To interrogate the substrate sequence preference of A3A, we systematically quantified the changes in binding affinity of catalytically inactive A3A bearing the mutation E72A to a library of labeled ssDNA sequences using a fluorescence anisotropy-based DNA binding assay28. First, to ensure that the affinity for substrate was due entirely to the sequence of interest and not due to nonspecific binding or undesired secondary structure effects, an appropriate control background sequence was identified. The dissociation constants (Kd’s) for homo-12-mer ssDNA sequences, Poly A, Poly T, Poly C, were determined (Fig. 1a). Poly G was not tested due its propensity to form secondary structure elements. Poly T (750 ± 44 nM), which had previously been used in background sequences28, bound to A3A with 2-fold higher affinity than Poly C (1,600 ± 117 nM). Thus without a greater context for A3A to target, Poly C was only weakly bound. A3A had the lowest affinity for Poly A with a Kd of >11,000 nM (Table 1). For all subsequent assays, Poly A was used as the background, as there is no detectable binding affinity of A3A to Poly A.Figure 1A3A specificity to ssDNA background and substrate. Fluorescence anisotropy of TAMRA-labeled ssDNA sequences binding to A3A(E72A). (a) Binding of A3A to poly nucleotide (12 mers): Poly A (blue), Poly T (red) and Poly C (green), (b) Binding to Poly A (blue), 5A-C-6A (red), 5A-U-6A (green), (c) Binding to Poly T (blue), 5T-C-6T (red), 5T-U-6T (green).Table 1A3A affinity for DNA sequences used in this analysis.DNA sequenceKd (nM)Poly C (12 C)1,568 ± 117Poly T (12 T)748 ± 44Poly T-C (5T-C-6T)35 ± 2Poly T-U (5T-U-6T)499 ± 23Poly A (12 A)>11,000Poly A-C (5A-C-6A)>5,000Poly A-U (5A-U-6A)>6,500Poly A-TC (4A-TC-6A)143 ± 4Poly A-CC (4A-CC-6A)250 ± 14Poly A-GC (4A-GC-6A)>6,500Poly A-ATUA (3A-ATUA-5A)>5,000Poly A-ATUG (3A-ATUG-5A)328 ± 42Poly A-TTUA (3A-TTUA-5A)306 ± 17Poly A-ATCA (3A-ATCA-5A)145 ± 2Poly A-ATCT (3A-ATCT-5A)209 ± 5Poly A-ATCC (3A-ATCC-5A)163 ± 3Poly A-ATCG (3A-ATCG-5A)154 ± 2Poly A-TTCA (3A-TTCA-5A)90 ± 1Poly A-TTCT (3A-TTCT-5A)127 ± 2Poly A-TTCC (3A-TTCC-5A)114 ± 2Poly A-TTCG (3A-TTCG-5A)92 ± 2Poly A-CTCA (3A-CTCA-5A)85 ± 1Poly A-CTCT (3A-CTCT-5A)122 ± 2Poly A-CTCC (3A-CTCC-5A)101 ± 2Poly A-CTCG (3A-CTCG-5A)86 ± 1Poly A-GTCA (3A-GTCA-5A)150 ± 3Poly A-GTCT (3A-GTCT-5A)218 ± 7Poly A-GTCC (3A-GTCC-5A)152 ± 2Poly A-GTCG (3A-GTCG-5A)150 ± 3Hairpin-TTC (G-CCATC-ATTC-GATGG-G)26 ± 2Hairpin-AAA (G-CCATC-AAAA-GATGG-G)676 ± 399