SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a key method for RNA secondary structure determination.
• Current SHAPE methods require normalization to a no-reagent control to mitigate background signals from reverse transcriptase premature termination.

Purpose of the Study:

• To introduce a novel SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA).
• To demonstrate the efficacy of SHAPES in reducing background noise and enriching probed RNAs for improved RNA structure analysis.

Main Methods:

• Development and application of the SHAPES reagent (NPIA) for RNA structure probing.
• Utilizing SHAPES for covalent coupling of the reagent to a biotin molecule.
• Employing streptavidin bead-based selection of cDNA-RNA hybrids to remove background signal.

Main Results:

• SHAPES-based selection significantly reduces background signal in SHAPE probing data.
• Sequencing-based SHAPES data retain the same RNA structure information as conventional SHAPE methods.
• The SHAPES strategy demonstrates efficient enrichment of probed RNAs.

Conclusions:

• The SHAPES reagent offers an effective solution for background reduction in RNA structure probing.
• SHAPES is particularly advantageous for applications with high background and low probing signals, such as in vivo RNA structure analysis.