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PreprintCited by 3Click Chemistry and ApplicationsBiotin and Related StudiesAdvanced biosensing and bioanalysis techniques

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Enhanced photo-crosslinking in living cells with high-intensity longwave ultraviolet light

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Trendel, Jakob, Prokofeva, Polina, Chen, Zhuo Angel, Horn, Lukas, Trendel, Simon, Mema, Mirea, Stuiver, Marchel, Rappsilber, Juri, Kuster, Bernhard·2025·Nucleic Acids Research
DOI 10.1101/2025.08.20.671286PMC PMC13096800

Preprint — published as 10.1093/nar/gkag339 · Paper page

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Abstract

The activation of chemical reactions in living cells using ultraviolet (UV) light enables the interrogation of biomolecules in their native environment with photoreactive probes or crosslinking reagents. Although numerous photo-crosslinking approaches have been successfully employed, they often suffer from common limitations, including low reaction yields, the need for long exposure times, and irradiation-induced cellular damage from heat, desiccation, or side reactions. We recently showed that 365 nm light-emitting diodes (LEDs) enable rapid, bioorthogonal protein-DNA crosslinking in living cells, incurring minimal photodamage. Here we generalize this approach and demonstrate that high-intensity, longwave UV light reduces the irradiation time for in-cell photo-crosslinking reactions by up to 1000-fold, allowing protein-drug, protein-protein, protein-DNA and protein-RNA interactions to be fixed within seconds. Benchmarking this rapid photo-activation for the analysis of RNA-interacting proteomes responding to RNA-binding drugs, we demonstrate both qualitative and quantitative advantages of controlled, high-intensity UV irradiation, uncovering emergent experimental opportunities that were previously inaccessible to light-activated chemistry in intact cells and tissues.

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Metadata

Authors
Trendel, J., Prokofeva, P., Chen, Z. A., Horn, L., Trendel, S., Mema, M., Stuiver, M., Rappsilber, J., & Kuster, B.
Affiliations
  • Technical University of Munich (DE)
  • Technische Universität Berlin (DE)
  • Wellcome Centre for Cell Biology (GB)
Corresponding author
Trendel, Jakob
ORCID
Venue
Nucleic Acids Research
Publisher
openRxiv
Published
2025-08-24
Volume
54
Issue
7
ISSN
0305-1048 (ISSN-L)
DOI
10.1101/2025.08.20.671286
PMC
PMC13096800
OpenAlex
W7155048661
Cited by
3
References
1
Citations by year
2026: 2 · 2025: 1
FWCI
3.50
Type
journal article
Language
en
Open access
Licence
Locations
  • Nucleic Acids Research (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
Funders
  • German Research Foundation (492625837)
  • Bavarian Research Foundation (AZ-1444-20C)
Topics
  • Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
Keywords
photocrosslinking, living cells, protein-DNA interactions, RNA-protein interactions, protein-protein interaction, drug-protein interactions, bioorthogonal chemistry, photodamage
MeSH terms
RNA Damage, Cross-Linking Reagents, DNA, Humans, RNA, Time Factors, Ultraviolet Rays, RNA-Binding Proteins, Photochemical Processes

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