Labeling Of Proteins Research Articles

Neural network-assisted single-molecule localization microscopy with a weak-affinity protein tag

Single-molecule localization microscopy achieves nanometer spatial resolution by localizing single fluorophores separated in space and time. A major challenge of single-molecule localization microscopy is the long acquisition time, leading to low throughput, as well as to a poor temporal resolution that limits its use to visualize the dynamics of cellular structures in live cells. Another challenge is photobleaching, which reduces information density over time and limits throughput and the available observation time in live-cell applications. To address both challenges, we combine two concepts: first, we integrate the neural network DeepSTORM to predict super-resolution images from high-density imaging data, which increases acquisition speed. Second, we employ a direct protein label, HaloTag7, in combination with exchangeable ligands (xHTLs), for fluorescence labeling. This labeling method bypasses photobleaching by providing a constant signal over time and is compatible with live-cell imaging. The combination of both a neural network and a weak-affinity protein label reduced the acquisition time up to ∼25-fold. Furthermore, we demonstrate live-cell imaging with increased temporal resolution, and capture the dynamics of the endoplasmic reticulum over extended time without signal loss.

Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins.

Photoaffinity labeling (PAL) has blossomed into a powerful and versatile tool for capture and identification of biomolecular targets. However, low labeling efficiency for specific targets such as lectins, the tedious process for protein purification, inevitable cellular photodamage, and less tissue penetration of UV light are significant challenges. Herein, we reported a near-infrared (NIR) light-driven photoaffinity labeling approach using upconverting nanoparticle (UCNP)-based photoactive probes, which were constructed by assembling photoactive groups and ligands onto NaYF4:Yb,Tm nanoparticles. The novel probes were easily prepared and functionalized, and the labeled proteins can be isolated and purified through simple centrifugation and washing. The advantages of this approach were demonstrated by labeling and isolation of peanut agglutinin (PNA), asialoglycoprotein receptor (ASGPR), and human carbonic anhydrase II (hCAII) from mixed proteins or cell lysates with good selectivity and efficiency, especially for PNA and ASGPR, two lectins that showed low binding affinity to their ligands. More importantly, successful labeling of PNA through pork tissues and ASGPR in mice strongly proved the good tissue penetrating capacity of NIR light and the application potential of UCNP-based photoactive probes for protein labeling in vivo. Biosafety of this approach was experimentally validated by enzyme, cell, and animal work, and we demonstrated that NIR light caused minimal photodamage to enzyme activity compared to UV light, and the UCNP-based photoactive probe presents good biosafety both in vitro and in vivo. We believe that this novel PAL approach will provide a promising tool for study of ligand-protein interactions and identification of biomolecular targets.

Proximity labeling and identification of endogenous client proteins recruited to Y15-based artificial granules tethering a bait protein.

Protein clustering is a ubiquitous event in diverse cellular processes. Self-association of proteins triggers recruitment of downstream proteins to regulate cellular signaling. To investigate the interactions in detail, chemical biology tools to identify proteins recruited to defined assemblies are required. Here, we exploit an identification of proteins recruited in artificial granules (IPRAG) platform that combines intracellular Y15-based supramolecule construction with a proximity labeling method. We validated the IPRAG tool using Nck1 as a target bait protein. We constructed Nck1-tethering granules, labeled the recruited proteins with biotin, and analyzed them by LC-MS/MS. As a result, we successfully identified proteins that directly or indirectly interact with Nck1.

In vivo fluorescent labeling and tracking of extracellular matrix.

Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.

Fluorescent Investigation of Proteins Using DNA-Synthetic Ligand Conjugates.

The unfathomable role that fluorescence detection plays in the life sciences has prompted the development of countless fluorescent labels, sensors, and analytical techniques that can be used to detect and image proteins or investigate their properties. Motivated by the demand for simple-to-produce, modular, and versatile fluorescent tools to study proteins, many research groups have harnessed the advantages of oligodeoxynucleotides (ODNs) for scaffolding such probes. Tight control over the valency and position of protein binders and fluorescent dyes decorating the polynucleotide chain and the ability to predict molecular architectures through self-assembly, inherent solubility, and stability are, in a nutshell, the important properties of DNA probes. This paper reviews the progress in developing DNA-based, fluorescent sensors or labels that navigate toward their protein targets through small-molecule (SM) or peptide ligands. By describing the design, operating principles, and applications of such systems, we aim to highlight the versatility and modularity of this approach and the ability to use ODN-SM or ODN-peptide conjugates for various applications such as protein modification, labeling, and imaging, as well as for biomarker detection, protein surface characterization, and the investigation of multivalency.

