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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

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Combining Biophysical Methods for Structure-Function Analyses of RNA in Solution.

Yun-Tzai Lee¹, Lixin Fan², Jienyu Ding¹

¹Protein-Nucleic Acid Interaction Section, Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)

|October 13, 2022

Summary

This summary is machine-generated.

This study explores riboswitch RNA regulation using a hybrid approach. Combining isothermal titration calorimetry, small-angle X-ray scattering, and atomic force microscopy offers a comprehensive analysis of ligand-induced conformational changes.

Keywords:

Atomic force microscopy Isothermal titration calorimetry RNA structure and dynamics Riboswitch Small-angle x-ray scattering

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Area of Science:

Biochemistry
Biophysics
Molecular Biology

Background:

Riboswitches regulate gene expression through metabolite binding to RNA aptamers.
Ligand binding induces conformational changes affecting transcription or translation.
Studying these dynamic RNA molecules requires multifaceted biophysical approaches.

Purpose of the Study:

To outline procedures for the biochemical and biophysical characterization of RNA.
To highlight the utility of hybrid methods for studying riboswitches.
To elucidate ligand-induced conformational changes in RNA regulatory mechanisms.

Main Methods:

Utilizing a combination of solution-based biochemical and biophysical techniques.
Employing isothermal titration calorimetry (ITC) for thermodynamic analysis.
Applying small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) for structural insights.

Main Results:

Demonstrating the synergistic power of combining ITC, SAXS, and AFM.
Providing a semi-quantitative assessment of the thermodynamics of ligand binding.
Characterizing conformational changes in RNA aptamers upon metabolite interaction.

Conclusions:

Hybrid biophysical methods are essential for comprehensive riboswitch characterization.
Integrated approaches yield deeper insights into RNA regulatory mechanisms.
This study provides a framework for analyzing ligand-induced RNA dynamics.