Abstract
There are large differences between the cellular environment and the conditions widely used to study RNA in vitro. SHAPE RNA structure probing in Escherichia coli cells has shown that the cellular environment stabilizes both long-range and local tertiary interactions in the adenine riboswitch aptamer domain. Synthetic crowding agents are widely used to understand the forces that stabilize RNA structure and in efforts to recapitulate the cellular environment under simplified experimental conditions. Here, we studied the structure and ligand binding ability of the adenine riboswitch in the presence of the macromolecular crowding agent, polyethylene glycol (PEG). Ethylene glycol and low-molecular mass PEGs destabilized RNA structure and caused the riboswitch to sample secondary structures different from those observed in simple buffered solutions or in cells. In the presence of larger PEGs, longer-range loop-loop interactions were more similar to those in cells than in buffer alone, consistent with prior work showing that larger PEGs stabilize compact RNA states. Ligand affinity was weakened by low-molecular mass PEGs but increased with high-molecular mass PEGs, indicating that PEG cosolvents exert complex chemical and steric effects on RNA structure. Regardless of polymer size, however, nucleotide-resolution structural characteristics observed in cells were not recapitulated in PEG solutions. Our results reveal that the cellular environment is difficult to recapitulate in vitro; mimicking the cellular state will likely require a combination of crowding agents and other chemical species.