Optimizing the specificity of nucleic acid hybridization
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...
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| Published in | Nature chemistry Vol. 4; no. 3; pp. 208 - 214 |
|---|---|
| Main Authors | , , |
| Format | Journal Article |
| Language | English |
| Published |
London
Nature Publishing Group UK
22.01.2012
Nature Publishing Group |
| Subjects | |
| Online Access | Get full text |
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| 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.
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. |
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| AbstractList | 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. 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. 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 degree C to 37 degree C, from 1 mM Mg super(2+) to 47 mM Mg super(2+), and with nucleic acid concentrations from 1 nM to 5 mu M. Experiments with RNA also showed effective single-base change discrimination. 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. 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.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. |
| Author | Yin, Peng Chen, Sherry Xi Zhang, David Yu |
| AuthorAffiliation | 3 Department of Electrical Engineering, University of Washington, Seattle, Washington, USA 2 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA 1 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA |
| AuthorAffiliation_xml | – name: 2 Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA – name: 1 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA – name: 3 Department of Electrical Engineering, University of Washington, Seattle, Washington, USA |
| Author_xml | – sequence: 1 givenname: David Yu surname: Zhang fullname: Zhang, David Yu email: david.zhang@wyss.harvard.edu organization: Department of Systems Biology, Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Harvard University – sequence: 2 givenname: Sherry Xi surname: Chen fullname: Chen, Sherry Xi organization: Department of Electrical Engineering, University of Washington – sequence: 3 givenname: Peng surname: Yin fullname: Yin, Peng email: py@hms.harvard.edu organization: Department of Systems Biology, Harvard Medical School, Wyss Institute for Biologically Inspired Engineering, Harvard University |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/22354435$$D View this record in MEDLINE/PubMed |
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| Title | Optimizing the specificity of nucleic acid hybridization |
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