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.