To determine the structural basis for the A3A consensus sequence (T/C)TC(A/G), crystal structures of A3A bound to ssDNA recently determined by our group and others (PDB ID: 5KEG and 5SWW) were analyzed20,22. The target deoxycytidine is well coordinated and buried within the active site of A3A (Supplementary Fig. 2A) in these structures. The thymidine at position −1 has extensive contacts with loop 7 (Y130, D131 and Y132), and van der Waals contacts with loop 5 (W98) (Supplementary Fig. 2B). The Watson-Crick edge of the thymidine base faces the loop 7 residues, and makes three hydrogen bonds: one with the backbone nitrogen of Y132 and the other two, one being water mediated, are with the D131 sidechain. The D131 sidechain further forms a salt bridge to the R189, which stabilizes the overall hydrogen-bonding configuration of loop 7 to the thymine base. This coordination appears critical, as residue 189 is conserved as a basic residue (Arg/Lys) in catalytically active A3 domains. This coordination also explains why −1 nucleotide must be a thymidine. If the −1 position is modeled as a cytidine, the N3 atom lacks the proton to hydrogen bond with D131 (Supplementary Fig. 2C) and would not be as well coordinated thus would be less preferable. Residues Y130 and D131 in loop 7 physically would preclude a larger purine base from fitting in this position (as modelled in Supplementary Fig. 2D). Thus the T specificity at the −1 position is consistent with the crystal structures.