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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
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RNA structure analyzed by chemical probing and mutational profiling with sequencing (MaPseq).

Eva Edelson1, Nicholas M Forino2, Michael D Stone3

  • 1Department of Microbiology & Environmental Toxicology, University of California, Santa Cruz, United States; UCSC RNA Center, University of California, Santa Cruz, United States.

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|December 3, 2025
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Summary

RNA chemical probing with mutational profiling read out by sequencing (MaPseq) reveals RNA folding properties. This protocol details methods for analyzing complex RNA structures and heterogeneity, crucial for understanding biological function.

Keywords:
DeconvolutionMutational profilingNext-generation sequencingRNA probingRNA structure

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genomics

Background:

  • RNA structure probing has advanced understanding of cellular RNA functions for decades.
  • Methods traditionally provided ensemble average structures, but newer techniques reveal heterogeneous folding populations.
  • RNA structural heterogeneity is critical for biological function.

Purpose of the Study:

  • To present detailed protocols for RNA chemical probing, library preparation, sequencing, and bioinformatic analysis.
  • To illustrate and compare experimental strategies using factor VIII pre-mRNA and human telomerase RNA.
  • To provide a flexible framework for investigating structural properties of diverse RNA molecules.

Main Methods:

  • RNA chemical probing combined with mutational profiling read out by sequencing (MaPseq).
  • Next-generation sequencing for accelerated workflows and analysis of complex RNA conformation mixtures.
  • Detailed bioinformatic analysis protocols.

Main Results:

  • Demonstrated the power of MaPseq for investigating complex RNA folding properties.
  • Successfully applied protocols to factor VIII pre-mRNA and human telomerase RNA.
  • Illustrated different experimental strategies for RNA structure analysis.

Conclusions:

  • MaPseq is a powerful approach for studying RNA folding and structural heterogeneity.
  • The presented protocols offer a flexible framework for RNA structure investigation.
  • Understanding RNA structural heterogeneity is key to elucidating RNA functions.