Selective binding of 2′-F-c-di-GMP to Ct-E88 and Cb-E43, new class I riboswitches from Clostridium tetani and Clostridium botulinum respectively

C-di-GMP is a second messenger in bacteria and partly regulates bacterial physiology by binding to class I and II riboswitches. Four class I c-di-GMP riboswitch aptamer candidates, Ct-E88, Cb-17B, Cb-E43 and Cd-630 RNAs, selected from a GEMM RNA sequence motif in the Rfam database, were expressed and experimentally verified to bind to c-di-GMP. The two newly characterized c-di-GMP riboswitches, Ct-E88 and Cb-E43, bound c-di-GMP with nanomolar K-d whereas the affinities of Cb-17B and Cd-630 for c-di-GMP were at least a 100-fold weaker. Interestingly, whereas the three riboswitches (Vc2, Et-E88 and Cb-E43) bound c-di-GMP with similar K-d values, 2'-modified analogs of c-di-GMP differentially bound to these three class I aptamers. For example, 2'-F-c-di-GMP bound Vc2 with a K-d value of 102 nM whereas the K-d value of 2'-F-c-di-GMP-Ct-E88 is 43 mu M (422 x higher than that for Vc2 RNA), revealing that there are differences in the binding sites of functional class I c-di-GMP riboswitches.