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Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct,
Matthew J Smola1, Greggory M Rice1, Steven Busan1
1Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina, USA.
Nature Protocols
|October 2, 2015
Summary
This study presents SHAPE-MaP, a method for RNA structure analysis. It accurately measures RNA structure across entire transcriptomes using chemical probing and sequencing.
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- RNA structure plays a critical role in biological processes.
- Accurate, high-resolution RNA structure probing is essential for understanding gene regulation and function.
- Existing methods have limitations in scalability and resolution for transcriptome-wide analysis.
Purpose of the Study:
- To describe a robust protocol for Selective 2'-hydroxyl acylation analyzed by primer extension and Mutational Profiling (SHAPE-MaP).
- To enable accurate, single-nucleotide resolution measurement of RNA structure across large biological systems and entire transcriptomes.
- To provide automated software tools for data processing and RNA secondary structure modeling.
Main Methods:
- Utilizing SHAPE chemistry to probe RNA 2'-hydroxyl groups with small electrophilic reagents.
- Employing Mutational Profiling (MaP) with reverse transcriptase to detect modified nucleotides.
- Performing massively parallel sequencing to quantify mutations and determine SHAPE reactivities.
- Implementing automated data analysis using ShapeMapper and SuperFold software for profile generation and structure modeling.
Main Results:
- The SHAPE-MaP approach achieves accuracy comparable to model RNAs for large and transcriptome-wide systems.
- A 3-day protocol is detailed for SHAPE probing and library construction for deep sequencing.
- Automated software enables rapid processing of sequencing data, generation of reactivity plots, and secondary structure modeling.
- SHAPE-MaP facilitates biophysical measurements of RNA motifs, complex ensembles, and entire transcriptomes at nucleotide resolution.
Conclusions:
- SHAPE-MaP is a powerful, scalable method for high-resolution RNA structure determination.
- The protocol and associated software streamline the analysis of RNA structure from individual motifs to whole transcriptomes.
- This technique significantly advances the ability to study RNA structure-function relationships in complex biological contexts.
