Stepwise Assembly Research Articles

Stepwise Monomicelle Assembly for Highly Ordered Mesoporous TiO2 Membranes with Precisely Tailored Mesophase and Porosity.

Mesoporous materials with crystalline frameworks have been acknowledged as very attractive materials in various applications. Nevertheless, due to the cracking issue during crystallization and incompatible hydrolysis and assembly, the precise control for crystalline mesoscale membranes is quite infertile. Herein, we presented an ingenious stepwise monomicelle assembly route for the syntheses of highly ordered mesoporous crystalline TiO2 membranes with delicately controlled mesophase, mesoporosity, and thickness. Such a process involves the preparation of monomicelle hydrogels and follows self-assembly by stepwise solvent evaporation, which enables the sensitive hydrolysis of TiO2 oligomers and dilatory micelle assembly to be united. In consequence, the fabricated mesoporous TiO2 membranes exhibit a broad flexibility, including tunable ordered mesophases (worm-like, hexagonal p6mm to body-centered cubic Im3̅m), controlled mesopore sizes (3.0-8.0 nm), and anatase grain sizes (2.3-8.4 nm). Besides, such mesostructured crystalline TiO2 membranes can be extended to diverse substrates (Ti, Ag, Si, FTO) with tailored thickness. The great mesoporosity of the in situ fabricated mesoscopic membranes also affords excellent pseudocapacitive behavior for sodium ion storage. This study underscores a novel pathway for balancing the interaction of precursors and micelles, which could have implications for synthesizing crystalline mesostructures in higher controllability.

Total Synthesis of Starfish Cyclic Steroid Glycosides. Part 1, Synthesis of the Aglycone and Attempts at Installation of the Sugar Units

Comprehensive SummarySepositoside A (1) is the major and first cyclic steroid glycoside isolated from starfishes, which features a strained 16‐membered ring formed by tethering C3 and C6 of the steroid aglycone with a linear trisaccharide. We conceived a convergent synthetic plan in which sepositoside A was disconnected into a disaccharide and a steroid‐glucose hybrid ether. The desired 3β,6β‐dihydroxy‐Δ7,8‐23‐ one aglcyone was readily prepared from dehydroepiandrosterone in 12 steps. The etherification of the 6β‐hydroxy‐Δ7,8‐steroid with a variety of hexopyranoside‐C6‐electrophiles and CO2‐extrusion of a steroid‐pyranose hybrid carbonate were extensively studied; however, formation of the desired steroid‐pyranose etherate linkage has not been successful. Model studies on the β‐selective installation of the (1→2)‐linked disaccharide onto the steroidal C3β‐OH with the corresponding disaccharide α‐imidates were also examined, however, formation of the α‐glycosides prevailed. The present results force us to explore alternative approaches to construct the steroid‐pyranose etherate linkage and stepwise assembly of the glycans en route to the starfish cyclic steroid glycosides, which will be presented in two articles that follow.

Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly

The SNARE complex assembles from vesicular Synaptobrevin-2 as well as Syntaxin-1 and SNAP25 both anchored to the presynaptic membrane. It mediates fusion of synaptic vesicles with the presynaptic plasma membrane resulting in exocytosis of neurotransmitters. While the general sequence of SNARE complex formation is well-established, our knowledge on possible intermediates and stable off-pathway complexes is incomplete. We, therefore, follow the stepwise assembly of the SNARE complex and target individual SNAREs, binary sub-complexes, the ternary SNARE complex as well as interactions with Complexin-1. Using native mass spectrometry, we identify the stoichiometry of sub-complexes and monitor oligomerisation of various assemblies. Importantly, we find that interactions with Complexin-1 reduce multimerisation of the ternary SNARE complex. Chemical cross-linking provides detailed insights into these interactions suggesting a role for membrane fusion. In summary, we unravel the stoichiometry of intermediates and off-pathway complexes and compile a road map of SNARE complex assembly including regulation by Complexin-1.

