Bridging the gap betweenin vitroandin vivoRNA folding

Deciphering the folding pathways and predicting the structures of complex three-dimensional biomolecules is central to elucidating biological function. RNA is single-stranded, which gives it the freedom to fold into complex secondary and tertiary structures. These structures endow RNA with the ability to perform complex chemistries and functions ranging from enzymatic activity to gene regulation. Given that RNA is involved in many essential cellular processes, it is critical to understand how it folds and functionsin vivo. Within the last few years, methods have been developed to probe RNA structuresin vivoand genome-wide. These studies reveal that RNA often adopts very different structuresin vivoandin vitro, and provide profound insights into RNA biology. Nonetheless, bothin vitroandin vivoapproaches have limitations: studies in the complex and uncontrolled cellular environment make it difficult to obtain insight into RNA folding pathways and thermodynamics, and studiesin vitrooften lack direct cellular relevance, leaving a gap in our knowledge of RNA foldingin vivo. This gap is being bridged by biophysical and mechanistic studies of RNA structure and function under conditions that mimic the cellular environment. To date, most artificial cytoplasms have used various polymers as molecular crowding agents and a series of small molecules as cosolutes. Studies under suchin vivo-likeconditions are yielding fresh insights, such as cooperative folding of functional RNAs and increased activity of ribozymes. These observations are accounted for in part by molecular crowding effects and interactions with other molecules. In this review, we report milestones in RNA foldingin vitroandin vivoand discuss ongoing experimental and computational efforts to bridge the gap between these two conditions in order to understand how RNA folds in the cell.