The key role of glutamine for protein expression and isotopic labeling in insect cells

Nuclear magnetic resonance studies of many physiologically important proteins have long been impeded by the necessity to express such proteins in isotope-labeled form in higher eukaryotic cells and the concomitant high costs of providing isotope-labeled amino acids in the growth medium. Economical routes use isotope-labeled yeast or algae extracts but still require expensive isotope-labeled glutamine. Here, we have systematically quantified the effect of 15N2-glutamine on the expression and isotope labeling of different proteins in insect cells. Sufficient levels of glutamine in the medium increase the protein expression by four to five times relative to deprived conditions. 1H-15N nuclear magnetic resonance spectroscopy shows that the 15N atoms from 15N2-glutamine are scrambled with surprisingly high (60-70%) efficiency into the three amino acids alanine, aspartate, and glutamate. This phenomenon gives direct evidence that the high energy demand of insect cells during baculovirus infection and concomitant heterologous protein expression is predominantly satisfied by glutamine feeding the tricarboxylic acid cycle. To overcome the high costs of supplementing isotope-labeled glutamine, we have developed a robust method for the large-scale synthesis of 15N2-glutamine and partially deuterated 15N2-glutamine-α,β,β-d3 from inexpensive precursors. An application is shown for the effective large-scale expression of the isotope-labeled β1-adrenergic receptor using the synthesized 15N2-glutamine-α,β,β-d3.

Naphthyridine-Based Electron Push-Pull-Type Amine-Reactive Fluorescent Probe for Sensing Amines and Proteins in Aqueous Media.

In bioengineering, fluorescent amine-reactive probes are invaluable for the detection of amine species. In particular, targeting probes for lysine, which has a free amino group in amino acids, are a valid method for protein detection. For this purpose, many fluorescent "turn-on type" probes with amine reactivity have been developed; however, they require improvements. In the typical florescence probes, BODIPY and NBD analogs have small Stokes shifts based on absorption and emission and lability in an aqueous environment, respectively. In this study, a new class of fluorescent probes, 1,8-Nap-F, based on the electron push-pull-type 1,8-naphthyridine framework, was designed and investigated as an amine-reactive probe. Generally, electron push-pull-type fluorophores exhibit a large Stokes shift at the expense of fluorescent enhancement in aqueous media; thus, there is a trade-off between possessing a large Stokes shift and intense emission. However, 1,8-Nap-F reacts with primary amines, yielding emissive amine products with a large Stokes shift (>70 nm) without fluorescence quenching and side products, even in an aqueous environment, thereby overcoming the disadvantages of electron push-pull-type fluorophores and lability in aqueous conditions. By applying the specific features of 1,8-Nap-F, we achieved selective lysine detection and fluorescence bioimaging, such as endoplasmic reticulum-selective protein labeling and organelle staining, in living cells by utilizing amine-substituted derivatives.

Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable ‘cargo’ , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

Fluorogenic and Cell‐Permeable Rhodamine Dyes for High‐Contrast Live‐Cell Protein Labeling in Bioimaging and Biosensing

AbstractThe advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small‐molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high‐contrast live‐cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell‐permeable dyes has been developed, which have substantially improved live‐cell protein labeling by fine‐tuning the reversible equilibrium between a cell‐permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein‐bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell‐permeable rhodamines, as well as their applications in bioimaging and biosensing.

Fluorogenic and Cell-Permeable Rhodamine Dyes for High-Contrast Live-Cell Protein Labeling in Bioimaging and Biosensing.

The advancement of fluorescence microscopy techniques has opened up new opportunities for visualizing proteins and unraveling their functions in living biological systems. Small-molecule organic dyes, which possess exceptional photophysical properties, small size, and high photostability, serve as powerful fluorescent reporters in protein imaging. However, achieving high-contrast live-cell labeling of target proteins with conventional organic dyes remains a considerable challenge in bioimaging and biosensing due to their inadequate cell permeability and high background signal. Over the past decade, a novel generation of fluorogenic and cell-permeable dyes has been developed, which have substantially improved live-cell protein labeling by fine-tuning the reversible equilibrium between a cell-permeable, nonfluorescent spirocyclic state (unbound) and a fluorescent zwitterion (protein-bound) of rhodamines. In this review, we present the mechanism and design strategies of these fluorogenic and cell-permeable rhodamines, as well as their applications in bioimaging and biosensing.

Chemoselective Fluorogenic Bioconjugation of Vicinal Dithiol-Containing Proteins for Live Cellular Imaging via Small Molecular Conjugate Acceptors.