Stepwise Growth of Copper-Based Coordination Polymer on α-MnO2 Nanorods for Sustainable Reduction of Nitroarenes

Metal oxide/coordination polymer composites have gained great attraction due to their synergistic effects to increase their efficiency in various fields. In this work, a Cu-based coordination polymer (Cu-CP) was grown on the surface of α-MnO2 nanorods via stepwise assembly of benzene-1,4-dicarboxylic acid (H2BDC) as a ligand and a Cu(II) ion as a metal center. Comprehensive characterization techniques confirmed the successful growth of Cu-CP on the surface of α-MnO2 without affecting the morphology of pristine α-MnO2 nanorods. The obtained nanocomposite (α-MnO2/Cu-CP) revealed outstanding activity in the catalytic reduction of nitroarenes to aminoarenes under mild reaction conditions and afforded wide substrate applicability (more than 99% for nitrobenzene and 70–99% for other substituted nitroarenes during 1 h at room temperature). Importantly, α-MnO2/Cu-CP served as a better catalyst than bare α-MnO2 and Cu-CP due to the synergistic effects between building components. In addition, α-MnO2/Cu-CP was easily separable by centrifugation and its catalytic activity remained unaffected over five cycles. The use of water as a sustainable and green solvent and non-toxic and inexpensive metals used for the synthesis of the catalyst along with easy recovery and stability of the catalyst make the present protocol economical and sustainable. The time-dependent reduction kinetics followed pseudo-first-order kinetics with respect to the substrates.

Stepwise assembly of the active site of [NiFe]-hydrogenase.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2e- and 2H+ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN- molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

Novel biomimetic Prussian blue nanocubes-based biosensor for Tau-441 protein detection

Tau protein is a promising biomarker for early diagnosis of Alzheimer's disease. Therefore, there is an urgent need to develop a simple and effective method for its detection. To this end, an innovative sensing device was developed using a carbon screen-printed electrode (C-SPE) decorated with graphene oxide/Prussian Blue nanocubes (GO/PBNCs) for the selective and sensitive determination of Tau-441 protein. The molecular imprinting polymer (MIP) was built on the GO/PBNCs/C-SPE by electropolymerizing 3-aminophenol (3-AMP) in the presence of the target protein using chronoamperometry, and the template was subsequently removed from the polymer matrix with oxalic acid. In parallel, a non-imprinted material (NIP) was also prepared in the absence of the target for comparison purposes. Scanning electron microscopy and transmission electron microscopy, were used to study the morphology of the modified electrode and electrochemical techniques were used to monitor the stepwise assembly of the sensor. Under optimized conditions, the sensing platform exhibited a linear range within 1.09 and 2.18 nmol/L and a detection limit of 0.01 pmol/L in spiked phosphate buffer solution (PBS). The MIP sensor showed minimal interference with uric acid and bovine albumin. The simplicity of production, affordable cost and promising performance make this sensor a potential strategic sensing platform for the detection of chemical and biological molecules.

High Seebeck coefficient from isolated oligo-phenyl arrays on single layered graphene via stepwise assembly

Organic thin films composed of highly ordered molecular arrays hold tremendous potential for thermoelectric energy harvesting.

Synthesis of the diazatricycloundecane scaffold via gold(I)-catalysed Conia-ene-type 5-exo-dig cyclization and stepwise substituent assembly for the construction of an sp3-rich compound library.

The bridged diazatricycloundecane sp3-rich scaffold was synthesised via the gold(I)-catalysed Conia-ene reaction. The electron-donating property of the siloxymethyl group on alkyne 1 enabled 6-endo-dig cyclization, whereas the ethoxy carbonyl group on alkyne 4 led to 5-exo-dig cyclization with complete regioselectivity in the Conia-ene reaction. The resulting bridged diazatricycloundecane scaffold 5 allowed the construction of a library of sp3-rich compounds. Among the compounds synthesised, compounds 6e and 6f inhibited the hypoxia inducible factor 1 (HIF-1) downstream signaling pathway without affecting HIF-1α mRNA expression.

