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Continuously tunable nucleic acid hybridization probes

Nature Methods volume 12, pages 1191–1196 (2015)Cite this article

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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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Figure 1: Tuning the affinity-selectivity tradeoff for hybridization probes.
Figure 2: Stoichiometric tuning for capture-yield uniformity.
Figure 3: Optimizing SNV discrimination using stoichiometric tuning.
Figure 4: Independent stoichiometric tuning in multiplex assays.
Figure 5: Multiplexed RNA quantitation with the Nanostring nCounter.

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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.

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Author notes

  1. Lucia R Wu and Juexiao Sherry Wang: These authors contributed equally to this work.

Authors and 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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  1. Lucia R Wu
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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.

Corresponding author

Correspondence to David Yu Zhang.

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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.

Supplementary information

Supplementary Text and Figures

Supplementary Notes 1–15 (PDF 12845 kb)

Supplementary Software

NABLab Probe Designer manual and Matlab source code (PDF 701 kb)

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Wu, L., Wang, J., Fang, J. et al. Continuously tunable nucleic acid hybridization probes. Nat Methods 12, 1191–1196 (2015). https://doi.org/10.1038/nmeth.3626

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