New preprint: We isolate peptide–RNA photo-crosslinks with tunable RNA chains from living cells for mass spec. This maps over 4,700 crosslinking sites across 744 proteins and offers the first glimpse into the RNA sequences in crosslinks by MS. Read here: doi.org/10.1101/2025...

New Preprint: High-intensity 365 nm irradiation accelerates photoreactions in living cells by up to 1000×. We show protein–drug 💊, –protein 💪, –DNA 🧬, –RNA 🧬 crosslinking within seconds, and analysis by #massspec for #proteomics, #chembio, #chromatin and #RNAbiology. www.biorxiv.org/content/10.1...

Thanks for your help with the first trials. Initially cells had to be irradiated quickly, before the LEDs desoldered 😂

Thanks for your contribution, was great to collaborate on this.

🚨Our new paper is online🚨
We use zero-distance⚡photo-crosslinking⚡to reveal direct protein-DNA interactions in living cells, enabling quantitative analysis of the DNA-interacting proteome on a timescale of minutes. #DNA #Chromatin #Proteomics
www.cell.com/cell/fulltex...

Very happy we pushed this across the finish line. Huge thanks to all the co-authors: Simon, Shuyao @shashuyao.bsky.social, the Greulich lab @franzigmuc.bsky.social, Susi, Karin @baybioms.bsky.social, Sandra, the Kubicek lab, the Uhlenhaut lab, and especially Bernhard @kusterlab.bsky.social. (11/11)

Overall, we present a resource of nearly 50 photo-crosslinked, i.e. direct physical, DNA interactomes and identify 688 peptide-DNA crosslinks. These crosslinks mark known but also unknown protein-DNA interaction sites, some overlapping with variant sites linked to genetic or somatic disease. (10/n)

DNA photo-crosslinking takes 1 minute on ice, while formaldehyde requires 10 minutes at room temperature. We record a time series of photo-crosslinked DNA interactomes during acute BAF inhibition and identify fast-acting restrictors of chromatin accessibility missed by conventional methods. (9/n)

We use these quantitative powers to profile three genotoxic drugs and find very distinct reactions in the DNA interactome of cancer cells. This reveals drug-specific vulnerabilities that can be exploited for combination treatments. (8/n)

This also works in primary cells, where we see recruitment of inflammation-associated transcription factors to DNA in macrophages challenged with LPS. (7/n)

To identify transcription factors or chromatin modifiers responding to changes in a cell, DNA interactomes can be compared between conditions. Treating breast cancer cells with increasing estrogen doses, we find the estrogen receptor and other regulators recruited to DNA dose-dependently. (6/n)

In contrast to formaldehyde crosslinking, photo-crosslinking only captures direct interactions (‘zero-distance’). The proteins we find in close proximity to DNA show very high degrees of intrinsic disorder. In fact, intrinsic disorder rises the closer proteins localize to DNA. (5/n)

XDNAX extracts are very clean and we discover previously elusive peptide-DNA crosslinks within them. They pinpoint protein-DNA interaction sites to individual amino acids. (4/n)

To analyze protein photo-crosslinked to DNA by proteomics, we develop XDNAX: protein-crosslinked DNA extraction. (3/n)

Unlike natural DNA, 4ST can be activated by wavelengths far outside the absorption range of most natural biomolecules. This avoids photodamage, allowing us to use high-power LEDs, with ~1000-fold higher intensity than conventional bulbs, for activating 4ST in living cells (www.uven.org). (2/n)

UV crosslinking works great for protein-RNA complexes, but DNA is much less photo-reactive and photo-crosslinking not commonly used. Analogous, we find that the photo-activatable nucleotide 4ST (DNA) is about ten times less reactive than the commonly used 4SU (RNA). (1/n)

Excited our paper is out in Cell @cp-cell.bsky.social!
🧬⚡ DNA photo-crosslinking proteomics in living cells
🎯 Pinpoints protein-DNA interactions to single amino acids
🌎 Globally quantifies DNA binding for >1800 proteins at a timescale of minutes
🔗 www.cell.com/cell/fulltex...
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