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Systematic reconstruction of RNA functional motifs with high-throughput microfluidics

Nature Methods volume 9, pages 11921194 (2012) | Download Citation

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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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.

Author information

Author notes

    • Matthias Meier

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

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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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stephen R Quake or Howard Y Chang.

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–10, Supplementary Tables 1–4, Supplementary Notes 1–2

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DOI

https://doi.org/10.1038/nmeth.2225

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