TY - JOUR T1 - Directed evolution of split APEX peroxidase JF - bioRxiv DO - 10.1101/452888 SP - 452888 AU - Yisu Han AU - Jeffrey D. Martell AU - Tess C. Branon AU - Daniela Boassa AU - David M. Shechner AU - Mark H. Ellisman AU - Alice Y. Ting Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/10/31/452888.abstract N2 - APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called “AP” and a 50-amino acid C-terminal fragment called “EX”. AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites. ER -