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High-throughput cellular RNA device engineering

Nature Methods volume 12, pages 989–994 (2015)Cite this article

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Abstract

Methods for rapidly assessing sequence-structure-function landscapes and developing conditional gene-regulatory devices are critical to our ability to manipulate and interface with biology. We describe a framework for engineering RNA devices from preexisting aptamers that exhibit ligand-responsive ribozyme tertiary interactions. Our methodology utilizes cell sorting, high-throughput sequencing and statistical data analyses to enable parallel measurements of the activities of hundreds of thousands of sequences from RNA device libraries in the absence and presence of ligands. Our tertiary-interaction RNA devices performed better in terms of gene silencing, activation ratio and ligand sensitivity than optimized RNA devices that rely on secondary-structure changes. We applied our method to build biosensors for diverse ligands and determine consensus sequences that enable ligand-responsive tertiary interactions. These methods advance our ability to develop broadly applicable genetic tools and to elucidate the underlying sequence-structure-function relationships that empower rational design of complex biomolecules.

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Figure 1: High-throughput RNA device engineering method.
Figure 2: GFP/mCherry activity ratios (μ) for all members of the theophylline aptamer libraries based on FACS-seq assays.
Figure 3: Validation of theophylline-responsive tertiary-interaction switches identified through the FACS-seq method.
Figure 4: Extension of the FACS-seq method to identifying tertiary-interaction switches for other aptamer-target pairs.

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Acknowledgements

We thank M. McKeague and C. Schmidt for valuable feedback in the preparation of this manuscript; and C. Crumpton, M. Bigos and B. Gomez of the Stanford Shared FACS facility. This work was supported by funds from the US National Institutes of Health (grants GM086663, AT007886 to C.D.S. and Shared Instrumentation Grant S10RR025518-01), Defense Advanced Research Projects Agency (grant HR0011-11-2-0002 to C.D.S.), the Human Frontiers Science Program (grant RGP0054/2013 to C.D.S.), Natural Sciences and Engineering Research Council of Canada (fellowship to A.B.K.) and Agency for Science, Technology and Research (fellowship to J.S.X.).

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Author notes

  1. Andrew B Kennedy

    Present address: Present address: Agilent Technologies, Santa Clara, California, USA.,

  2. Brent Townshend and Andrew B Kennedy: These authors contributed equally to this work; ordering chosen at random.

Authors and Affiliations

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

    Brent Townshend, Andrew B Kennedy, Joy S Xiang & Christina D Smolke

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Contributions

A.B.K. and B.T. have contributed equally to this work. The author order was chosen at random. A.B.K., B.T. and C.D.S. conceived the project and wrote the manuscript. A.B.K., B.T. and J.S.X. conducted the experiments. B.T. developed the software to analyze the cytometry and NGS data. B.T., A.B.K., J.S.X. and C.D.S. designed the experiments and analyzed the results.

Corresponding author

Correspondence to Christina D Smolke.

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Competing interests

A.B.K. and C.D.S. are named on a pending patent application relating to RNA devices with ligand-responsive tertiary interactions (US application serial no. 62/186,767).

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Townshend, B., Kennedy, A., Xiang, J. et al. High-throughput cellular RNA device engineering. Nat Methods 12, 989–994 (2015). https://doi.org/10.1038/nmeth.3486

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