Direct Mapping of Higher-Order RNA Interactions by SHAPE-JuMP

  • Biochemistry. 2021 Jun 29;60(25):1971-1982. doi: 10.1021/acs.biochem.1c00270.
Thomas W Christy  1  2 ,  Catherine A Giannetti  1 ,  Gillian Houlihan  3 ,  Matthew J Smola  1 ,  Greggory M Rice  1 ,  Jian Wang  4 ,  Nikolay V Dokholyan  4  5 ,  Alain Laederach  6 ,  Philipp Holliger  3 ,  Kevin M Weeks  1
Affiliations
  • 1. Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, United States.
  • 2. Curriculum in Bioinformatics and Computational Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
  • 3. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, U.K.
  • 4. Department of Pharmacology and Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, Pennsylvania 17033, United States.
  • 5. Department of Chemistry and Department of Biomedical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.
  • 6. Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
PMID: 34121404 DOI: 10.1021/acs.biochem.1c00270
Abstract

Higher-order structure governs function for many RNAs. However, discerning this structure for large RNA molecules in solution is an unresolved challenge. Here, we present SHAPE-JuMP (selective 2'-hydroxyl acylation analyzed by primer extension and juxtaposed merged pairs) to interrogate through-space RNA tertiary interactions. A bifunctional small molecule is used to chemically link proximal nucleotides in an RNA structure. The RNA cross-link site is then encoded into complementary DNA (cDNA) in a single, direct step using an engineered Reverse Transcriptase that "jumps" across cross-linked nucleotides. The resulting cDNAs contain a deletion relative to the native RNA sequence, which can be detected by Sequencing, that indicates the sites of cross-linked nucleotides. SHAPE-JuMP measures RNA tertiary structure proximity concisely across large RNA molecules at nanometer resolution. SHAPE-JuMP is especially effective at measuring interactions in multihelix junctions and loop-to-helix packing, enables modeling of the global fold for RNAs up to several hundred nucleotides in length, facilitates ranking of structural models by consistency with through-space restraints, and is poised to enable solution-phase structural interrogation and modeling of complex RNAs.

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