Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq)

Area of Science:

• Molecular Biology
• Biochemistry
• Genomics

Background:

• Selective 2 -hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a key method for RNA structure determination.
• SHAPE reagents modify structurally flexible RNA nucleotides, enabling reactivity mapping.
• Previous methods required sequence-specific priming, limiting broader applications.

Purpose of the Study:

• To present an updated protocol for in vitro RNA structure probing using SHAPE chemistry coupled with next-generation sequencing (SHAPE-Seq v2.0).
• To enable high-throughput, consistent, and multiplexed RNA structure analysis.
• To facilitate sequence-independent RNA structural analysis.

Main Methods:

• In vitro RNA structure probing using SHAPE reagents.
• Quantification of modified RNA via reverse transcriptase (RT) primer extension.
• Next-generation sequencing (NGS) to analyze cDNA pools and determine nucleotide reactivities.
• Ligation of a universal reverse transcription priming site to modified RNA.

Main Results:

• SHAPE-Seq v2.0 provides high-throughput and consistent estimation of RNA nucleotide reactivities.
• The protocol allows for RNA structure analysis independent of its specific sequence.
• NGS enables accurate quantification of cDNA pools, reflecting SHAPE modification sites.

Conclusions:

• SHAPE-Seq v2.0 is a powerful and versatile tool for detailed in vitro RNA structure mapping.
• The sequence-independent priming site significantly enhances the applicability of SHAPE-Seq.
• This method advances the characterization of RNA structure in diverse biological contexts.