Selective deuteration of an RNA:RNA complex for structural analysis using small-angle scattering

Area of Science:

• Structural Biology
• Biophysics
• Molecular Biology

Background:

• Protein-free RNA:RNA complexes are crucial for biological processes but are structurally underrepresented.
• Understanding RNA:RNA complex dynamics is essential for deciphering their functions.

Purpose of the Study:

• To develop and demonstrate a joint small-angle X-ray and neutron scattering (SAXS/SANS) approach for interrogating RNA:RNA complex structures.
• To validate and refine computational models like AlphaFold 3 using experimental scattering data.

Main Methods:

• Utilized SAXS to determine solution structures of individual RNAs and their complexes.
• Employed SANS with isotope labeling and contrast matching (CM) to probe selectively deuterated RNA:RNA complexes.
• Integrated in silico modeling with experimental SAXS/SANS data.

Main Results:

• Measured solution structures of free RNAs and the overall RNA:RNA complex using SAXS.
• Demonstrated SANS with CM and isotope labeling to analyze bound RNA structures within complexes.
• Showcased how experimental data can validate and improve AlphaFold 3 predictions for RNA:RNA complexes.

Conclusions:

• The combined SAXS/SANS approach, alongside in silico modeling, effectively analyzes conformational changes in RNA:RNA complexes.
• This integrated strategy enhances the understanding of RNA structure within functional assemblies.
• The method provides a powerful tool for studying dynamic RNA structures in solution.