To develop small molecular fluorogenic tools for the chemoselective labeling of vicinal dithiol-containing proteins (VDPs) in live cells is important for studying intracellular redox homeostasis. With this research, we developed small molecule-based fluorescent probes, achieving selective labeling of VDPs through thiol-thiol substitutions on bisvinylogous thioester conjugated acceptors (IDAs). Initially, IDAs demonstrated its ability to bridge vicinal cysteine-sulfhydryls on a peptide as a mimic. Then, the peptide complex could be decoupled to recover the original peptide-SH in the presence of dithiothreitol. Furthermore, fluorometric signal amplification of the fluorescent probes occurred with high sensitivity, low limit of detection, and selectivity toward vicinal dithiols on reduced bovine serum albumin, as an example of real world VDPs. More importantly, the probes were utilized successfully for labeling of endogenous VDPs at different redox states in live cells. Thus, the bisvinylogous thioester-based receptor as a functional probe represents a new platform for uncovering the function of VDPs in live cells.

Sequential Azidation/Azolation of Prenylated Derivatives and a Click Reaction Enable Selective Labeling and Degradation of RAS Protein.

We propose the introduction of the azido and azo-functionalities into prenylated derivatives under mild conditions in a selective and efficient way. Upon protocol establishment and substrate scope determination, we apply this method to prenylated protein (citronellol-BSA) labeling, chemical pulldown, and enrichment. Eventually, we achieve the degradation of RAS on MCF-7 and HeLa cell lines by employing the well-designed probe von Hippel-Lindau derivatives C4 through the sequential azidation/azolation and click-reaction (SACR) pathway targeting the prenyl functionality attached to the Caax motif of the tested RAS protein. This method displays great potential in regulation of prenylated molecules.

Genetically Defined Subtypes of Somatostatin-Containing Cortical Interneurons.

Inhibitory interneurons play a crucial role in proper development and function of the mammalian cerebral cortex. Of the different inhibitory subclasses, dendritic-targeting, somatostatin-containing (SOM) interneurons may be the most diverse. Earlier studies used GFP-expressing and recombinase-expressing mouse lines to characterize genetically defined subtypes of SOM interneurons by morphologic, electrophysiological, and neurochemical properties. More recently, large-scale studies classified SOM interneurons into 13 morpho-electric transcriptomic (MET) types. It remains unclear, however, how these various classification schemes relate to each other, and experimental access to MET types has been limited by the scarcity of specific mouse driver lines. To address these issues, we crossed Flp and Cre driver lines with a dual-color intersectional reporter, allowing experimental access to several combinatorially defined SOM subsets. Brains from adult mice of both sexes were retrogradely dye labeled from the pial surface to identify layer 1-projecting neurons and immunostained against several marker proteins, revealing correlations between genetic label, axonal target, and marker protein expression in the same neurons. Lastly, using whole-cell recordings ex vivo, we analyzed and compared electrophysiological properties between different intersectional subsets. We identified two layer 1-targeting subtypes with nonoverlapping marker protein expression and electrophysiological properties, which, together with a previously characterized layer 4-targeting subtype, account for >50% of all layer 5 SOM cells and >40% of all SOM cells, and appear to map onto 5 of the 13 MET types. Genetic access to these subtypes will allow researchers to determine their synaptic inputs and outputs and uncover their roles in cortical computations and animal behavior.

Systematic analysis of the molecular and biophysical properties of key DNA damage response factors.

Repair of DNA double strand breaks (DSBs) is integral to preserving genomic integrity. Therefore, defining the mechanisms underlying DSB repair will enhance our understanding of how defects in these pathways contribute to human disease and could lead to the discovery of new approaches for therapeutic intervention. Here, we established a panel of HaloTagged DNA damage response factors in U2OS cells which enables concentration-dependent protein labeling by fluorescent HaloTag ligands. Genomic insertion of HaloTag at the endogenous loci of these repair factors preserves expression levels and proteins retain proper subcellular localization, foci-forming ability, and functionally support DSB repair. We systematically analyzed total cellular protein abundance, measured recruitment kinetics to laser-induced DNA damage sites, and defined the diffusion dynamics and chromatin binding characteristics by live-cell single-molecule imaging. Our work demonstrates that the Shieldin complex, a critical factor in end-joining, does not exist in a preassembled state and that relative accumulation of these factors at DSBs occurs with different kinetics. Additionally, live-cell single-molecule imaging revealed the constitutive interaction between MDC1 and chromatin mediated by its PST repeat domain. Altogether, our studies demonstrate the utility of single-molecule imaging to provide mechanistic insights into DNA repair, which will serve as a powerful resource for characterizing the biophysical properties of DNA repair factors in living cells.

