Table 2. Dissociation Constants (K_d^ITC) of ssDNA from A3 Enzymes Obtained by Isothermal Titration Calorimetry in Different Buffers^a.

	DNA sequence (5′–3′)	buffer	enzyme	KdITC (μM)
Oligo-1	AAAAAAAATTCAAAAAAAAA	high-salt	A3A-E72A	24 ± 7
		medium-salt	A3A-E72A	0.11 ± 0.05
Oligo-2	ATTTCATTT	high-salt	A3A-E72A	25.3 ± 0.9
		medium-salt	A3A-E72A	0.20 ± 0.04
Oligo-3*b	ATTCCCAATT	medium-salt	A3A-E72A	0.24 ± 0.10
Oligo-4*	TTCCC	medium-salt	A3A-E72A	5.0 ± 0.4
Oligo-5*	CCCAA	medium-salt	A3A-E72A	3.1 ± 0.4
Oligo-6	TTCAT	medium-salt	A3A-E72A	0.48 ± 0.10
Oligo-7	ATTCCdZAATT	medium-salt	A3A-E72A	0.97 ± 0.15
Oligo-8	ATTCCdZMeAATT	medium-salt	A3A-E72A	1.7 ± 0.3
Oligo-9	ATTTdZATTT	activity assay	A3Bc-QM-ΔL3-AL1swap	5.5 ± 0.6
Oligo-10*	TTTTCAT	med. salt	A3A-E72A	0.27 ± 0.04
Oligo-16	5′-(6-FAM)TTT TCAT	med. salt	A3A-E72A	0.41 ± 0.04

^aThe high-salt buffer consisted of 25 mM sodium phosphate, 500 mM NaCl, 300 mM choline acetate, 5 mM β-mercaptoethanol, and 0.2 mM Na₂-EDTA (pH 6.0). The medium-salt buffer consisted of 50 mM MES, 100 mM NaCl, and 2.0 mM tris(2-carboxyethyl)phosphine (pH 6.0). The activity assay buffer consisted of 50 mM citrate-phosphate buffer, 200 mM NaCl, and 2 mM β-mercaptoethanol (pH 5.5). Means ± the standard deviation (SD) are shown. Uncertainties (SD) in K_d^ITC were calculated using standard error propagation methods from partial derivatives.

^bAn asterisk denotes an oligo evaluated in both ITC and FP experiments. A3A prefers to deaminate the third C in the CCC motif.³⁶