Glycosylated Cell‐Penetrating Poly(disulfide)s: Multifunctional Cellular Uptake at High Solubility

The glycosylation of cell‐penetrating poly(disulfide)s (CPDs) is introduced to increase the solubility of classical CPDs and to achieve multifunctional cellular uptake. With the recently developed sidechain engineering, CPDs decorated with α‐d ‐glucose (Glu), β‐d ‐galactose (Gal), d ‐trehalose (Tre), and triethyleneglycol (TEG) were readily accessible. Confocal laser scanning microscopy images of HeLa Kyoto cells incubated with the new CPDs at 2.5 μm revealed efficient uptake into cytosol and nucleoli of all glycosylated CPDs, whereas the original CPDs and TEGylated CPDs showed much precipitation into fluorescent aggregates at these high concentrations. Flow cytometry analysis identified Glu‐CPDs as most active, closely followed by Gal‐CPDs and Tre‐CPDs, and all clearly more active than non‐glycosylated CPDs. In the MTT assay, all glyco‐CPDs were non‐toxic at concentrations as high as 2.5 μm . Consistent with thiol‐mediated uptake, glycosylated CPDs remained dependent on thiols on the cell surface for dynamic covalent exchange, their removal with Ellman's reagent DTNB efficiently inhibited uptake. Multifunctionality was demonstrated by inhibition of Glu‐CPDs with d ‐glucose (IC₅₀ ca. 20 mm ). Insensitivity toward l ‐glucose and d ‐galactose and insensitivity of conventional CPDs toward d ‐glucose supported that glucose‐mediated uptake of the multifunctional Glu‐CPDs involves selective recognition by glucose receptors at the cell surface. Weaker but significant sensitivity of Gal‐CPDs toward d ‐galactose but not d ‐glucose was noted (IC₅₀ ca. 110 mm ). Biotinylation of Glu‐CPDs resulted in the efficient delivery of streptavidin together with a fluorescent model substrate. Protein delivery with Glu‐CPDs was more efficient than with conventional CPDs and remained sensitive to DTNB and d ‐glucose, i.e., multifunctional.     

A Suite of “Minimalist” Photo‐Crosslinkers for Live‐Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors

Affinity‐based probes (Af BPs) provide a powerful tool for large‐scale chemoproteomic studies of drug–target interactions. The development of high‐quality probes capable of recapitulating genuine drug–target engagement, however, could be challenging. “Minimalist” photo‐crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine‐containing photo‐crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of Af BPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug–target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC‐based chemoproteomic studies have led to the discovery of a novel off‐target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up‐regulation of endogenous DNMT1 expression under normoxia conditions.     

A Double‐Imprinted Diffraction‐Grating Sensor Based on a Virus‐Responsive Super‐Aptamer Hydrogel Derived from an Impure Extract

The detection of viruses is of interest for a number of fields including biomedicine, environmental science, and biosecurity. Of particular interest are methods that do not require expensive equipment or trained personnel, especially if the results can be read by the naked eye. A new “double imprinting” method was developed whereby a virus‐bioimprinted hydrogel is further micromolded into a diffraction grating sensor by using imprint‐lithography techniques to give a “Molecularly Imprinted Polymer Gel Laser Diffraction Sensor” (MIP‐GLaDiS). A simple laser transmission apparatus was used to measure diffraction, and the system can read by the naked eye to detect the Apple Stem Pitting Virus (ASPV) at concentrations as low as 10 ng mL⁻¹, thus setting the limit of detection of these hydrogels as low as other antigen‐binding methods such as ELISA or fluorescence‐tag systems.     

Boronic Acid Functionalized Core–Satellite Composite Nanoparticles for Advanced Enrichment of Glycopeptides and Glycoproteins

A core–satellite‐structured composite material has been successfully synthesized for capturing glycosylated peptides or proteins. This novel hybrid material is composed of a silica‐coated ferrite “core” and numerous “satellites” of gold nanoparticles with lots of “anchors”. The anchor, 3‐aminophenylboronic acid, designed for capturing target molecules, is highly specific toward glycosylated species. The long organic chains bridging the gold surface and the anchors could reduce the steric hindrance among the bound molecules and suppress nonspecific bindings. Due to the excellent structure of the current material, the trap‐and‐release enrichment of glycosylated samples is quite simple, specific, and effective. Indeed, the composite nanoparticles could be used for enriching glycosylated peptides and proteins with very low concentrations, and the enriched samples can be easily separated from bulk solution by a magnet. By using this strategy, the recovery of glycopeptides and glycoproteins after enrichment were found to be 85.9 and 71.6 % separately, whereas the adsorption capacity of the composite nanoparticles was proven to be more than 79 mg of glycoproteins per gram of the material. Moreover, the new composite nanoparticles were applied to enrich glycosylated proteins from human colorectal cancer tissues for identification of N‐glycosylation sites. In all, 194 unique glycosylation sites mapped to 155 different glycoproteins have been identified, of which 165 sites (85.1 %) were newly identified.     

