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RNA Secondary Structure Prediction Using High-throughput SHAPE
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In-cell RNA structure probing with SHAPE-MaP.

Matthew J Smola1, Kevin M Weeks1

  • 1Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Nature Protocols
|May 5, 2018
PubMed
Summary
This summary is machine-generated.

This study extends the Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) protocol for analyzing RNA structures in live cells. This method provides nucleotide-resolution RNA structure information, crucial for understanding RNA function and interactions.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • RNA structure is critical for its function, processing, and interactions with proteins and ligands.
  • Existing methods like in vitro SHAPE-MaP provide nucleotide-resolution RNA structure data.
  • Analyzing RNA structure within live cells is essential for understanding cellular processes.

Purpose of the Study:

  • To extend the in vitro SHAPE-MaP protocol for application in live cells.
  • To provide detailed guidance for performing and analyzing in-cell SHAPE-MaP experiments.
  • To enable the study of RNA structure in low-to-moderate abundance cellular RNAs.

Main Methods:

  • In-cell SHAPE-MaP combines chemical probing of RNA 2 eal;-hydroxyl groups with mutational profiling via reverse transcription and sequencing.
  • The protocol is adaptable to various cell types, from bacteria to mammalian cells.
  • It can be performed in approximately 3 days and is compatible with multiple probing reagents.

Main Results:

  • The in-cell SHAPE-MaP strategy successfully quantifies RNA structure at single-nucleotide resolution within living cells.
  • This method is effective for both abundant and low-to-moderate abundance cellular RNAs.
  • The protocol facilitates the identification of sequence and structure motifs involved in RNA-protein interactions.

Conclusions:

  • In-cell SHAPE-MaP is a powerful tool for investigating RNA structure and function in a native cellular environment.
  • This technique can generate new biological hypotheses by revealing RNA structural dynamics.
  • It enhances our understanding of RNA-mediated cellular processes and molecular interactions.