Figure 2.

Qualitative activity of A3A using a restriction-enzyme based assay. (A) 3΄-FAM-labeled single-stranded oligonucleotides containing one substrate cytosine in an ATT^xCAAAT sequence context are treated with A3A. A complementary strand is annealed, with G paired across from the substrate ^xC (mismatched if deaminated to ^xU). SwaI recognizes and specifically cleaves only the 5΄-ATT^xUAAAT in this context, allowing the labeled, unreacted 35-mer substrate (S35) to be separated from cleaved, deaminated 16-mer product (P16) on a denaturing polyacrylamide gel imaged with FAM filters. (B) A3A titrations on substrates containing C, mC or ox-mCs. In each gel, from left to right are incubations of each substrate (500 nM) with 0, 1, 10, 100 and 1000 nM A3A (37°C for 30 min). When noted, the rightmost lane is a product control without A3A. Product controls for the fC/caC reactions are shown in Supplementary Figure S1. Degradation of the fC substrate with prolonged incubation produces a band (*) at the same size as P16 product, requiring background correction in quantification of deamination. Deamination of fC is above background at 1000 nM A3A. (C) A3A reactions with substrates containing with 5-halogenated cytosines are shown in an analogous format.