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Updated: May 12, 2025

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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
Published on: December 9, 2022
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Investigating the interplay between RNA structural dynamics and RNA chemical probing experiments
Ethan B Arnold1, Daniel Cohn1, Emma Bose1
1Department of Chemistry, New York University, 31 Washington Place, NY 10003, United States.
Nucleic Acids Research
|April 16, 2025
Summary
Chemical probes reveal complex RNA dynamics. Unexpected reactivity shifts with probe concentration suggest cooperative binding and can help infer nucleotide pairing interactions, impacting RNA structure.
Area of Science:
- Molecular Biology
- Biophysics
- Chemical Biology
Background:
- Small molecule chemical probes are essential for RNA structure determination.
- Recent simulations suggest cooperative RNA-probe binding influences reactivity.
- Understanding this relationship is key to accurate RNA structure analysis.
Purpose of the Study:
- To investigate the interplay between RNA structural dynamics and chemical probe reactivity.
- To explore how probe concentration affects nucleotide modification rates.
- To determine if observed reactivity shifts can infer RNA pairing interactions.
Main Methods:
- Utilized selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) chemical probing.
- Employed nuclear magnetic resonance (NMR) spectroscopy, including chemical exchange experiments.
- Analyzed RNA nucleotide reactivity across varying probe concentrations.
Main Results:
- NMR revealed high imino proton exchange rates in SHAPE-reactive, base-paired nucleotides.
- Observed unexpected shifts in nucleotide modification rates with increasing probe concentration.
- Demonstrated that some base-paired nucleotides become reactive at higher probe concentrations, correlating with complementary nucleotide changes.
Conclusions:
- RNA conformational dynamics intricately influence chemical probe reactivity.
- Cooperative binding effects can lead to non-linear reactivity trends.
- Harnessing concentration-dependent reactivity shifts may enable inference of RNA pairing interactions and structural ensembles.
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