Simulation-guided DNA probe design for consistently ultraspecific hybridization


Hybridization of complementary sequences is one of the central tenets of nucleic acid chemistry; however, the unintended binding of closely related sequences limits the accuracy of hybridization-based approaches to analysing nucleic acids. Thermodynamics-guided probe design and empirical optimization of the reaction conditions have been used to enable the discrimination of single-nucleotide variants, but typically these approaches provide only an approximately 25-fold difference in binding affinity. Here we show that simulations of the binding kinetics are both necessary and sufficient to design nucleic acid probe systems with consistently high specificity as they enable the discovery of an optimal combination of thermodynamic parameters. Simulation-guided probe systems designed against 44 sequences of different target single-nucleotide variants showed between a 200- and 3,000-fold (median 890) higher binding affinity than their corresponding wild-type sequences. As a demonstration of the usefulness of this simulation-guided design approach, we developed probes that, in combination with PCR amplification, detect low concentrations of variant alleles (1%) in human genomic DNA.

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Figure 1: Detection of rare nucleic acid variants by competitive compositions.
Figure 2: Kinetic simulations of competitive compositions.
Figure 3: The X-Probe is a dissociative probe that conditionally fluoresces on hybridization to its DNA target.
Figure 4: Design workflow and experimental demonstration of competitive composition.
Figure 5: Summary of competitive composition experimental results on synthetic targets.
Figure 6: Competitive composition assays on PCR amplicons of human genomic DNA samples.


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This work was funded by the Rice University Department of Bioengineering start-up fund to D.Y.Z., a John S. Dunn award to D.Y.Z. and a National Institutes of Health Grant (EB015331) to D.Y.Z.

Author information

J.S.W. and D.Y.Z. conceived the project, performed the theoretical analysis, designed the experiments, conducted the experiments, analysed the data and wrote the paper.

Correspondence to David Yu Zhang.

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Competing interests

Two patents are pending on this work. J.S.W. and D.Y.Z. are equity holders of Searna Technologies, a startup aiming to commercialize the presented technology.

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Wang, J., Zhang, D. Simulation-guided DNA probe design for consistently ultraspecific hybridization. Nature Chem 7, 545–553 (2015) doi:10.1038/nchem.2266

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