利用分裂西兰花RNA报告细胞在 体外 RNA簇中分子间RNA碱基配对的可视化与定量

Overview

This article presents a protocol for directly detecting and quantifying intermolecular base pairing in RNA clusters in vitro using a split Broccoli RNA reporter assay. The method leverages fluorescently labeled RNAs and a split aptamer system to visualize and measure RNA–RNA interactions, providing a quantitative alternative to chemical probing and computational predictions.

Key Study Components

Area of Science

• Molecular biology
• RNA biochemistry
• Fluorescence microscopy

Background

• RNA clustering is driven by multivalent intermolecular RNA–RNA interactions.
• Traditional methods like chemical probing and computational simulations infer but do not directly measure these interactions.
• Direct assessment of intermolecular base pairing within RNA clusters has been limited.
• The split Broccoli RNA aptamer system enables fluorescence-based detection of RNA dimerization.

Purpose of Study

• To develop and demonstrate a visual assay for direct detection of intermolecular base pairing in RNA clusters.
• To quantify how in vitro clustering conditions affect RNA–RNA interactions.
• To provide a complementary approach to existing RNA interaction assays.

Methods Used

• Preparation of DNA templates and in vitro transcription of RNAs labeled with Cy5.
• Purification of transcribed RNAs using phenol-chloroform extraction and ethanol precipitation.
• Folding of RNAs under co-folding and separate folding conditions with crowding reagents (spermine, PEG 8000).
• Assembly of split Broccoli RNA reporters and addition of DFHBI-1T fluorophore.
• Fluorescence imaging using confocal microscopy and quantitative image analysis.

Main Results

• DFHBI-1T fluorescence was substantially higher in co-folded RNA samples compared to separately folded or single RNA controls.
• Co-folding of complementary split Broccoli RNAs resulted in up to 82.6-fold increases in fluorescence, indicating robust intermolecular base pairing.
• Mixed orientation pairs (shu-top with shu-anti-bottom, etc.) showed the highest fluorescence increases upon co-folding.
• Robust signal generation depended on crowding reagents and efficient RNA dimerization.

Conclusions

• The split Broccoli RNA reporter assay enables direct, quantitative assessment of intermolecular base pairing in RNA clusters.
• This method complements traditional RNA interaction assays such as pull-downs and gel electrophoresis.
• The assay can be used to study the effects of RNA sequence, helicases, and ions on RNA–RNA interactions in vitro.

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