a, ITC binding curves of ydaO riboswitch bound to c-di-AMP, c-di-dAMP, c-di-GMP and c-di-IMP. b, Comparative ITC binding curves for complex formation between the sensing domain of the ydaO riboswitch with c-di-AMP, and cGAMP linkage isomers containing both 2′,5′ linkages (designated 2′,2′-cGAMP), both 3′,5′ linkages (designated 3′,3′-cGAMP) and G(2′,5′)A/A(3′,5′)G linkages (designated 2′,3′-cGAMP). c, ITC binding curves for mutation G39C (disruption of G39-C94 pair), G96C (disruption of C32-G96 pair), A73C (disruption of A73•U112 pair) and U98C (disruption of Mg2+ coordination). d, A triple mutant where C39-G94, C32-G96 and A73•U112 were simultaneously replaced either by three C•C pairs (designated 3CC) or three A•A pairs (designated 3AA). In panels a, b, c and d, the cyclic dinucleotides were added gradually to the T. tengcongensis ydaO riboswitch and mutants.