Directed Evolution Improves the Catalytic Efficiency of APEX2-Mediated Proximity-Dependent RNA Labeling

Engineered ascorbate peroxidase APEX2 has been widely used for spatially restricted profiling of subcellular biomolecules, but its catalytic efficiency toward newly developed probes such as biotin-aniline (Btn-An) remains suboptimal. To overcome this limitation, we performed yeast surface display-based directed evolution to enhance APEX2 activity toward Btn-An. The resulting variant, ^L242FAPEX2, exhibits an approximately two-fold improvement in labeling efficiency, likely through enhanced enzyme-substrate interactions. This increased activity enables rapid and efficient proximity labeling while maintaining spatial specificity. After validating its performance at the ER membrane, we applied ^L242FAPEX2 to profile the transcriptome proximal to the midbody and identified ANLN as a previously unreported midbody-localized mRNA during telophase. Drug perturbation and reporter assays further revealed that ANLN mRNA targeting to the midbody occurs co-translationally and depends on its nascent N-terminal peptide. Together, this work establishes ^L242FAPEX2 as an improved and versatile tool for spatially resolved transcriptomics in complex subcellular contexts.