CODMA-A3: A Competitive Oligo-based DNA Modification Assay Applied to APOBEC Enzymes

Abstract

Quantifying the functional effects of human variation in proteins is a challenge because bioinformatic pipelines allow variant detection to outpace experimental characterization. This results in a practical problem of resource allocation to identify the most scientifically significant variants to prioritize for mechanistic investigation. There are many approaches appropriate depending on class of enzymatic activity being investigated. However, fundamental to many of these approaches is a trade-off between mechanistic insight and throughput. In seeking to understand how activity changes across human variants of the APOBEC3 family of proteins we sought to create an early screening assay to prioritize variants for mechanistic study. We wanted an assay that was not dependent on protein purification, allowed for multiple-substrate competition and could be executed in a microtiter plate allowing for high throughput and approximate normalized substrate turnover without requiring classic Michaelis-Menten approaches. Here we describe CODMA (Competitive Oligo-based DNA Modification Assay) and applied it to A3 enzymology as a method to rapidly screen A3 constructs for changes in substrate utilization fingerprints. CODMA-A3 detected effects of substrate competition, distinguished fluorophore identity from substrate sequence effects, resolved activity changes caused by a single nucleotide substitution within a competing substrate, and identified shifts in substrate utilization following modification of lysate composition, oligonucleotide architecture, or antibody-mediated protein perturbation. CODMA-A3 occupies a methodological niche between low-throughput mechanistic assays and sequencing-intensive approaches by providing a rapid, inexpensive screening platform for comparative characterization of 20 to 40 enzyme variants prior to detailed mechanistic investigation.