1583-P: Dynamic Tracking and Identification of Liver-Specific Secretory Proteins in the Circulation of Live Mice

Secretory proteins are an essential component of interorgan communication networks that regulate animal physiology. Current approaches for identifying secretory proteins from specific cell and tissue types are largely limited to in vitro or ex vivo models which often fail to recapitulate in vivo biology. As such, there is mounting interest in developing in vivo analytical tools that can provide accurate information on the origin, identity, and spatiotemporal dynamics of secretory proteins. Here, we describe iSLET (in situ Secretory protein Labeling via ER-anchored TurboID) which selectively labels proteins that transit through the classical secretory pathway via catalytic actions of Sec61b-TurboID, a proximity labeling enzyme anchored in the ER lumen. To validate iSLET in a whole-body system, we express iSLET in the mouse liver and demonstrate efficient labeling of liver secretory proteins which could be tracked and identified within circulating blood plasma. Furthermore, proteomic analysis of the labeled liver secretome enriched from liver iSLET mouse plasma is highly consistent with previous reports of liver secretory protein profiles. Taken together, iSLET is a versatile and powerful tool for studying spatiotemporal dynamics of secretory proteins, a valuable class of biomarkers and therapeutic targets. Disclosure K.Kim: None. J.Suh: None. Funding National Research Foundation of Korea (2018R1A2A3075389, 2016M3A9B6902871, 2017K1A1A2013124, 2021R1A2C2007573, 2020R1C1C1013927); KAIST Key Research Institutes Project; Bio and Medical Technology Development Program (2019M3E5D3073104); Ministry of Science

Enrichment and Delivery of Target Proteins into the Cell Cytosol via Outer Membrane Vesicles.

Advanced intracellular delivery of proteins has profound applications in both scientific investigations and therapies. However, existing strategies relying on various chemical and physical methods have drawbacks such as the requirement of high concentrations of in vitro prepared target proteins and difficulty in labeling target proteins. Developing new delivery systems integrating the enveloping and labeling of target proteins would bring great advantages for efficient protein transfections. Here, we enriched a high concentration (62 mg/mL) of several target proteins into the outer membrane vesicles (OMVs) of Escherichia coli to employ the native property of OMVs to deliver proteins into the cytosol of eukaryotic cells. The results revealed a high protein transfection efficiency from 90 to 97% for different cell lines. Moreover, the free penetration of molecules less than 600 Da across the membrane of OMVs allows direct labeling of target proteins within OMVs, facilitating the visualization of target proteins. Importantly, the nanobody delivered intracellularly by OMVs retains the biological activity of binding with its target, highlighting the advantages of OMVs as an emerging tool for efficient intracellular delivery of proteins.

TgMORN2, a MORN Family Protein Involved in the Regulation of Endoplasmic Reticulum Stress in Toxoplasma gondii.

MORN proteins play a key role in the cytoskeletal structure of eukaryotes and are essential for the close arrangement of the endoplasmic reticulum and plasma membrane. A gene with nine MORN motifs (TGGT1_292120, named TgMORN2) was identified in the Toxoplasma gondii genome; it was presumed to belong to the MORN protein family and to have the function of forming the cytoskeleton, which affects the survival of T. gondii. However, the genetic deletion of MORN2 did not noticeably affect parasite growth and virulence. Using adjacent protein labeling techniques, we identified a network of TgMORN2 interactions, which mainly included endoplasmic reticulum stress (ER stress)-related proteins. In exploring these data, we found that the pathogenicity of the KO-TgMORN2 strain was significantly reduced in the case of tunicamycin-induced ER stress. Reticulon TgRTN (TGGT1_226430) and tubulin β-Tubulin were identified as interaction proteins of TgMORN2. Collectively, TgMORN2 plays a role in ER stress, which lays a foundation for further research on the function of the MORN protein in T. gondii.

First person – Nevena Stajković

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Nevena Stajković is first author on ‘ Direct fluorescent labeling of NF186 and NaV1.6 in living primary neurons using bioorthogonal click chemistry’, published in JCS. Nevena is a PhD student in the lab of Ivana Nikić-Spiegel at Centre for Integrative Neuroscience (CIN), Tübingen, Germany, investigating neuronal biology, axon initial segment components (particularly voltage-gated sodium channels) and their role in neurological diseases, fluorescent protein labeling and advanced microscopy methods, such as super-resolution microscopy.