Stepwise assembly of thiacalix[4]arene-protected Ag/Ti bimetallic nanoclusters: accurate identification of catalytic Ag sites in CO2 electroreduction.

The accurate identification of catalytic sites in heterogeneous catalysts poses a significant challenge due to the intricate nature of controlling interfacial chemistry at the molecular level. In this study, we introduce a novel strategy to address this issue by utilizing a thiacalix[4]arene (TC4A)-protected Ti-oxo core as a template for loading Ag1+ ions, leading to the successful synthesis of a unique Ag/Ti bimetallic nanocluster denoted as Ti8Ag8. This nanocluster exhibits multiple surface-exposed Ag sites and possesses a distinctive "core-shell" structure, consisting of a {Ti4@Ag8(TC4A)4} core housing a {Ti2O2@Ag4(TC4A)2} motif and two {Ti@Ag2(TC4A)} motifs. To enable a comprehensive analysis, we also prepared a Ti2Ag4 cluster with the same {Ti2O2@Ag4(TC4A)2} structure found within Ti8Ag8. The structural disparities between Ti8Ag8 and Ti2Ag4 provide an excellent platform for a comparison of catalytic activity at different Ag sites. Remarkably, Ti8Ag8 exhibits exceptional performance in the electroreduction of CO2 (eCO2RR), showcasing a CO faradaic efficiency (FECO) of 92.33% at -0.9 V vs. RHE, surpassing the FECO of Ti2Ag4 (69.87% at -0.9 V vs. RHE) by a significant margin. Through density functional theory (DFT) calculations, we unveil the catalytic mechanism and further discover that Ag active sites located at {Ti@Ag2(TC4A)} possess a higher εd value compared to those at {Ti2O2@Ag4(TC4A)2}, enhancing the stabilization of the *COOH intermediate during the eCO2RR. This study provides valuable insights into the accurate identification of catalytic sites in bimetallic nanoclusters and opens up promising avenues for efficient CO2 reduction catalyst design.

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion-exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen evolving activity of approx. 6000 μmol O2·(mg Chl·h)−1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen evolving activity of approx. 3000 μmol O2·(mg Chl·h)−1 and a manganese content of 2.7 (±0.2) per reaction center compared to 3.7 (±0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role of this complex in PSII repair.

The origin of a land flora.

The origin of a land flora fundamentally shifted the course of evolution of life on earth, facilitating terrestrialization of other eukaryotic lineages and altering the planet's geology, from changing atmospheric and hydrological cycles to transforming continental erosion processes. Despite algal lineages inhabiting the terrestrial environment for a considerable preceding period, they failed to evolve complex multicellularity necessary to conquer the land. About 470 million years ago, one lineage of charophycean alga evolved complex multicellularity via developmental innovations in both haploid and diploid generations and became land plants (embryophytes), which rapidly diversified to dominate most terrestrial habitats. Genome sequences have provided unprecedented insights into the genetic and genomic bases for embryophyte origins, with some embryophyte-specific genes being associated with the evolution of key developmental or physiological attributes, such as meristems, rhizoids and the ability to form mycorrhizal associations. However, based on the fossil record, the evolution of the defining feature of embryophytes, the embryo, and consequently the sporangium that provided a reproductive advantage, may have been most critical in their rise to dominance. The long timeframe and singularity of a land flora were perhaps due to the stepwise assembly of a large constellation of genetic innovations required to conquer the terrestrial environment.

Cms1 coordinates stepwise local 90S pre-ribosome assembly with timely snR83 release.

