The specificity of A3A for substrate versus product was measured by binding to Poly A with a single C versus Poly A with a single U (Fig. 1b). Surprisingly, the presence of a single deoxycytidine in a Poly A background was not sufficient for binding with appreciable affinity. The energetics of free ssDNA conformations in solution for Poly A sequences and base stacking propensity29 might be unaltered upon the introduction of a single C. The affinity of A3A for the Poly A-C (5A-1C-6A) (>5,000 nM) is similar to the affinity for Poly A-U (5A-1U-6A) (>6,500 nM) and even the background Poly A. This is in contrast to A3A’s specificity for binding a single C over U in a Poly T background, which is more than ten-fold (35 ± 2 nM and 500 ± 23 nM respectively) (Fig. 1c), as we previously measured28. This strong context dependence differentiating substrate C versus product U within the background of Poly A versus Poly T indicates that A3A heavily relies on the identity of the surrounding nucleotide sequence to recognize and bind substrate deoxycytidine.