Schematic comparison of SreA MaP approaches. A schematic comparing the proposed Switch-MaP approach (left) with traditional primer-based (center) and cassette-MaP (right). SHAPE reagent and SHAPE-acylated nucleotides are indicated by red pentagons and circles, respectively. In the Switch-MaP approach, the RT primer-binding site is added postprobing via ligation of a 5′-5′ adenylated oligo containing a dideoxy (H) 3′ terminal nucleotide (green). Subsequent G-capping (orange) supports template switching with a template-switching oligo (TSO, violet) during the MaP RT step. Post-RT, the resulting cDNA is amplified with primers specific for these appended sequences, preserving reactivity information in the native sequence. Indexing PCR, generating the sequencing library, results in full capture of nucleotide reactivities for the native RNA producing an accurate and unambiguous structure model. In comparison, primer-based MaP approaches lose reactivity information due to primer masking on the 5′ and 3′ ends of the modified RNA (indicated by open circles). This information loss introduces ambiguity in the resulting structural models. The use of structured cassettes (right) provides sequence handles for downstream processing but may introduce nonnative structure to the RNA of interest (cassette interference).
