Table 1.

Binding affinities of analogs measured for the class I riboswitch (G94(2AP) RNA).

		Competition			Fluorescence
	Analog	Kd (nM)	Fold Loss	ΔΔGbind (kcal/mol)	Kd (nM)	Fold Loss	ΔΔGbind (kcal/mol)
	c-di-GMP	1.4 ± 0.1a	-	-	16b	-	-
Base	c-GMP-IMP	39 ± 2.4	27	2.0	75 ± 14	5	0.9
	c-di-IMP	1100 ± 210	790	3.9	≥ 15,000	≥ 940	≥ 4.1
	c-c7G-GMP	1100 ± 80	790	3.9	≥ 15,000	≥ 940	≥ 4.1
	c-di-c7GMP	n.b.c	-	-	n.b.	-	-
	c-GMP-AMP	1600 ± 160	1200	4.0	-	-	-
	c-di-AMP	n.b.	-	-	n.b.	-	-
	c-N1mG-GMP	420 ± 16	300	3.4	-	-	-
Ribose	c-dG-GMP	51 ± 5.4	37	2.1	140 ± 24	9	1.3
	c-di-dGMP	2600 ± 88	1800	4.4	≥ 20,000	≥ 1300	≥ 4.2
	c-2’F-G-GMP	-	-	-	25 ± 3.7	2	0.3
	c-di-2’F-GMP	56 ± 9.0	40	2.2	180 ± 15	11	1.4
	c-2’OMe-G-GMP	200 ± 31	150	2.9	-	-	-
	c-di-2’OMe-GMP	≥ 30,000	≥ 21,000	5.9	n.b.	-	-
Phos.	c-(Rp)-Gps-GMP	-	-	-	67 ± 11	4	0.8
	c-(Sp)-Gps-GMP	-	-	-	280 ± 40	18	1.7
	c-(RpRp)-di-Gps	150 ± 33	100	2.8	320 ± 70	20	1.8
	c-(RpSp)-di-Gps	750 ± 63	540	3.7	1700 ± 160	110	2.8

^aMeasured by direct binding.

^bValue determined by K_d = k_off/k_on (see supplemental info, Table S2).

^cNo detectable binding at highest ligand concentration tested (250 μM by competition, 20 μM by fluorescence).