Finally, to identify if there was any interdependency between nucleotide identity at −2 and +1 positions, the affinity of A3A for (3A)-NTCN-(5 A) was determined (Fig. 3, Table 1). A3A displayed preference for pyrimidines at −2 position regardless of the nucleotide at +1. A3A also disfavored T at +1 position regardless of the nucleotide identity at −2. Most interestingly, A3A preferred a pyrimidine at −2 when there was a purine at +1 position. However, the reverse was not true; purine at −2 position with pyrimidine at +1 position did not result in comparable affinities. In fact, the worst binders (ATCT and GTCT) were those that contained purines at −2 with pyrimidines at +1 position. Thus, the substrates can be broadly classified as high (80–130 nM), medium (150–165 nM), and weak (210–220 nM) affinity binders, with (T/C)TC(A/G) identified as the preferred sequence for ssDNA recognition by A3A.Figure 3A3A specificity for poly A NTCN. Binding affinity of A3A(E72A) to TAMRA-labeled ssDNA sequences in a Poly A background. Gray boxes bin sequences by −2 nucleotide identity. Colors represent +1 nucleotide identity: A (blue), T (red), C (green), G (orange). Consensus sequence derived from these Kd values is shown above the graph.