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

ABSTRACT 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, ^L242F APEX2, 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 ^L242F APEX2 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 ^L242F APEX2 as an improved and versatile tool for spatially resolved transcriptomics in complex subcellular contexts.