Effects of Refolding on Large-Scale RNA Structure

Area of Science:

• Molecular Biology
• Virology
• Structural Biology

Background:

• RNA structure is crucial for RNA-mediated functions.
• Structure probing experiments often use refolded RNA, which may not reflect native conformations.

Purpose of the Study:

• To compare the structure of Dengue virus (DENV) RNA in its native, ex virion state versus after heat denaturation and refolding.
• To evaluate the utility of a high-structure, low-entropy analysis framework for identifying native-like RNA structures.

Main Methods:

• Selective 2 omino-hydroxyl acylation analyzed by primer extension, read out by mutational profiling (SHAPE-MaP) was used to probe RNA structure.
• DENV RNA was analyzed in both its native ex virion state and after refolding.
• SHAPE reactivity and Shannon entropy were used to characterize RNA structural features.

Main Results:

• Refolded DENV RNA was more highly structured and sampled fewer conformations compared to ex virion RNA.
• Regions with low SHAPE reactivity and low Shannon entropy (lowSS regions) represent well-determined structures.
• The high-structure, low-entropy framework successfully identified structural elements in refolded RNA that likely represent native ex virion structures.

Conclusions:

• Refolding alters RNA conformation, leading to increased structure and reduced conformational sampling compared to native ex virion RNA.
• The high-structure, low-entropy analysis framework is effective for de novo identification of RNA structural elements that are likely to be present in the native state.
• Less well-defined structural regions, potentially involved in RNA switching, are more prevalent in native-like ex virion RNA and are difficult to recapitulate in refolded RNA.