Ribosome synthesis begins in the nucleolus with 90S pre-ribosome construction, but little is known about how the many different snoRNAs that modify the pre-rRNA are timely guided to their target sites. Here, we report a role for Cms1 in such a process. Initially, we discovered CMS1 as a null suppressor of a nop14 mutant impaired in Rrp12-Enp1 factor recruitment to the 90S. Further investigations detected Cms1 at the 18S rRNA 3' major domain of an early 90S that carried H/ACA snR83, which is known to guide pseudouridylation at two target sites within the same subdomain. Cms1 co-precipitates with many 90S factors, but Rrp12-Enp1 encircling the 3' major domain in the mature 90S is decreased. We suggest that Cms1 associates with the 3' major domain during early 90S biogenesis to restrict premature Rrp12-Enp1 binding but allows snR83 to timely perform its modification role before the next 90S assembly steps coupled with Cms1 release take place.

Catalytic hairpin assembly-mediated Cu2O nanocubes as the competitive dual-quenching tags for photoelectrochemical bioassay of miR-141

MicroRNAs (miR) as newly identified biomarkers are closely associated with various cancers, and sensitive monitoring of abnormal miR is great importance in cancer diagnosis. In this work, a novel photoelectrochemical biosensor was developed to sensitively detect miR-141 by the competitive dual-quenching of catalytic hairpin assembly (CHA)-dependent Cu2O nanocubes (NCs) toward β-CD-functionalized CdS nanorods (β-CD@CdS NRs). The biosensing platform was constructed by the stepwise assembly of β-CD@CdS NRs and adamantane-labeled hairpin DNA probe 1 on ITO electrode. In the presence of miR-141, CHA recycling was triggered to immobilize the capture DNA-labeled Cu2O NCs with the aid of hairpin DNA probe 2. The assembly of Cu2O NCs could largely reduce the photocurrent response of CdS NRs based on the competitive consumption of light energy and electron donor. The designed biosensor showed a wide linear range (1.0 pM–100 nM) and a detection limit of 0.47 pM, as well as excellent selectivity and satisfactory stability. Based on the above outcomes, the proposed biosensing device was also applied to miR-141 analysis in human serum with the recoveries ranging from 95.9 % to 104.7 %, achieving a great prospect in clinical application.

Single-molecule tracking of perfringolysin O assembly and membrane insertion uncoupling.

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore formation of the archetypical cholesterol-dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 ms temporal resolution and 20 nm spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes.

Optically Responsive Protein Coating of DNA Origami for Triggered Antigen Targeting.

DNA nanostructures have emerged as modular building blocks in several research fields including biomedicine and nanofabrication. Their proneness to degradation in various environments has led to the development of a variety of nature-inspired protection strategies. Coating of DNA origami nanostructures with proteins can circumvent degradation and alter their properties. Here, we have used a single-chain variable antibody fragment and serum albumin to construct positively charged and stimuli-responsive protein-dendron conjugates, which were complexed with DNA origami through electrostatic interactions. Using a stepwise assembly approach, the coated nanostructures were studied for their interaction with the corresponding antigen in fluorescence-based immunoassays. The results suggest that the antibody–antigen interaction can be disturbed by the addition of the bulky serum albumin. However, this effect is fully reversible upon irradiation of the structures with an optical stimulus. This leads to a selective dissociation of the serum albumin from the nanostructure due to cleavage of a photolabile group integrated in the dendron structure, exposing the antibody fragment and enabling triggered binding to the antigen, demonstrating that serum albumin can be considered as an externally controlled “camouflaging” agent. The presented stimuli-responsive complexation approach is highly versatile regarding the choice of protein components and could, therefore, find use in DNA origami protection, targeting, and delivery as well as their spatiotemporal control.

