A Genetically Encoded Diazirine Analogue for RNA–Protein Photo‐crosslinking

Abstract

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.

Bifunctional Non-Canonical Amino Acids: Combining Photo-Crosslinking with Click Chemistry

“…Proof of principle experiments in E. coli and human cell culture were conducted and recently it was used to study protein-RNA interactions. This experiment also showed that the position of the diazirine is of great importance for the crosslinking efficiency [44]. In addition, PrDiAzK was incorporated and labeled proteome wide, using sense codon competition.…”

Visualizing and trapping transient oligomers in amyloid assembly pathways

“…Although diazirine groups have to be introduced into the target protein or peptide by synthesis [ 25 , 241 , 253 ], covalent modification (e.g. via cysteine using methanethiosulfonate-functionalized diazirines [ 254 ]), or the use of unnatural amino acids during protein expression [ 255 , 256 ], the advantages offered by the short lifetime of the carbene, the high yield of potential crosslinks formed, and the rapidity of their formation when using LED illumination (reducing irradiation times from minutes or hours to seconds) [ 254 ] make diazirines attractive tools for capturing transient amyloidogenic interactions [ 25 , 241 , 244 , 253 ].…”

Synthetic Biology: Bottom-Up Assembly of Molecular Systems

“…Through incorporation of non-native reactive groups, such as ketones, proteins could be selectively targeted even in vivo by suitable chemical probes, such as hydrazine derivatives of fluorescent dyes . A wide variety of chemistries has since been introduced into proteins, including ring-substituted aromatics, − halogenated derivatives, − β- and γ-amino acids, − photo- − or redox-reactive − groups, metal binding moieties, − fluorescent probes, − and many more . The introduction of p -aminophenylalanine into the Lactococcus lactis multidrug resistance regulator protein significantly increased the hydrazine and oxime formation and showcases how catalytic activities can be improved using UAAs .…”