A Genetically Encoded Diazirine Analogue for RNA–Protein Photo‐crosslinking
| Autor: | Matthias W. Hentze, Dmytro Dziuba, Carsten Schultz, Jan Erik Hoffmann |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Ultraviolet Rays
macromolecular substances 010402 general chemistry 01 natural sciences Biochemistry chemistry.chemical_compound Humans Nucleotide Molecular Biology Expanded genetic code chemistry.chemical_classification amino acids Molecular Structure Photoaffinity labeling 010405 organic chemistry Communication Organic Chemistry RNA-Binding Proteins RNA Combinatorial chemistry Communications protein modifications 3. Good health 0104 chemical sciences Amino acid Cross-Linking Reagents Diazomethane chemistry Covalent bond click chemistry Diazirine Click chemistry Molecular Medicine photoaffinity labeling |
| Zdroj: | Chembiochem |
| ISSN: | 1439-7633 1439-4227 |
| DOI: | 10.1002/cbic.201900559 |
| Popis: | Ultraviolent crosslinking is a key experimental step in the numerous protocols that have been developed for capturing and dissecting RNA–protein interactions in living cells. UV crosslinking covalently stalls dynamic interactions between RNAs and the directly contacting RNA‐binding proteins and enables stringent denaturing downstream purification conditions needed for the enrichment and biochemical analysis of RNA–protein complexes. Despite its popularity, conventional 254 nm UV crosslinking possesses a set of intrinsic drawbacks, with the low photochemical efficiency being the central caveat. Here we show that genetically encoded photoreactive unnatural amino acids bearing a dialkyl diazirine photoreactive group can address this problem. Using the human iron regulatory protein 1 (IRP1) as a model RNA‐binding protein, we show that the photoreactive amino acids can be introduced into the protein without diminishing its RNA‐binding properties. A sevenfold increase in the crosslinking efficiency compared to conventional 254 nm UV crosslinking was achieved using the diazirine‐based unnatural amino acid DiAzKs. This finding opens an avenue for new applications of the unnatural amino acids in studying RNA–protein interactions. UV irradiation is widely used in molecular biology to covalently crosslink RNA with RNA‐binding proteins; however, the yield of this process is usually low. We show that site‐selective labeling of an RNA‐binding protein with a diazirine‐based unnatural amino acid gives higher yields of the photo‐crosslinking product under milder irradiation conditions. |
| Databáze: | OpenAIRE |
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