Optimizing the specificity of nucleic acid hybridization

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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.

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Figure 1: Hybridization specificity of nucleic acids.
Figure 2: Toehold exchange probes.
Figure 3: Experimental demonstration of toehold exchange probes.
Figure 4: Results for additional DNA and RNA targets.
Figure 5: Performance of the 7/5 probe for the X1 target at different conditions.


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The authors thank M. Dai and P-S. Loh for assistance with mathematical analysis and J. Aliperti, E. Haney, R. Jungmann and T. Schaus for helpful suggestions during manuscript preparation. This work was funded by a Wyss Institute for Biologically Inspired Engineered faculty start-up fund, an NIH Director's New Innovator Award (1DP2OD007292), an NSF CAREER Award (CCF1054898) and an Office of Naval Research grant (N000141010827) to P.Y. D.Y.Z. is a Howard Hughes Medical Institute postdoctoral fellow, as part of the Life Sciences Research Foundation programme. There is a patent pending on the methods described in this work.

Author information

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.

Correspondence to David Yu Zhang or Peng Yin.

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

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Zhang, D., Chen, S. & Yin, P. Optimizing the specificity of nucleic acid hybridization. Nature Chem 4, 208–214 (2012) doi:10.1038/nchem.1246

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