# Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety

## TL;DR

Researchers created a new amino acid that can be used to study protein interactions in cells, with features that allow capturing and releasing proteins for detailed analysis.

## Contribution

A novel trifunctional photo-cross-linker with an alkaline-cleavable alkyl ester is genetically encoded for enhanced protein interaction studies.

## Key findings

- DiZAAsu was successfully genetically encoded in mammalian cells using an engineered archaeal tRNA synthetase.
- The trifunctional DiZAAsu enabled cross-linking, biotinylation, and alkaline-based cleavage of protein complexes.
- The system reduced nonspecific contamination during purification of cross-linked complexes.

## Abstract

Genetically encoded photo‐cross‐linkable amino acids (PAAs) are powerful tools for analyzing direct protein–protein interactions (PPIs) in mammalian cells. Cleavable PAAs are particularly useful, enabling covalent capture and subsequent release of interacting partners, which facilitates the characterization of interaction interfaces using mass spectrometry. However, the limited options for cleavable linker structures have restricted the design of PAAs. In this study, we genetically encoded a novel trifunctional PAA, DiZAAsu, which contains three distinct chemical groups: diazirine, alkyne, and alkaline‐cleavable alkyl ester moieties. An archaeal pyrrolysyl‐tRNA synthetase was engineered to incorporate DiZAAsu efficiently into proteins in mammalian cells. We demonstrated the in‐cell photoreactive function of diazirine by cross‐linking the DiZAAsu‐introduced GRB2 protein to its binding partner, SHC. Using the alkyne group for biotinylation, we established a tandem affinity purification strategy that enabled efficient enrichment of the cross–linked complex, thereby reducing nonspecific protein contamination. The alkaline‐based cleavage of the ester group in DiZAAsu was also demonstrated, confirming its potential for the dissociation of covalently linked complexes. This system thus expands the design space of multifunctional PAAs and adds alkaline‐based dissociation to the limited repertoire of available cleavage strategies.

A novel noncanonical amino acid, DiZAAsu, possessing photo‐cross‐linking, biotinylation, and alkaline‐cleavable functionalities, is genetically encoded by an engineered pyrrolyl‐tRNA synthetase mutant. The alkyl ester moiety expands the design space of cleavable photo‐cross‐linkable amino acids.© 2026 WILEY‐VCH GmbH