A3A is a single-domain enzyme with the highest catalytic activity among human APOBEC3 proteins23 and a known restriction factor for the retroelement LINE-1 and HPV24,25. A3A can also contribute to carcinogenesis with increased expression or defective regulation26. A3A is the only A3 where both the intact apo and substrate-bound structures have been determined19,20,22,27,28. Initial substrate specificity studies have shown selectivity for DNA over RNA, suggested by NMR chemical shift perturbation19. Since A3A is the best biochemically characterized A3 human cytidine deaminase and thus a critical benchmark within the family, we chose A3A to elucidate the extended characteristics of ssDNA specificity.