Key result
Heat denaturation and refolding of Dengue virus RNA yields more highly structured conformations than native RNA.
Why the study?
RNA structure probing experiments are often performed on refolded RNA, which may cause conformation to differ from native RNA.
High-structure and low-entropy analysis allows de novo identification of structural elements in refolded RNA that likely recapitulate native ex virion RNA structures.
Highlights potential artifacts in RNA structure studies; leaves open functional impact of native Dengue conformations in vivo.
Understanding RNA-mediated functions requires a detailed characterization of the underlying RNA structure. In many cases, structure probing experiments are performed on RNA that has been "refolded" in some way, which may cause the conformation to differ from that of the native RNA. We used SHAPE-MaP (selective 2'-hydroxyl acylation analyzed by primer extension, read out by mutational profiling), to probe the structure of the Dengue virus (DENV) RNA genome after gentle extraction of the native RNA from intact virions (ex virion) and after heat denaturation and refolding. Comparison of mutiple SHAPE-informed structural features revealed that refolded RNA is more highly structured and samples fewer conformations than the ex virion RNA. Regions with similar structural features are generally those with low SHAPE reactivity and low Shannon entropy (lowSS regions), which correspond to elements with high levels of well-determined structure. This high-structure and low-entropy analysis framework, previously shown to make possible discovery of functional RNA structures, is thus now shown to allow de novo identification of structural elements in a refolded RNA that are likely to recapitulate RNA structures in the ex virion RNA state. Regions with less well-defined structures, which occurred more frequently in the more native-like ex virion RNA and may contain RNA switches, are challenging to recapitulate using refolded RNA.
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Dethoff et al. (2019) studied Dengue virus (DENV) RNA genome. Heat denaturation and refolding vs. Native RNA from intact virions (ex virion) was evaluated on SHAPE-informed structural features. Heat denaturation and refolding of Dengue virus RNA resulted in a more highly structured conformation that sampled fewer conformations compared to native ex virion RNA.
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