OpenEMMU: a versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

Abstract

5-Ethynyl-2-deoxyuridine (EdU) has transformed DNA replication and cell cycle analyses through fast and efficient click chemistry detection. However, commercial EdU kits are expensive, contain proprietary reagents, are suboptimal for antibody multiplexing, and are challenging to use with larger biological specimens, highlighting the need for innovation. Here, we report Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics (OpenEMMU), an optimized, affordable, and user-friendly click chemistry resource using off-the-shelf reagents. OpenEMMU improves the efficiency, brightness, and multiplexing capabilities of EdU thymidine analog staining with both non-conjugated and conjugated antibodies across various cell types. We validated its effectiveness for fluorescent imaging of nascent DNA in developing embryos and organs, including the embryonic heart and forelimbs, and in 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled 3D imaging of DNA synthesis in zebrafish larvae at fine resolution. This opens new avenues for insights into organismal development and growth, cell proliferation, and DNA replication, with precision and flexibility that surpass traditional methods. TeaserAn open-source and cost-effective resource for DNA replication profiling and cell cycle analysis using click chemistry.