title: Optimizing the specificity of nucleic acid hybridization.
abstract: 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 Mg(2+) to 47 mM Mg(2+), and with nucleic acid concentrations from 1 nM to 5 µM. Experiments with RNA also showed effective single-base change discrimination.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. david.zhang@wyss.harvard.edu
authors.forename: David Yu
authors.initials: DY
authors.lastname: Zhang
authors.forename: Sherry Xi
authors.initials: SX
authors.lastname: Chen
authors.forename: Peng
authors.initials: P
authors.lastname: Yin
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015341:Nucleic Acid Probes / Q000737:chemistry* / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D015341:Nucleic Acid Probes; D012313:RNA; D004247:DNA