A dual-photoreactive adapter enables one-step DNA barcoding of antibody panels for multiplexed imaging

Abstract

DNA-barcoded antibodies are central to a broad range of spatial and dissociative assays and applications, including multiplexed imaging, single-cell profiling, and proximity detection. Direct modification of primary antibodies with defined DNA barcodes enables flexible panel design for multiplexed labeling of proteins. However, conventional antibody-oligonucleotide conjugation methods are inefficient, low-throughput, and prone to batch variability, limiting the reliable generation of orthogonally barcoded antibody panels. These challenges are particularly acute during initial panel development, where lengthy protocols, conjugation failures, large antibody input requirements, and the need for custom antibody formulations increase experimental cost and effort. Site-specific conjugation strategies based on antibody Fc-domain binders offer a promising alternative. We streamlined this foundational approach to establish its compatibility with multiplexed imaging in cells and tissues; however, generating a full barcoded library is still resource intensive. This is because every unique DNA barcode must first be chemically linked to a separate binder before it can be attached to an antibody. To overcome the prominent bottleneck of rapidly and reliably generating DNA-barcoded antibody panels, we introduce the universal oligo adapter (UnO) strategy. UnO fundamentally changes the workflow from barcode-specific conjugation to universal barcode conversion. We build on the established photoreactive protein G binder and combine it with a universal oligonucleotide carrying a second ultrafast photocrosslinking group, 3-cyanovinylcarbazole (cnvK). This creates a dual-functional adapter: one photoreactive group enables covalent attachment to the antibody, while the cnvK-containing universal oligo simultaneously captures a user-defined DNA barcode through hybridization and UV crosslinking in a single step. Rather than preparing separate conjugation reactions for dozens of barcodes, UnO acts as a single reagent that covalently couples any desired barcode onto small quantities of off-the-shelf primary antibodies in minutes. We validate the generalizability and modularity of this approach across subcellular Immuno-SABER and tissue-based CODEX workflows for multiplex immunostaining. By converting antibody barcoding into a modular, one-step nucleic-acid adapter workflow, UnO reduces the cost, time, and complexity of generating DNA-barcoded antibody panels and provides an efficient, accessible solution to a central bottleneck in DNA-enabled multiplexed protein detection.