The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.
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All sequencing datasets have been deposited in ArrayExpress under accession number E-MTAB-6427. Base-pairing prediction, structure prediction, and clustering data are available in the Supplementary Data files. Source data for Figs. 1 and 3b are available online. Additional data that support the findings of this study are available from the corresponding authors upon request. A step-by-step protocol is available as a Supplementary Protocol and will be provided as an open resource in Protocol Exchange42.
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The authors thank A. Kohl (Centre for Virus Research, University of Glasgow, Glasgow, UK) and L.J. Pena and R.F. França (Fiocruz Recife, Pernambuco, Brazil) for providing the PE243 ZIKV RNA used to generate the virus stock. We thank Y. Galanty and F.M. Martínez for assisting with CRISPR–Cas9 knockout; T.D. Domenico and W. Matsushima for collapsing UMIs; G. Sanguinetti for suggesting the RNA-folding strategy; A.M. Sharkey and L. Gardner (University of Cambridge, Cambridge, UK) for providing the JEG-3 cells; S. Moss for assisting with risk assessment; M.S. Diamond, T. Sweeney, A. Firth, D. Jordan, A. Zeisel, and members of the Miska group for their comments; and C. Flandoli for illustrations. This work was supported by Cancer Research UK (C13474/A18583 and C6946/A14492 to E.A.M.; award no. A17197 to J.C.M.), the Wellcome Trust (104640/Z/14/Z and 092096/Z/10/Z to E.A.M.; 207507 to G.K. and M.M.G.; Senior Fellowship in Basic Biomedical Science 207498/Z/17/Z to I.G.), the Human Frontier Science Program (HFSP; LT000558/2015 to O.Z.), the European Molecular Biology Organization (EMBO; ALTF1622-2014 to O.Z.), the Blavatnik Family Foundation (postdoctoral fellowship to O.Z.), the UK Medical Research Council (G.K. and M.M.G.), EMBL (core funding to J.C.M.), NIGMS (grants R01GM104475 and R01GM115649 to I.J.M., L.F.R.G., and J.S.-G.), City University of Hong Kong (projects 9610363 and 7200520 to C.K.K.), the Croucher Foundation (project 9500030 to C.K.K.), the Hong Kong RGC (projects 9048103 and 9054020 to C.K.K.), the NSFC (Excellent Young Scientist Fund 81522025 to C.-F.Q.), and the Academy of Medical Sciences, UK (Newton Advanced Fellowship to C.-F.Q.).