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Recombinant RNA technology: the tRNA scaffold

Nature Methods volume 4, pages 571–576 (2007)Cite this article

Abstract

RNA has emerged as a major player in most cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA have been in vitro syntheses. Here we describe a generic approach for expressing and purifying structured RNA in Escherichia coli, using tools that parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the 'tRNA scaffold', in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular RNases. This opens the way to large-scale structural and molecular investigations of RNA function.

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Figure 1: The tRNA scaffold strategy.
Figure 2: Using tRNA chimeras in structural studies.
Figure 3: Structures of the several RNA chimera that were successfully expressed using the tRNA scaffold strategy.
Figure 4: Expression and purification of recombinant RNA.
Figure 5: NMR analysis of RNA inserts cleaved with RNase H.

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Acknowledgements

This research was supported by the 6th framework program of the European Union (grant FSG-V-RNA). We thank F. Allemand (Institut de Biologie Physico-Chimique, Paris) for the kind gift of purified E. coli L20 protein and C. Tisné and S. Nonin for assistance with the NMR experiments.

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Authors and Affiliations

  1. Cristallographie & RMN Biologiques, Université Paris Descartes, CNRS, 4 avenue de l'Observatoire, Paris, 75006, France

    Luc Ponchon & Frédéric Dardel

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  1. Luc Ponchon
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  2. Frédéric Dardel
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Contributions

F.D. was responsible for project planning. L.P. performed the experiments. F.D. and L.P. discussed the results, drafted the paper and revised the manuscript.

Corresponding author

Correspondence to Frédéric Dardel.

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The authors declare no competing financial interests.

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Supplementary Figures 1-5, Supplementary Table 1. (PDF 1921 kb)

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Ponchon, L., Dardel, F. Recombinant RNA technology: the tRNA scaffold. Nat Methods 4, 571–576 (2007). https://doi.org/10.1038/nmeth1058

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