Structural probing of RNA hairpins quantifies protein occupancy on RNA and links it to function in human cells

RNA structures that form inside living cells govern processes ranging from translation to RNA decay, many of which are controlled by RNA-binding proteins (RBPs). Because RBP specificity can depend on both RNA sequence and folding, traditional pulldown-based methods often obscure the structural context of bound RNAs. Here, a quantitative framework based on dimethyl sulfate mutational profiling and sequencing was established to directly measure RNA structure and RBP binding in human cells at single-nucleotide resolution. Applied to a 1600-member MS2 hairpin library, this approach revealed that RNA folding is the strongest determinant of MS2 coat protein (MCP) recognition in the MS2-MCP system, requiring formation of a stable hairpin and a consensus loop sequence, with stem length or GC content contributing primarily through their effect on hairpin folding. An in-cell occupancy model was derived to quantify fractional binding from structural readouts. Complementary systems using degron-tuned MCP and a downregulatory MCP fusion demonstrated binding behavior under subsaturating protein concentrations and showed that inferred occupancy quantitatively predicts RNA degradation efficiency. Together, these results establish a quantitative strategy for measuring RBP-RNA specificity and affinity directly in cells, using the MS2-MCP system to link RNA structure, protein occupancy, and regulatory outcome.