ITRAQ-Based Quantitative Proteomics Unveils Protein Dynamics in the Root of Solanum melongena L. under Waterlogging Stress Conditions.

Waterlogging poses significant abiotic stress that endangers the survival of plants, including crops. In response, plants dramatically change their physiology to enhance their tolerance to waterlogging, such as proteome reconfiguration. Here, we utilized isobaric tags for the relative and absolute quantitation (iTRAQ)-based protein labeling technique to examine the proteomic changes induced by waterlogging in the roots of Solanum melongena L., a solanaceous plant. The plants were subjected to 6, 12, and 24 h of waterlogging stress at the flowering stage. Of the 4074 identified proteins, compared to the control, the abundance of the proteins increased and decreased in 165 and 78 proteins, respectively, in 6 h of treatments; 219 and 89 proteins, respectively, in 12 h of treatments; and 126 and 127 proteins, respectively, in 24 h of treatments. The majority of these differentially regulated proteins participated in processes such as energy metabolism, amino acid biosynthesis, signal transduction, and nitrogen metabolism. Fructose-bisphosphate aldolase and three alcohol dehydrogenase genes, in particular, were up- or down-regulated in waterlogging-treated Solanum melongena roots, suggesting that some proteins related to anaerobic metabolism (glycolysis and fermentation) may play vital roles in protecting its roots from waterlogging stress to enable long-term survival. Overall, this research not only offers a comprehensive dataset of protein alterations in waterlogged Solanum melongena roots but also insights into the mechanisms by which solanaceous plants adapt to waterlogging stress.

Reversible control of tetrazine bioorthogonal reactivity by naphthotube-mediated host-guest recognition

•Naphthotubes can recognize phenyltetrazine-modified biomolecules with high affinity •Reversible control of phenyltetrazine reactivity through molecular recognition •Selective tetrazine residue caging enables sequential IEDDA reactions on proteins •Reversible caging of phenyltetrazine activity in living cells and animals Tetrazine-mediated inverse electron demand Diels-Alder (IEDDA) reactions are widely used ultrafast bioorthogonal reactions, and methods to control such reactions would be extremely useful. Here, we identified a high-affinity host-guest pair between synthetic macrocyclic naphthotubes and phenyltetrazine and developed a molecular-recognition strategy for controlling tetrazine reactions by host caging. Naphthotubes can recognize phenyltetrazine on various biomolecules with low-micromolar and sub-micromolar binding affinities and thus cage their IEDDA reactivity efficiently in a reversible manner. For tetrazine residues on proteins encoded by non-canonical amino acid mutagenesis, their positions could be computationally designed such that exposed residues could be reversibly caged, whereas semiburied residues that were resistant to caging retained their reactivity, thus allowing preparation of site-specific protein dual conjugates and dual protein labeling on living cells with IEDDA reactions. The reactivity of tetrazine can be further regulated in living animals. Our strategy is thus expected to expand the applications of tetrazine chemistry. Tetrazine-mediated inverse electron demand Diels-Alder (IEDDA) reactions are widely used ultrafast bioorthogonal reactions, and methods to control such reactions would be extremely useful. Here, we identified a high-affinity host-guest pair between synthetic macrocyclic naphthotubes and phenyltetrazine and developed a molecular-recognition strategy for controlling tetrazine reactions by host caging. Naphthotubes can recognize phenyltetrazine on various biomolecules with low-micromolar and sub-micromolar binding affinities and thus cage their IEDDA reactivity efficiently in a reversible manner. For tetrazine residues on proteins encoded by non-canonical amino acid mutagenesis, their positions could be computationally designed such that exposed residues could be reversibly caged, whereas semiburied residues that were resistant to caging retained their reactivity, thus allowing preparation of site-specific protein dual conjugates and dual protein labeling on living cells with IEDDA reactions. The reactivity of tetrazine can be further regulated in living animals. Our strategy is thus expected to expand the applications of tetrazine chemistry. Bioorthogonal chemistry: Bridging chemistry, biology, and medicineHartung et al.ChemJune 5, 2023In BriefBioorthogonal chemistry has become a flourishing toolbox of approaches for performing selective reactions in complex biological systems without perturbing natural processes. Through the implementation of this chemistry, chemical biologists have answered previously unanswerable questions, made new and exciting discoveries, and even begun to have an impact on important therapies, including implementing a bioorthogonal reaction at the center of a new cancer-treatment clinical trial. We highlight some key advances and anticipate next steps within this latest Nobel Prize-winning field. Full-Text PDF