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Designing and using RNA scaffolds to assemble proteins in vivo

Nature Protocols volume 7, pages 1797–1807 (2012)Cite this article

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Abstract

RNA scaffolds are synthetic noncoding RNA molecules with engineered 3D folding harnessed to spatially organize proteins in vivo. Here we provide a protocol to design, express and characterize RNA scaffolds and their cognate proteins within 1 month. The RNA scaffold designs described here are based on either monomeric or multimeric units harboring RNA aptamers as protein docking sites. The scaffolds and proteins are cloned into inducible plasmids and expressed to form functional assemblies. RNA scaffolds find applications in many fields in which in vivo organization of biomolecules is of interest. RNA scaffolds provide extended flexibility compared with DNA or protein scaffolding strategies through programmed modulation of multiple protein stoichiometry and numbers, as well as the proteins' relative distances and spatial orientations. For synthetic biology, RNA scaffolds provide a new platform that can be used to increase yields of sequential metabolic pathways.

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Figure 1: General workflow for design, induction, expression and experimental testing of the RNA scaffold.
Figure 2: Designing and optimizing RNA scaffolds using RNA Designer.
Figure 3: Compatible aptamer pair.
Figure 4: Examples of RNA scaffold folding scheme designs.
Figure 5: Modular cloning approach to the design of RNA scaffolds.
Figure 6: Example of RNA Designer input and output sequences and structures.

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Acknowledgements

We are indebted to F. Aldaye who had a leading role in this work. This work was supported by the Enerbio-Tuck Foundation and the Institut Français du Pétrole Energies Nouvelles (to C.J.D.); by the Agence Nationale de la Recherche France, Institut National de la Santé et de la Recherche Médicale (Unité 1001)–Institut National de Recherche en Informatique et en Automatique projet d'envergure, and an Axa Foundation Chair on Longevity (to A.B.L.); and by the Wyss Institute for Biologically Inspired Engineering and support from the Department of the Army W911NF-09-1-00226 (to P.A.S.).

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Authors and Affiliations

  1. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Camille J Delebecque & Pamela A Silver

  2. Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA

    Camille J Delebecque & Pamela A Silver

  3. Institut National de la Santé et de la Recherche Médicale, Paris, France

    Camille J Delebecque & Ariel B Lindner

  4. Faculty of Medicine, Paris Descartes University, Paris, France

    Camille J Delebecque & Ariel B Lindner

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  1. Camille J Delebecque
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All authors participated extensively in developing the protocol described in this paper.

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Correspondence to Pamela A Silver or Ariel B Lindner.

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Delebecque, C., Silver, P. & Lindner, A. Designing and using RNA scaffolds to assemble proteins in vivo. Nat Protoc 7, 1797–1807 (2012). https://doi.org/10.1038/nprot.2012.102

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