Graphical abstract. From Trendel et al., 2026. “Rapid photo-crosslinking in living cells reveals protein–nucleic acid dynamics on a timescale of minutes.“ Nucleic Acids Research (2026). doi: https://doi.org/10.1093/nar/gkag339; Licensed under the terms of the Creative Commons CC-BY 4.0 license.

Photo-crosslinking is a key technique for capturing protein interactions with DNA, RNA, and drugs in living cells, but conventional UV bulb-based systems are too slow, requiring minutes of irradiation, to accurately freeze rapid molecular events. This limits the study of interactions that change on a timescale of seconds to minutes, such as those occurring during translation, splicing, or acute cellular stress responses. Trendel et al. set out to address these gaps by demonstrating that a high-intensity LED-based irradiation device (UVEN) can accelerate photo-crosslinking across multiple proteomic applications by several orders of magnitude.

Using UVEN, the researchers from the Kuster lab performed protein–drug, protein–protein, protein–DNA, and protein–RNA crosslinking in living cells within seconds, reducing standard irradiation times from 10–20 minutes to under one second in some cases. For protein–RNA crosslinking (XRNAX), samples were analysed using DIA-based label-free proteomics. Aurora® Ultimate™ 25×75 CSI C18 UHPLC columns were coupled to a timsTOF HT via a Vanquish Neo with a CaptiveSpray source. This configuration, using a 22-minute active gradient, identified more than double the number of RNA-interacting proteins compared to conventional DDA analysis on an Orbitrap Eclipse with a 60-minute gradient, including lower-abundance proteins involved in chromatin organisation and mitochondrial translation.

Leveraging this accelerated workflow, the researchers captured the earliest-ever proteomic snapshot of the ribotoxic stress response, detecting ribosome rescue machinery engagement and key ubiquitination events just two minutes after UV-induced RNA damage – previously impossible with bulb-based systems. This advance opens new possibilities for studying rapid molecular events in drug action, DNA repair, and RNA biology at unprecedented temporal resolution.

Publication
Nucleic Acids Research

Authors

Jakob Trendel, Polina Prokofeva, Zhuo Angel Chen, Lukas Horn, Simon Trendel, Mirea Mema, Marchel Stuiver, Juri Rappsilber, & Bernhard Kuster;

Title

Rapid photo-crosslinking in living cells reveals protein–nucleic acid dynamics on a timescale of minutes