title: Simulation-guided DNA probe design for consistently ultraspecific hybridization.
abstract: 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.
authors.affiliation: 1] Systems, Synthetic, and Physical Biology, Rice University, Houston, Texas 77030, USA [2] Department of Bioengineering, Rice University, Houston, Texas 77030, USA.
authors.forename: Juexiao Sherry
authors.initials: JS
authors.lastname: Wang
authors.affiliation: 1] Systems, Synthetic, and Physical Biology, Rice University, Houston, Texas 77030, USA [2] Department of Bioengineering, Rice University, Houston, Texas 77030, USA.
authors.forename: David Yu
authors.identifier: 0000000202137663
authors.initials: DY
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D015342:DNA Probes / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D015342:DNA Probes