Rational Engineering of Photoconvertible Fluorescent Proteins for Dual‐Color Fluorescence Nanoscopy Enabled by a Triplet‐State Mechanism of Primed Conversion

Green‐to‐red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super‐resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400‐nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far‐red/near‐infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate “primed” state to be a triplet dark state formed by intersystem crossing. We show that formation of this state can be influenced by the introduction of serine or threonine at sequence position 69 (Eos notation) and use this knowledge to create “pr”‐ (for primed convertible) variants of most known green‐to‐red pcFPs.     

Redesign of a Fluorogenic Labeling System To Improve Surface Charge,Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains

Protein labeling with fluorogenic probes is a powerful method for the imaging of cellular proteins. The labeling time and fluorescence contrast of the fluorogenic probes are critical factors for the precise spatiotemporal imaging of protein dynamics in living cells. To address these issues, we took mutational and chemical approaches to increase the labeling kinetics and fluorescence intensity of fluorogenic PYP‐tag probes. Because of charge‐reversal mutations in PYP‐tag and probe redesign, the labeling reaction was accelerated by a factor of 18 in vitro, and intracellular proteins were detected with an incubation period of only 1 min. The brightness of the probe both in vitro and in living cells was enhanced by the mutant tag. Furthermore, we applied this system to the imaging analysis of bromodomains. The labeled mutant tag successfully detected the localization of bromodomains to acetylhistone and the disruption of the bromodomain–acetylhistone interaction by a bromodomain inhibitor.     

A Strategy for Creating Organisms Dependent on Noncanonical Amino Acids

The use of noncanonical amino acids (ncAAs) to control the viability of an organism provides a strategy for the development of conditional “kill switches” for live vaccines or engineered human cells. We report an approach inspired by the posttranslational acetylation/deacetylation of lysine residues, in which a protein encoded by a gene with an in‐frame nonsense codon at an essential lysine can be expressed in its native state only upon genetic incorporation of N ‐ϵ ‐acetyl‐l ‐Lys (AcK), and subsequent enzymatic deacetylation in the host cell. We applied this strategy to two essential E. coli enzymes: the branched‐chain aminotransferase BCAT and the DNA replication initiator protein DnaA. We also devised a barnase‐based conditional suicide switch to further lower the escape frequency of the host cells. This strategy offers a number of attractive features for controlling host viability, including a single small‐molecule‐based kill switch, low escape frequency, and unaffected protein function.     

Development of Ubiquitin‐Based Probe for Metalloprotease Deubiquitinases

Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn²⁺ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn²⁺ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes.     

Protein glycoengineering enabled by the versatile synthesis of aminooxy glycans and the genetically encoded aldehyde tag

Homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development. The use of unnatural protein-glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins. Here we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages. We employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site. Complex aminooxy glycans were synthesized using a new route that features N-pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically. We demonstrated the method by constructing site-specifically glycosylated variants of the human growth hormone.     

Traceless Purification and Desulfurization of Tau Protein Ligation Products

We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin–protein conjugate on streptavidin‐coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On‐bead desulfurization is followed by delivery of the final tag‐free protein product. The strategy is demonstrated in the isolation of a tag‐free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C‐terminus of tau.     

NMR Analysis of Apo Glutamine‐Binding Protein Exposes Challenges in the Study of Interdomain Dynamics

Glutamine‐binding protein (GlnBP) displays an apo, “open” and a holo, “closed” crystal form, mutually related by a rigid‐body reorientation of its domains. A fundamental question about such large‐scale conformational transitions, whether the closed state exists in the absence of ligand, is controversial in the case of GlnBP. NMR observations have indicated no evidence of the closed form, whereas experimentally validated computations have suggested a remarkable ca. 40 % population. Herein, a paramagnetic NMR strategy designed to detect the putative apo‐closed species shows that a major population of the latter is highly improbable. Further, NMR residual dipolar couplings collected under three anisotropic conditions do not reveal differential domain alignment and establish that the average solution conformation is satisfied by the apo‐open crystal structure. Our results indicate that the computational prediction of large‐scale interdomain motions is not trivial and may lead to erroneous conclusions without proper experimental validation.     

Direct Evidence for the Polymeric Nature of Polydopamine

Inspired by the adhesive proteins of mussels, polydopamine (pDA) has emerged as one of the most widely employed materials for surface functionalization. Despite numerous attempts at characterization, little consensus has emerged regarding whether pDA is a covalent polymer or a noncovalent aggregate of low molecular weight species. Here, we employed single‐molecule force spectroscopy (SMFS) to characterize pDA films. Retraction of a pDA‐coated cantilever from an oxide surface shows the characteristic features of a polymer with contour lengths of up to 200 nm. pDA polymers are generally weakly bound to the surface through much of their contour length, with occasional “sticky” points. Our findings represent the first direct evidence for the polymeric nature of pDA and provide a foundation upon which to better understand and tailor its physicochemical properties.     

