Effects of Refolding on Large-Scale RNA Structure

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.