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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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.
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). https://doi.org/10.1038/nchem.1246
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