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Systematic reconstruction of RNA functional motifs with high-throughput microfluidics

Nature Methods volume 9, pages 1192–1194 (2012)Cite this article

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Abstract

We present RNA–mechanically induced trapping of molecular interactions (RNA-MITOMI), a microfluidic platform that allows integrated synthesis and functional assays for programmable RNA libraries. The interaction of a comprehensive library of RNA mutants with stem-loop–binding protein precisely defined the RNA structural and sequence features that govern affinity. The functional motif reconstructed in a single experiment on our platform uncovers new binding specificities and enriches interpretation of phylogenetic data.

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Figure 1: RNA-MITOMI is a microfluidic platform for integrated synthesis and affinity measurement across a programmable RNA library.
Figure 2: SLBP binding landscape across the library of stem-loop sequence and structure variants.
Figure 3: Reconstruction and validation of the stem-loop functional motif.

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Acknowledgements

We thank R.C. Spitale and C. Chu for helpful discussions, J.C. Liang, R.J. Bloom and C.D. Smolke for helpful discussions and assistance with Biacore, and A. Clore of Integrated DNA Technologies for assistance with probe designs. L.M. was funded by the Department of Defense National Defense Science & Engineering Graduate Fellowship. Our work was supported by US National Institutes of Health R01-HG004361 (to H.Y.C.) and R01GM29832 (to W.F.M.). H.Y.C. and S.R.Q. are funded by the Howard Hughes Medical Institute.

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Author notes

  1. Matthias Meier

    Present address: Present address: Department of Microsystems Engineering and Center for Biological Signaling Studies, University of Freiburg, Freiburg, Germany.,

Authors and Affiliations

  1. Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Lance Martin & Howard Y Chang

  2. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Lance Martin, Matthias Meier, Rene V Sit, Stephen R Quake & Howard Y Chang

  3. Department of Bioengineering, Stanford University, Stanford, California, USA

    Lance Martin, Matthias Meier, Rene V Sit & Stephen R Quake

  4. Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, North Carolina, USA

    Shawn M Lyons & William F Marzluff

  5. Department of Applied Physics, Stanford University, Stanford, California, USA

    Stephen R Quake

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  1. Lance Martin
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Contributions

L.M., S.R.Q. and H.Y.C. conceived the study. L.M., M.M., S.M.L. and R.V.S. performed experiments. L.M., M.M., S.M.L., W.F.M., S.R.Q. and H.Y.C. discussed the results and analyzed the data. L.M. and H.Y.C. wrote the paper.

Corresponding authors

Correspondence to Stephen R Quake or Howard Y Chang.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10, Supplementary Tables 1–4, Supplementary Notes 1–2 (PDF 5979 kb)

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Martin, L., Meier, M., Lyons, S. et al. Systematic reconstruction of RNA functional motifs with high-throughput microfluidics. Nat Methods 9, 1192–1194 (2012). https://doi.org/10.1038/nmeth.2225

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