Multi-site DMS probing reveals higher-order structure of RNA-protein complexes in living cells

Identifying tertiary structures and protein binding sites in RNA molecules remains a key challenge in RNA biology. We describe multi-site dimethyl sulfate (DMS)-mutational profiling (MaP) (msDMS-MaP), a strategy that enables simultaneous measurement of RNA secondary, tertiary, and quaternary structures via a single DMS chemical probing experiment. Optimized reverse transcription decodes typically invisible DMS N7-methylguanine (N7-G) modifications via a tautomer-induced mutational signature concurrent with N1 and N3 modifications. We show that N7-G reactivity reports on higher-order RNA structures, revealing key functional motifs such as pseudoknots and protein binding sites. Using msDMS-MaP, we find that E. coli ribosomal RNAs encode numerous independently folding tertiary structures that coincide with binding sites for primary assembly proteins. We further apply msDMS-MaP to define the quaternary structural ensemble of the 7SK small nuclear ribonucleoprotein particle (snRNP), revealing that each of the three 7SK structural isoforms possesses distinct protein binding profiles in cells. msDMS-MaP represents a broadly applicable strategy for enhanced RNA functional motif discovery and characterization.