The Base-Editing Enzyme APOBEC3A Catalyzes Cytosine Deamination in RNA with Low Proficiency and High Selectivity

Human
APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs
to the cytidine deaminase family. Canonically, A3A catalyzes the deamination
of cytosine into uracil in single-stranded DNA, an activity that makes
A3A both a critical antiviral defense factor and a useful tool for
targeted genome editing. However, mutagenesis by A3A has also been
readily detected in both cellular DNA and RNA, activities that have
been implicated in cancer. Given the importance of substrate discrimination
for the physiological, pathological, and biotechnological activities
of A3A, here we explore the mechanistic basis for its preferential
targeting of DNA over RNA. Using a chimeric substrate containing a
target ribocytidine within an otherwise DNA backbone, we demonstrate
that a single hydroxyl at the sugar of the target base acts as a major
selectivity determinant for deamination. To assess the contribution
of bases neighboring the target cytosine, we show that overall RNA
deamination is greatly reduced relative to that of DNA but can be
observed when ideal features are present, such as preferred sequence
context and secondary structure. A strong dependence on idealized
substrate features can also be observed with a mutant of A3A (eA3A,
N57G), which has been employed for genome editing due to altered selectivity
for DNA over RNA. Altogether, our work reveals a relationship between
the overall decreased reactivity of A3A and increased substrate selectivity,
and our results hold implications both for characterizing off-target
mutagenesis and for engineering optimized DNA deaminases for base-editing
technologies.