Small Molecule Modulation of APOBEC3A-Catalyzed Cytosine Deamination in CCG Repeat Deoxyribonucleic Acid via Stabilization of Hairpin Structures

Repeat expansions in the genome are associated with numerous genetic diseases. The instability of repeat sequences is driven in part by slipped-out structures, such as hairpins. APOBEC3A (A3A), a cytosine deaminase, preferentially targets single-stranded DNA, including repeat regions capable of forming such secondary structures. In this study, we investigated how small molecules that selectively bind C-C mismatches in CCG hairpin repeats modulate A3A-mediated deamination. Using model oligonucleotides containing (CCG)₉ repeats and mismatch-binding ligands (MBLs: AmND and AmBzND), we show that these ligands selectively stabilize the stem regions of hairpin structures, suppressing deamination in the stem and directing A3A activity to the loop regions. The inhibitory effect was dose-dependent, and deamination occurred preferentially at loop cytosines. These findings demonstrate that hairpin stabilization with small molecules can modulate A3A site-selectivity with implications for understanding repeat instability and its therapeutic control. Furthermore, this approach may serve as a basis for developing chemical tools to manipulate repeat-associated genome functions.