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

A dimethyl sulfate mutational profiling framework in human cells shows RNA folding is the strongest determinant of MS2 coat protein (MCP) recognition in the MS2-MCP system, requiring a stable hairpin and consensus loop sequence.

• Why it matters: Pulldown methods can blur the structural context of RNA bound by RBPs, limiting how well specificity and affinity can be measured in living cells.
• What they did: The authors built a quantitative approach combining dimethyl sulfate mutational profiling and sequencing at single-nucleotide resolution, applying it to a 1600-member MS2 hairpin library and deriving an in-cell occupancy model.
• The result: In-cell inferred fractional occupancy under degron-tuned MCP and a downregulatory MCP fusion is linked to RNA degradation efficiency, enabling quantitative prediction of degradation from structural readouts.

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