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bioRxiv · 10.1101/2025.02.07.637082

Versatile Modular Antibodies for Sensitive and Specific Detection of Poly-ADP-Ribose

Dauben, H.·Matic, I.
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Abstract

ADP-ribosylation is a chemically versatile modification of proteins, RNA and DNA that regulates various important signaling pathways, many of which are implicated in human diseases. Despite being discovered 60 years ago in the form of poly-ADP-ribosylation - the most studied form of this modification - investigating ADP-ribosylation at the molecular level has historically been challenging. By applying serine ADP-ribosylation-based antibody engineering technology, we have developed the first site-specific, as well as sensitive, mono-ADP-ribosylation modular antibodies. Here, we extend the scope of this technology to poly-ADP-ribosylation. By combining serine poly-ADP-ribosylated peptides as antigens, PARP1 serine mono- and poly-ADP-ribosylation for validation, phage display and the SpyTag protein ligation systems, we developed modular antibodies that are highly specific for poly-ADP-ribosylation. SpyTag-based coupling of horseradish peroxidase at specific positions, distant from the antigen-binding region of the antibody, yields a format that simplifies immunoblotting while dramatically enhancing poly-ADP-ribosylation detection sensitivity. Additionally, the creation of synthetic immunoglobulin formats - mouse, rabbit and human - enables straightforward co-detection of mono- and poly-ADP-ribosylation in cells by immunofluorescence. Our new tools improve and simplify the detection of poly-ADP-ribosylation, particularly in immunoblotting, enabling specific investigations of this key cellular signal in the context of other distinct forms of ADP-ribosylation.

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Dauben, H., Matic, I.. 2025-02-08. Versatile Modular Antibodies for Sensitive and Specific Detection of Poly-ADP-Ribose. https://doi.org/10.1101/2025.02.07.637082

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