Although A3A prefers (T/C)TC(A/G), neither of the co-crystal structures has the optimal nucleotide identity at the −2 and +1 positions20,22. Specificity for purine at the −2 position was not evident in the available A3A–ssDNA structures, presumably as neither structure contains an optimal ssDNA sequence. For instance, even though the 5KEG structure contains a preferred pyrimidine in the −2 position, the thymidine is disordered in this complex. However, in both structures20,22, the base at +1 position (pyrimidine T in 5KEG and a purine G in 5SWW) stacks with the critical His29 (Fig. 5a,b)20,22. This type of histidine π-π stacking can occur with either a purine or a pyrimidine. However, protonated histidine prefers to stack with a purine base over pyrimidine, with thymidine stacking being the least preferred33 at pH 6. Thus the base stacking potential with protonated His29 provides strong rationale for the specificity for purines and the disfavoring of thymidine at the +1 position relative to substrate deoxycytidine observed in our biochemical assays (Fig. 3).Figure 5ssDNA is bent within the complex with A3A. Crystal structure of A3A shown in surface and cartoon representation (gray) bound to ssDNA displayed as orange sticks; (a) +1 thymidine (light blue) is interacting with His29 (light green sticks) through aromatic stacking (PDB ID: 5KEG) (b) +1 guanine (light blue) also interacting with His29 through aromatic stacking (light green sticks) (PDB ID: 5SWW). (c) A3A(E72A/C171A) with TTTTTTTTCTTTTTT (PDB ID: 5KEG) (d) A3A(E72A) with AAAAAAATCGGGAAA (PDB ID: 5SWW). Other nucleotides are shown as orange sticks, while water (red), zinc (blue), and chloride (gray) in the active site are shown as spheres. Nitrogen and oxygen of residues and nucleic acids are in blue and red respectively. (e) A schematic of hydrogen bonding between pyrimidine (pink) at −2 and purine (light blue) at +1 position via bending of the DNA by A3A upon binding. (f) Model of inter-DNA base interactions through binding of A3A to ssDNA. A3A(E72A)–ssDNA complex (PDB ID: 5SWW) was used to model A3A signature sequence CTCG bound at the active site. A3A is shown as gray surface and cartoon, His29 as light green sticks, original ssDNA as orange sticks with +1 G in light blue. Adenosine at −1 position was switched to cytosine (pink) with hydrogen bonds to +1 G displayed as yellow dashes.