Glycans impact every aspect of physiology. Despite the rapid increase in relevance and translational potential, there remains a lack of tools for studying glycosylation in vivo. Here, we present FlyMOE, a chemical-genetic platform for targeted glycoprotein profiling in Drosophila melanogaster. Transgenic flies are equipped with biosynthetic capabilities to install bioorthogonally-tagged monosaccharides into the cellular glycoproteome. The genetic tractability of Drosophila allows for versatile application based on developmental stages, genetic drivers and glycosyltransferase enzymes. We employ FlyMOE to create an overview of the glycoproteome of Drosophila tissues and embryos based on mass spectrometry glycoproteomics, and investigate the substrates of individual members of the PGANT glycosyltransferase family in vivo. FlyMOE will allow the detailed investigation of the glycoproteome in Drosophila and similarly genetically modifiable in vivo model systems.

Generating teeth in vitro requires mimicking tooth developmental processes. Biomaterials are essential to support 3D tooth organoid formation, but their properties must be finely tuned to achieve the ...

Tracking the localization and mobility of individual proteins in live cells is key for understanding how they mediate their function. Such information can be obtained from single molecule imaging techniques such as Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM). Genetic code expansion (GCE) combined with bioorthogonal chemistry offers an elegant approach for direct labeling of proteins with fluorescent dyes, holding great potential for improving protein labeling in single molecule applications. Here we calibrated conditions for performing SPT and live-SMLM of bioorthogonally labeled plasma membrane proteins in live mammalian cells. Using SPT, the diffusion rates of bioorthogonally labeled EGF receptor and the prototypical Shaker voltage-activated potassium channel (Kv) were successfully measured. Applying live-SMLM to bioorthogonally labeled Shaker Kv channels enabled visualizing the plasma membrane distribution of the channel over time with ~30 nm accuracy. Finally, by competitive labeling with two Fl-dyes, SPT and live-SMLM were performed in a single cell and both the density and dynamics of the EGF receptor were measured at single molecule resolution in sub-regions of the cell. We conclude that GCE and bioorthogonal chemistry is a highly suitable, flexible approach for protein labeling in quantitative single molecule applications that outperforms current protein live cell labeling approaches.

Proteolysis-Targeting Chimeras (PROTACs) represent a transformative therapeutic platform for targeted protein degradation across diverse disease indications. However, their potent catalytic activity i...

In this work, we designed and synthesised a new cyclometallated iridium(iii) nitrone complex [Ir(bpz)2(bpy-nitrone)](PF6) (1) (Hbpz = benzo[a]phenazine; bpy-nitrone = 4-((methyl(oxido)imino)methyl)-4′...

Designing a targeted magnetic resonance imaging (MRI) probe is challenging due to the intrinsically micromolar-millimolar sensitivity of MRI and the high background signal associated with traditional...

Glycosylation is a ubiquitous modification of proteins, necessitating approaches for its visualization and characterization. Bioorthogonally tagged monosaccharides have been instrumental to this end, ...

Incorporating bioorthogonally-modified amino acids into newly synthesized proteins allows studying the nascent proteome, but current methods often require special conditions or are toxic to cells. Her...

The incorporation of non-canonical amino acids (ncAAs) enables customized chemistry to tailor protein functions1–3. Genetic code expansion offers a general approach for ncAA encoding by reassigning stop codons as the ‘blank’ codon; however, it is not completely orthogonal to translation termination for cellular transcripts. Here, to generate more bona fide blank codons, we developed an RNA codon-expansion (RCE) strategy that introduces and decodes bioorthogonally assignable pseudouridine (Ψ) codons (ΨGA, ΨAA or ΨAG) on specified mRNA transcripts to incorporate ncAAs in mammalian cells. The RCE strategy comprises a programmable guide RNA4, an engineered decoder tRNA, and aminoacyl-tRNA synthetase. We first developed the RCE(ΨGA) system, which incorporates functional ncAAs into proteins via the ΨGA codon, demonstrating a higher translatome-wide and proteomic specificity compared with the genetic code expansion system. We further expanded our strategy to produce the RCE(ΨAA) and RCE(ΨAG)