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A two-dimensional mutate-and-map strategy for non-coding RNA structure

Nature Chemistry volume 3, pages 954–962 (2011)Cite this article

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Abstract

Non-coding RNAs fold into precise base-pairing patterns to carry out critical roles in genetic regulation and protein synthesis, but determining RNA structure remains difficult. Here, we show that coupling systematic mutagenesis with high-throughput chemical mapping enables accurate base-pair inference of domains from ribosomal RNA, ribozymes and riboswitches. For a six-RNA benchmark that has challenged previous chemical/computational methods, this ‘mutate-and-map’ strategy gives secondary structures that are in agreement with crystallography (helix error rates, 2%), including a blind test on a double-glycine riboswitch. Through modelling of partially ordered states, the method enables the first test of an interdomain helix-swap hypothesis for ligand-binding cooperativity in a glycine riboswitch. Finally, the data report on tertiary contacts within non-coding RNAs, and coupling to the Rosetta/FARFAR algorithm gives nucleotide-resolution three-dimensional models (helix root-mean-squared deviation, 5.7 Å) of an adenine riboswitch. These results establish a promising two-dimensional chemical strategy for inferring the secondary and tertiary structures that underlie non-coding RNA behaviour.

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Figure 1: The mutate-and-map method gives an information-rich picture of RNA structure.
Figure 2: Mutate-and-map data and secondary structure.
Figure 3: Comparison of chemical/computational modelling approaches on tRNAphe.
Figure 4: Accurate secondary structure models for non-coding RNAs.
Figure 5: Two states of a glycine-binding riboswitch.
Figure 6: Three-dimensional modelling from mutate-and-map data.

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Acknowledgements

The authors thank A. Laederach and J. Lucks for comments on the manuscript and the authors of RNAstructure for making their source code freely available. This work was supported by the Burroughs-Wellcome Foundation (CASI to R.D.), the National Institutes of Health (T32 HG000044 to C.C.V.) and a Stanford Graduate Fellowship (to P.C.).

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Authors and Affiliations

  1. Department of Biochemistry, Stanford University, Stanford, 94305, California, USA

    Wipapat Kladwang & Rhiju Das

  2. Department of Chemical Engineering, Stanford University, Stanford, 94305, California, USA

    Christopher C. VanLang

  3. Program in Biomedical Informatics, Stanford University, Stanford, 94305, California, USA

    Pablo Cordero & Rhiju Das

  4. Department of Physics, Stanford University, Stanford, 94305, California, USA

    Rhiju Das

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  1. Wipapat Kladwang
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Contributions

R.D. conceived and designed the experiments. W.K., C.C.V. and R.D. performed the experiments. C.C.V., P.C. and R.D. analysed the data. R.D. wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Rhiju Das.

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Kladwang, W., VanLang, C., Cordero, P. et al. A two-dimensional mutate-and-map strategy for non-coding RNA structure. Nature Chem 3, 954–962 (2011). https://doi.org/10.1038/nchem.1176

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