Directed evolution improves the catalytic efficiency of APEX2-mediated proximity-dependent RNA labeling

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.