res. 28, 19291934 (2000).
  31. Dirks, R. M., Bois, J. S., Schaeffer, J. M., Winfree, E. & Pierce, N. A. Thermodynamic analysis of interacting nucleic acid strands. SIAM Rev. 49, 6588 (2007).
  32. Zhang, D. Y. & Winfree, E. Robustness and modularity properties of a non-covalent DNA catalytic reaction. Nucleic Acids Res. 38, 41824197 (2010).
  33. Temsamani, J., Kubert, M. & Agrawal, S. Sequence identity of the n–1 product of a synthetic oligonucleotide. Nucleic Acids Res. 23, 18411844 (1995).
  34. Marras, S. A., Kramer, F. R. & Tyagi S. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30, e122 (2002).
  35. Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834838 (2005).
  36. Li, Q., Luan, G., Guo, Q. & Liang, J. A new class of homogeneous nucleic acid probe based on specific displacement hybridization. Nucleic Acids Res. 30, e5 (2002).
  37. Subramanian, H. K. K., Chakraborty, B., Sha, R. & Seeman, N. C. The label-free unambiguous detection and symbolic display of single nucleotide polymorphisms on DNA origami. Nano Lett. 11, 910913 (2010).
  38. 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).
  39. Kim, S. & Misra A. SNP genotyping: technologies and biomedical applications. Annu. Rev. Biomed. Eng. 9, 289320 (2007).
  40. Lizardi, P. M. et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nature Genet. 19, 225232 (1998).
  41. Isaacs, F. J. , et al. Engineered riboregulators enable post-transcriptional control of gene expression. Nature Biotechnol. 22, 841847 (2004).
  42. Venkataraman, S., Dirks, R. M., Ueda, C. T. & Pierce, N. Selective cell death mediated by small conditional RNAs. Proc. Natl Acad. Sci. USA 107, 1677716782 (2010).

Download references

Author information


  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    • David Yu Zhang &
    • Peng Yin
  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    • David Yu Zhang &
    • Peng Yin
  3. Department of Electrical Engineering, University of Washington, Seattle, Washington, USA

    • Sherry Xi Chen


D.Y.Z. conceived the project, designed and conducted the experiments, analysed the data and wrote the manuscript. S.X.C. conducted experiments, analysed the data and edited the manuscript. P.Y. conceived, designed and supervised the study, analysed the data and wrote the manuscript.

Competing financial interests

The authors have a patent pending on the methods described in the manuscript.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (747 KB)

    Supplementary information

Additional data