An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic,Anionic, Non‐Viral Transfection Method

Circulating nucleic acids, such as short interfering RNA (siRNA), regulate many biological processes; however, the mechanism by which these molecules enter the cell is poorly understood. The role of extracellular‐matrix‐derived polymers in binding siRNAs and trafficking them across the plasma membrane is reported. Thermal melting, dynamic light scattering, scanning electron microscopy, and computational analysis indicate that hyaluronic acid can stabilize siRNA via hydrogen bonding and Van der Waals interactions. This stabilization facilitated HA size‐ and concentration‐dependent gene silencing in a CD44‐positive human osteosarcoma cell line (MG‐63) and in human mesenchymal stromal cells (hMSCs). This native HA‐based siRNA transfection represents the first report on an anionic, non‐viral delivery method that resulted in approximately 60 % gene knockdown in both cell types tested, which correlated with a reduction in translation levels.     

The Dual‐Stereocontrol Mechanism: Heteroselective Polymerization of rac‐Lactide and Syndioselective Polymerization of meso‐Lactide by Chiral Aluminum Salan Catalysts

We describe alkoxo‐aluminum catalysts of chiral bipyrrolidine‐based salan ligands that follow the dual‐stereocontrol mechanism wherein a given combination of stereogeneities at the metal site and the proximal center of the last inserted lactidyl (“match”) is active towards lactide having a proximal stereogenic center of the opposite configuration, while the diastereomeric combination of stereogeneities (“mismatch”) is inactive towards any lactide. Polymerization of rac‐LA by the enantiomerically pure catalysts was sluggish and gave stereoirregular poly(lactic acid) (PLA) because selective insertion to a match diastereomer gives a mismatch diastereomer. The racemic catalysts showed higher activity and led to highly heterotactic PLA following polymeryl exchange between two mismatched catalyst enantiomers. A succession of match diastereomers in selective meso‐LA insertions led to syndiotactic PLAs reaching a syndiotacticity degree of α=0.96. This polymer featured a T_m of 153 °C matching the highest reported value, and the highest crystallinity (ΔH_m=56 J g^?1) ever reported for syndiotactic PLA.     

Enhanced Fluorescence Imaging and Photodynamic Cancer Therapy Using Hollow Mesoporous Nanocontainers

Here we show that “off‐on” type of photodynamic therapy agents could be developed using hollow mesoporous silica nanoparticles (HMSNPs), which can be used not only for enhancing delivery of photosensitizers to cancer cells but also for enabling switchable optical properties of the photosensitizers. Fluorescence and singlet oxygen generation of the photosensitizer‐loaded HMSNP are turned off in its native state. In vitro cell studies showed that this HMSNP‐based “off‐on” agent may have potential utility in selective fluorescence detection and photodynamic therapy of cancers.     

Developing A “Polysulfide‐Phobic” Strategy to Restrain Shuttle Effect in Lithium–Sulfur Batteries

Inspired by hydrophobic interface, a novel design of “polysulfide‐phobic” interface was proposed and developed to restrain shuttle effect in lithium–sulfur batteries. Two‐dimensional VOPO₄ sheets with adequate active sites were employed to immobilize the polysulfides through the formation of a V?S bond. Moreover, owing to the intrinsic Coulomb repulsion between polysulfide anions, the surface anchored with polysulfides can be further evolved into a “polysulfide‐phobic” interface, which was demonstrated by the advanced time/space‐resolved operando Raman evidences. In particular, by introducing the “polysulfide‐phobic” surface design into separator fabrication, the lithium–sulfur battery performed a superior long‐term cycling stability. This work expands a novel strategy to build a “polysulfide‐phobic” surface by “self‐defense” mechanism for suppressing polysulfides shuttle, which provides new insights and opportunities to develop advanced lithium–sulfur batteries.     

Recognition‐then‐Reaction Enables Site‐Selective Bioconjugation to Proteins on Live‐Cell Surfaces

Site‐selective protein modification is a key step in facilitating protein functionalization and manipulation. To accomplish this, genetically engineered proteins were previously required, but the procedure was laborious, complex, and technically challenging. Herein we report the development of aptamer‐based recognition‐then‐reaction to guide site‐selective protein/DNA conjugation in a single step with outstanding selectivity and efficiency. As models, several proteins, including human thrombin, PDGF‐BB, Avidin, and His‐tagged recombinant protein, were studied, and the results showed excellent selectivity under mild reaction conditions. Taking advantage of aptamers as recognition elements with extraordinary selectivity and affinity, this simple preparation method can tag a protein in a complex milieu. Thus, with the aptamer obtained from cell‐SELEX, real‐time modification of live‐cell membrane proteins can be achieved in one step without any pre‐treatment.