Article

Continuously tunable nucleic acid hybridization probes

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Abstract

In silico–designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0–100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

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References

  1. 1.

    et al. Target-enrichment strategies for next-generation sequencing. Nat. Methods 7, 111–118 (2010).

  2. 2.

    et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503–2516 (1989).

  3. 3.

    et al. Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping. J. Mol. Diagn. 15, 171–176 (2013).

  4. 4.

    A decade's perspective on DNA sequencing technology. Nature 470, 198–203 (2011).

  5. 5.

    & The expanding scope of DNA sequencing. Nat. Biotechnol. 30, 1084–1094 (2012).

  6. 6.

    1000 Genomes Project Consortium. et al. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).

  7. 7.

    , & Thermodynamic optimization of nucleic acid hybridization specificity. Nat. Chem. 4, 208–214 (2012).

  8. 8.

    & Simulation-guided probe design for consistently ultraspecific hybridization. Nat. Chem. 7, 545–553 (2015).

  9. 9.

    , , & Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Natl. Acad. Sci. USA 96, 6171–6176 (1999).

  10. 10.

    , , , & Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry 47, 5336–5353 (2008).

  11. 11.

    et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 40, e115 (2012).

  12. 12.

    et al. Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res. 20, 1420–1431 (2010).

  13. 13.

    et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat. Med. 20, 1479–1484 (2014).

  14. 14.

    , , & Accurate SHAPE-directed RNA structure determination. Proc. Natl. Acad. Sci. USA 106, 97–102 (2009).

  15. 15.

    & The thermodynamics of DNA structural motifs. Annu. Rev. Biophys. Biomol. Struct. 33, 415–440 (2004).

  16. 16.

    & Measuring the thermodynamics of RNA secondary structure formation. Biopolymers 44, 309–319 (1997).

  17. 17.

    Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

  18. 18.

    et al. NUPACK: analysis and design of nucleic acid systems. J. Comput. Chem. 32, 170–173 (2011).

  19. 19.

    et al. Performance comparison of exome DNA sequencing technologies. Nat. Biotechnol. 29, 908–914 (2011).

  20. 20.

    & SNP genotyping: technologies and biomedical applications. Annu. Rev. Biomed. Eng. 9, 289–320 (2007).

  21. 21.

    & Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

  22. 22.

    et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat. Biotechnol. 26, 317–325 (2008).

  23. 23.

    et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 39, D945–D950 (2011).

  24. 24.

    & Nucleic acid hybridization: robust sequence discrimination. Nat. Chem. 4, 155–157 (2012).

  25. 25.

    & Control of DNA strand displacement kinetics using toehold exchange. J. Am. Chem. Soc. 131, 17303–17314 (2009).

  26. 26.

    et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat. Biotechnol. 27, 182–189 (2009).

  27. 27.

    , & Multifunctional encoded particles for high-throughput biomolecule analysis. Science 315, 1393–1396 (2007).

  28. 28.

    et al. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. Nat. Chem. 7, 569–575 (2015).

  29. 29.

    , , , & Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor. Anal. Chem. 77, 6976–6984 (2005).

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Acknowledgements

The authors thank J.H. Bae for assisting with qPCR instrument correction. This work was partially funded by the Rice University Department of Bioengineering startup fund (to D.Y.Z.), the US National Institutes of Health (grant EB015331 to D.Y.Z.) and the Nanostring Technologies R&D team.

Author information

Author notes

    • Lucia R Wu
    •  & Juexiao Sherry Wang

    These authors contributed equally to this work.

Affiliations

  1. Department of Bioengineering, Rice University, Houston, Texas, USA.

    • Lucia R Wu
    • , Juexiao Sherry Wang
    • , John Z Fang
    • , Emily R Evans
    • , Alessandro Pinto
    •  & David Yu Zhang
  2. Systems, Synthetic, and Physical Biology, Rice University, Houston, Texas, USA.

    • Juexiao Sherry Wang
    •  & David Yu Zhang
  3. Nanostring Technologies, Seattle, Washington, USA.

    • Irena Pekker
    • , Richard Boykin
    • , Celine Ngouenet
    • , Philippa J Webster
    •  & Joseph Beechem

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Contributions

L.R.W., J.S.W. and D.Y.Z. conceived the project and performed theoretical analysis on stoichiometric tuning. L.R.W., J.S.W., E.R.E., J.Z.F., A.P., I.P., R.B., C.N., P.J.W. and D.Y.Z. designed and conducted experiments. L.R.W., J.S.W., J.Z.F., A.P., I.P., R.B., C.N., P.J.W., J.B. and D.Y.Z. analyzed the data. L.R.W., J.S.W. and D.Y.Z. wrote the paper with input from all authors.

Competing interests

There are two patents pending on this work (PCT/US14/52827 and US provisional 62/148,555). J.S.W. and D.Y.Z. are significant equity holders of Searna Technologies. I.P., R.B., C.N., P.J.W. and J.B. are employees of Nanostring Technologies.

Corresponding author

Correspondence to David Yu Zhang.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Notes 1–15

  2. 2.

    Supplementary Software

    NABLab Probe Designer manual and Matlab source code