An MCIA-like complex is required for mitochondrial complex I assembly and seed development in maize

During adaptive radiation, mitochondria have co-evolved with their hosts, leading to gain or loss of subunits and assembly factors of respiratory complexes. Plant mitochondrial complex I harbors ∼40 nuclear- and 9 mitochondrial-encoded subunits, and is formed by stepwise assembly during which different intermediates are integrated via various assembly factors. In mammals, the mitochondrial complex I intermediate assembly (MCIA) complex is required for building the membrane arm module. However, plants have lost almost all of the MCIA complex components, giving rise to the hypothesis that plants follow an ancestral pathway to assemble the membrane arm subunits. Here, we characterize a maize crumpled seed mutant, crk1, and reveal by map-based cloning that CRK1 encodes an ortholog of human complex I assembly factor 1, zNDUFAF1, the only evolutionarily conserved MCIA subunit in plants. zNDUFAF1 is localized in the mitochondria and accumulates in two intermediate complexes that contain complex I membrane arm subunits. Disruption of zNDUFAF1 results in severe defects in complex I assembly and activity, a cellular bioenergetic shift to aerobic glycolysis, and mitochondrial vacuolation. Moreover, we found that zNDUFAF1, the putative mitochondrial import inner membrane translocase ZmTIM17-1, and the isovaleryl-coenzyme A dehydrogenase ZmIVD1 interact each other, and could be co-precipitated from the mitochondria and co-migrate in the same assembly intermediates. Knockout of either ZmTIM17-1 or ZmIVD1 could lead to the significantly reduced complex I stability and activity as well as defective seeds. These results suggest that zNDUFAF1, ZmTIM17-1 and ZmIVD1 probably form an MCIA-like complex that is essential for the biogenesis of mitochondrial complex I and seed development in maize. Our findings also imply that plants and mammals recruit MCIA subunits independently for mitochondrial complex I assembly, highlighting the importance of parallel evolution in mitochondria adaptation to their hosts.

In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains.

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

Development of Precisely Controlled Structures Containing Main Group Elements for Preparing Superatoms

Abstract This review summarizes our recent studies of main group elements of boron, aluminum, gallium and bismuth units with dendrimer templates. Stepwise assembly of bismuth salts realized tunable photoluminescent materials according to the introduced amounts. Solid-state emission and switching of optical properties were demonstrated by using a shell-effect of the dendrimer. BH3 species placed in the dendrimer acted as a reductant in the nanosized space to form size-controlled metal particles. Atomicity control for AlCl3 or GaCl3 enabled a solution-phase synthesis of superatoms, which have been expected to be new building blocks for materials.

Automated Fabrication of Streptavidin-Based Self-assembled Materials for High-Content Analysis of Cellular Response to Growth Factors.

The automation of liquid-handling routines offers great potential for fast, reproducible, and labor-reduced biomaterial fabrication but also requires the development of special protocols. Competitive systems demand for a high degree in miniaturization and parallelization while maintaining flexibility regarding the experimental design. Today, there are only a few possibilities for automated fabrication of biomaterials inside multiwell plates. We have previously demonstrated that streptavidin-based biomimetic platforms can be employed to study cellular behaviors on biomimetic surfaces. So far, these self-assembled materials were made by stepwise assembly of the components using manual pipetting. In this work, we introduce for the first time a fully automated and adaptable workflow to functionalize glass-bottom multiwell plates with customized biomimetic platforms deposited in single wells using a liquid-handling robot. We then characterize the cell response using automated image acquisition and subsequent analysis. Furthermore, the molecular surface density of the biomimetic platforms was characterized in situ using fluorescence-based image correlation spectroscopy. These measurements were in agreement with standard ex situ spectroscopic ellipsometry measurements. Due to automation, we could do a proof of concept to study the effect of heparan sulfate on the bioactivity of bone morphogenetic proteins on myoblast cells, using four different bone morphogenetic proteins (BMPs) (2, 4, 6, and 7) in parallel, at five increasing concentrations. Using such an automated self-assembly of biomimetic materials, it may be envisioned to further investigate the role of a large variety of extracellular matrix (ECM) components and growth factors on cell signaling.

Innentitelbild: Anion‐Guided Stepwise Assembly of High‐Nuclearity Lanthanide Hydroxide Clusters (Angew. Chem. 33/2022)

Innentitelbild: Anion‐Guided Stepwise Assembly of High‐Nuclearity Lanthanide Hydroxide Clusters (Angew. Chem. 33/2022)