The prediction of RNA secondary structure is complex. The biomolecule can adopt or sample numerous stable conformations, can change structure in response to a stimulus such as a binding event, and does not simply obey thermodynamically favorable folding rules. Due to this, estimation of structure based on the primary sequence is unreliable and misleading. Probing RNA structure dramatically improves computational prediction. The examination of both, ex vivo and in cell RNA can provide important information regarding structural stability, the RNA interactome, and refolding effects.
Current structural probes for RNA selective 2′-hydroxy acylation analyzed by primer extension (SHAPE) rely on a reaction with the 2′-OH on the ribose sugar of residues that are not base paired. These flexible residues can then be determined using gel electrophoresis or quantified using next generation sequencing (NGS) and mutational profiling (MaP) to prepare a library of probing data. The advent of SHAPE technologies led to a rapid increase in the accessibility of RNA structural data. Several successful SHAPE probes have been previously demonstrated, but arguments regarding reactivity and cell permeability remain. In this work, the design and application of novel, variably reactive SHAPE probes is shown ex vivo and a novel in cell probe is demonstrated.