Similarly to the Fusobacterium nucleatum queC riboswitch, the B. subtilis queC aptamer forms kissing dimers, as observed in non-denaturing polyacrylamide gels (Supplementary Figure S1 ) (52) . To ensure that the dimer does not obstruct hairpin-tail interactions, we compared a mutant C14U/C17U construct characterized previously by Kang and coworkers to generate a ligandbound solution NMR structure (26) to the WT aptamer. MFold predicts the C14U/C17U mutations will reduce the dimer stability from À6.1 kcal/mol to À0.9 kcal/mol (53). While we observe removal of the kissing dimer, chemical shifts overall overlay extremely well between the WT queC aptamer and the C14U/C17U mutant (Supplementary Figure S1 ). Specifically, tail chemical shifts correspond extremely well to the 12 nt ssRNA, further suggesting that the tail does not participate in tertiary interactions in the absence of ligand under our NMR conditions. Strikingly, the spectra of the 12 nt ssRNA are well resolved, indicating that it does not adopt a completely random conformation ( Figure 1B and Supplementary Figure S2 ). This stands in stark contrast to corresponding spectra of a 12 nt polyuridine (polyU) ssRNA, well established to have a random-coil conformation (16) , which exhibits severe spectral overlap indicative of a highly disordered conformation (Supplementary Figure S2) . This structural order is observed in the ssRNA despite the lack of any observable imino protons and therefore any base pairing or secondary structure (Supplementary Figure S3) .