Investigating Protonation-Coupled Conformational Changes in Precursor microRNAs.

MicroRNAs (miRNAs or miRs) are tightly regulated non-coding RNAs that form distinct hairpin motifs which play important roles in their processing. Structure-dependent chemical probing, such as that with dimethyl sulfate (DMS), can inform computational structures of microRNAs to gauge various structural properties at nucleotide-level resolution. pH-dependent DMS probing data has previously been used to identify miRNA nucleotides with reactivities that vary with pH, but it remains unclear how these changes manifest in miRNA structure. By implementing a modified folding algorithm and providing information on nucleotide protonation status, as investigated here, structures of miRNAs can be better elucidated at low pH. Using a well-characterized miR-21 construct as a case study, we computationally recapitulated two previously discovered protonation-stabilized mismatches, A⁺:C and A⁺:G, and a general conformational ensemble. We further identified microRNAs potentially involved in protonation-coupled conformational changes and created a workflow for experimentally identifying these changes on a large scale. With a stronger ability to predict and understand structural changes of microRNAs in varying chemical environments, we can accelerate the usage of these molecules in therapeutics for disease among other health-related applications.