Table 1. Thermodynamic parameters of the WT and mutants of the APUM23 to different RNAs by ITC experiments.

Protein APUM23	RNA 5′ to 3′	ΔH kcal/mol	ΔS cal/mol/K	K D nM	K D (mean) nM	K rel #
		36	−85	21
WT	GGAAUUGACGG	38	−91	20	21 ± 2	1
		36	−84	23
		35	−82	27
WT	GAAUUGACGG	33	−76	29	32 ± 8	1.5
		38	−95	41
		39	−96	12
WT	GGAUUUGACGG	39	−94	11	13 ± 3	0.6
		39	−96	17
		24	−47	35
WT	GGAAAUGACGG	27	−58	44	43 ± 7	2.0
		25	−50	49
		26	−61	1236
F398A	GGAAUUGACGG	29	−71	2725	(1.9 ± 0.7) × 103	90
		28	−68	1783
		28	−67	1422
Q401E	GGAAUUGACGG	22	−48	1143	(1.1 ± 0.3) × 103	52
		21	−41	840
		27	−62	205
Delete	GGAAUUGACGG	29	−66	116	(0.160 ± 0.04) × 103	7.6
		28	−64	160
		34	−86	943
Delete	GAAUUGACGG	30	−73	1248	(1.1 ± 0.2) × 103	52
		32	−80	980

^# K _rel reports the affinity of WT or mutant APUM23 for the RNA sequence, relative to the affinity of WT APUM23 for the 11-mer WT RNA 5′-GGAAAUUGACGG-3′.

The ITC experiments were carried out at 293K and all proteins were in buffer conditions: 50 mM Tris (pH 7.5), 200 mM NaCl. We performed three trials for each ITC titration. WT, F398A, Q401E and Delete represent APUM23^85-655, APUM23^85-655 F398A mutant, APUM23^85-655 Q401E mutant and APUM23^85-655 without the insertion in PUF repeat R3, respectively. The average dissociation constant (K_D mean) values (±standard deviation) were derived from three independent experiments. The fitted curves are shown in Supplementary Figure S7.