A systematic measurement of A3A affinity in a broad range of pH values was performed to verify and quantify the pH dependence of A3A binding to substrate ssDNA21,26,30 and set a reference pH for subsequent experiments. The Kd of A3A for TTC in a Poly A background was determined at pH ranging from 4.0 to 9.0 in 0.5 pH increments (Supplementary Fig. 1 and Supplementary Table 1). A3A had the highest affinity for Poly A-TTC at pH 5.5 with a Kd of 68 ± 3 nM. The isotherms for A3A binding ssDNA at pHs below 6.0 show some secondary binding event that may be due to non-specific binding or aggregation (Supplementary Fig. 1a). A steady decrease was also observed for the affinity of A3A for ssDNA when pH was increased above 6 (Supplementary Fig. 1b), in agreement with decreased deamination activity at higher pH26. A3A affinity also overall correlated with reported deamination activity determined using a different assay at pH 7.531. Interestingly, A3A had no appreciable affinity for Poly A-TTC above pH 8.0. Since A3A is stable at these higher pH values, the lower affinity for ssDNA with increased pH is likely not due to aggregation but due to the protonation of His29, as previously described26 and reported to be responsible for coordinating ssDNA32. Therefore, all of the subsequent binding experiments were performed at pH 6.0 to avoid any potential for secondary binding events or aggregation of the protein.