Single-molecule correlated chemical probing of RNA

Significance RNA molecules function as the central conduit of information transfer in biology. To do this, they encode information both in their sequences and in their higher-order structures. Understanding the higher-order structure of RNA remains challenging. In this work we devise a simple, exper...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2014-09, Vol.111 (38), p.13858-13863
Main Authors: Homan, Philip J., Favorov, Oleg V., Lavender, Christopher A., Kursun, Olcay, Ge, Xiyuan, Busan, Steven, Dokholyan, Nikolay V., Weeks, Kevin M.
Format: Article
Language:English
Subjects:
Adducts
Biological Sciences
Correlation analysis
Escherichia coli - chemistry
Gene expression
Genetic mutation
Geobacillus stearothermophilus - chemistry
Ligands
Models, Molecular
Molecular structure
Molecules
Mutation
Nucleic Acid Conformation
Nucleotides
Reactivity
Ribonucleic acid
RNA
RNA, Bacterial - chemistry
RNA, Protozoan - chemistry
Sequencing
Spectroscopic analysis
Tetrahymena - chemistry
Three dimensional modeling
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Summary:Significance RNA molecules function as the central conduit of information transfer in biology. To do this, they encode information both in their sequences and in their higher-order structures. Understanding the higher-order structure of RNA remains challenging. In this work we devise a simple, experimentally concise, and accurate approach for examining higher-order RNA structure by converting widely used massively parallel sequencing into an easily implemented single-molecule experiment for detecting through-space interactions and multiple conformations. We then use this experiment to analyze higher-order RNA structure, detect biologically important hidden states, and refine accurate three-dimensional structure models. Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify through-space interactions, create 3D models for RNAs spanning 80–265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1407306111