Optimizing the specificity of nucleic acid hybridization

• Published in: Nature chemistry Vol. 4; no. 3; pp. 208 - 214
• Main Authors: Zhang, David Yu, Chen, Sherry Xi, Yin, Peng
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 22.01.2012; Nature Publishing Group
• Subjects: 631/1647/1888/1890; 631/1647/666; 639/638/45; 639/925/927; Acids; Analytical Chemistry; Base Pairing; Base Sequence; Biochemistry; Biology; Biotechnology; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - metabolism; Equilibrium; Humans; Hybridization; Inorganic Chemistry; Melting; Molecular Sequence Data; Nucleic Acid Hybridization - methods; Nucleic Acid Probes - chemistry; Nucleic Acid Probes - metabolism; Nucleic acids; Organic Chemistry; Physical Chemistry; Probes; RNA - chemistry; RNA - metabolism; Salinity; Substrate Specificity; Temperature; Thermodynamics; United States > US; Massachusetts
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/nchem.1246

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. High-fidelity pairing of nucleic acid polymers is important in the development of sensors and for the application of DNA nanotechnology. Here, a set of hybridization probes is described that discriminates single-base changes with high specificity. The probes function robustly across many different temperatures, salinities and nucleic acid concentrations.