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Protein–RNA footprinting: an evolving tool

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Abstract As more RNA molecules with important cellular functions are discovered, there is a strong need to characterize their structures, functions, and interactions. Chemical and enzymatic footprinting methods are used to map RNA secondary and tertiary structure, to monitor ligand interactions and conformational changes, and in the study of protein–RNA interactions. These methods provide data at single‐nucleotide resolution that nicely complements the structural information available from X‐ray diffraction, nuclear magnetic resonance spectroscopy (NMR), or cryo‐electron microscopy. Footprinting methods also complement the dynamic information derived from single‐molecule Förster resonance energy transfer. RNA footprinting tools have been used for decades, but we have recently seen spectacular advances, for instance, the use in combination with massive parallel sequencing techniques. Large libraries of RNA molecules (small or large in size) can now be probed in high‐throughput manner when RNA footprinting methods are combined with fluorescent probe technologies and automation. In this article, after a brief historical overview, we summarize recent advances in RNA–protein footprinting methodologies that now integrate tools for massive parallel analysis. WIREs RNA 2011, 3:557–566. doi: 10.1002/wrna.1119 This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes RNA Methods > RNA Analyses In Vitro and In Silico

Classification of RNA probes. The molecular weights are indicated in parentheses, as are specificities.

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Overview of the FragSeq and parallel analysis of RNA structure (PARS) methods.28,29 RNA molecules are extracted from cells, denatured, and renatured. Fragments that result from enzymatic probing (RNase V1, nuclease S1, or P1) are then analyzed by deep sequencing to determine the secondary structures of RNA molecules.

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Overview of SHAPE‐Seq. (a) Experimental pipeline. A DNA bar code is added to the 3′ end of template molecules, enabling SHAPE chemistry and subsequent sequencing to be performed on a mixture of bar‐coded RNAs. (b) Bioinformatics and analysis pipeline. The automated pipeline separates reads by handle pools and bar code and maps the reads onto RNA sequences. Raw read counts at each nucleotide position in the (+) and (−) channel are fed into a maximum likelihood (ML) estimation calculation to determine the reactivities at each nucleotide. Reactivities can be scaled and used in programs like RNA Structure90 to infer secondary structure from SHAPE‐Seq data. (Reprinted with permission from Ref 31. Copyright 2011 PNAS)

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The mechanism of RNA SHAPE chemistry with benzoyl cyanide (BzCN).85 BzCN reacts with 2′‐hydroxyl groups when the ribose is conformationally flexible to form a 2′‐O‐adduct.

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RNA Methods > RNA Analyses In Vitro and In Silico
RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Interactions with Proteins and Other Molecules > Protein–RNA Recognition

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