Figure 3: Modifying RNA structure in vitro and in vivo.

(A) In vitro, buffers that include magnesium ions (Mg²⁺) and a crowding agent and exclude the chelating agent EDTA, will support the formation of both strong RNA structures (structures with many base pairs) and weak RNA structures (fewer base pairs) at lower temperatures (left), whereas in the opposite buffer conditions or in higher temperature, the weaker RNA structures will unfold (middle). Extreme pH will denature and cause the degradation of even strong RNA structures (right). (B) In vitro transcription favours interactions at the 5’-end region, as this region is transcribed first. Melting and refolding of the in vitro transcribed RNA will allow and the formation of interactions between the 5’ and 3’ regions of the molecule. (C) In vivo, RNA structures are modulated by cellular RNAses, which actively remove particular RNA sequences and structures; by RNA helicases, which unwind double-stranded RNA; and by RNA-binding proteins, which can shield RNA sequences from RNA–RNA interaction or block structure formation. The cellular environment helps to explain the discrepancy between RNA-structure probing data obtained in vitro or from cells. This discrepancy may be explained also by RNA modifications (Figure 2).