Detection of TurboID fusion proteins by fluorescent streptavidin outcompetes antibody signals and visualises targets not accessible to antibodies.

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

A plug-and-play monofunctional platform for targeted degradation of extracellular proteins and vesicles.

Existing strategies use bifunctional chimaeras to mediate extracellular protein degradation. However, these strategies rely on specific lysosome-trafficking receptors to facilitate lysosomal delivery, which may raise resistance concerns due to intrinsic cell-to-cell variation in receptor expression and mutations or downregulation of the receptors. Another challenge is establishing a universal platform applicable in multiple scenarios. Here, we develop MONOTAB (MOdified NanOparticle with TArgeting Binders), a plug-and-play monofunctional degradation platform that can drag extracellular targets into lysosomes for degradation. MONOTAB harnesses the inherent lysosome-targeting ability of certain nanoparticles to obviate specific receptor dependency and the hook effect. To achieve high modularity and programmable target specificity, we utilize the streptavidin-biotin interaction to immobilize antibodies or other targeting molecules on nanoparticles, through an antibody mounting approach or by direct binding. Our study reveals that MONOTAB can induce efficient degradation of diverse therapeutic targets, including membrane proteins, secreted proteins, and even extracellular vesicles.

Proximity-driven site-specific cyclization of phage-displayed peptides.

Cyclization provides a general strategy for improving the proteolytic stability, cell membrane permeability and target binding affinity of peptides. Insertion of a stable, non-reducible linker into a disulphide bond is a commonly used approach for cyclizing phage-displayed peptides. However, among the vast collection of cysteine reactive linkers available, few provide the selectivity required to target specific cysteine residues within the peptide in the phage display system, whilst sparing those on the phage capsid. Here, we report the development of a cyclopropenone-based proximity-driven chemical linker that can efficiently cyclize synthetic peptides and peptides fused to a phage-coat protein, and cyclize phage-displayed peptides in a site-specific manner, with no disruption to phage infectivity. Our cyclization strategy enables the construction of stable, highly diverse phage display libraries. These libraries can be used for the selection of high-affinity cyclic peptide binders, as exemplified through model selections on streptavidin and the therapeutic target αvß3.

Redox-active polyoxovanadates as cofactors in the development of functional protein assemblies.

The interactions of polyoxovanadates (POVs) with proteins have increasingly attracted interest in recent years due to their potential biomedical applications. This is especially the case because of their redox and catalytic properties, which make them interesting for developing artificial metalloenzymes. Organic-inorganic hybrid hexavanadates in particular offer several advantages over all-inorganic POVs. However, they have been scarcely investigated in biological systems even though, as shown in this work, hybrid hexavanadates are highly stable in aqueous solutions up to relatively high pH. Therefore, a novel bis-biotinylated hexavanadate was synthesized and shown to selectively interact with two biotin-binding proteins, avidin and streptavidin. Bridging interactions between multiple proteins led to their self-assembly into supramolecular bio-inorganic hybrid systems that have potential as artificial enzymes with the hexavanadate core as a redox-active cofactor. Moreover, the structure and charge of the hexavanadate core were determined to enhance the binding affinity and slightly alter the secondary structure of the proteins, which affected the size and speed of formation of the assemblies. Hence, tuning the polyoxometalate (POM) core of hybrid POMs (HPOMs) with protein-binding ligands has been demonstrated to be a potential strategy for controlling the self-assembly process while also enabling the formation of novel POM-based biomaterials that could be of interest in biomedicine.

Phage-induced "one-to-many" FRET sensor for highly sensitive detection of Escherichia coli O157:H7.

As a foodborne pathogen capable of causing severe illnesses, early detection of Escherichia coli O157:H7 (E. coli O157:H7) is crucial for ensuring food safety. While Förster resonance energy transfer (FRET) is an efficient and precise detection technique, there remains a need for amplification strategies to detect low concentrations of E. coli O157:H7. In this study, we presented a phage (M13)-induced "one to many" FRET platform for sensitively detecting E. coli O157:H7. The aptamers, which specifically recognize E. coli O157:H7 were attached to magnetic beads as capture probes for separating E. coli O157:H7 from food samples. The peptide O157S, which specifically targets E. coli O157:H7, and streptavidin binding peptide (SBP), which binds to streptavidin (SA), were displayed on the P3 and P8 proteins of M13, respectively, to construct the O157S-M13K07-SBP phage as a detection probe for signal output. Due to the precise distance (≈3.2 nm) between two neighboring N-terminus of P8 protein, the SA-labeled FRET donor and acceptor can be fixed at the Förster distance on the surface of O157S-M13K07-SBP via the binding of SA and SBP, inducing FRET. Moreover, the P8 protein, with ≈2700 copies, enabled multiple FRET (≈605) occurrences, amplifying FRET in each E. coli O157:H7 recognition event. The O157S-M13K07-SBP-based FRET sensor can detect E. coli O157:H7 at concentration as low as 6 CFU/mL and demonstrates excellent performance in terms of selectivity, detection time (≈3 h), accuracy, precision, practical application, and storage stability. In summary, we have developed a powerful tool for detecting various targets in food safety, environmental monitoring, and medical diagnosis.

Signal-On Detection of Caspase-3 with Methylene Blue-Loaded Metal-Organic Frameworks as Signal Reporters.

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal-organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.

Copolymer-Coated Gold Nanoparticles: Enhanced Stability and Customizable Functionalization for Biological Assays.

Gold nanoparticles (AuNPs) play a vital role in biotechnology, medicine, and diagnostics due to their unique optical properties. Their conjugation with antibodies, antigens, proteins, or nucleic acids enables precise targeting and enhances biosensing capabilities. Functionalized AuNPs, however, may experience reduced stability, leading to aggregation or loss of functionality, especially in complex biological environments. Additionally, they can show non-specific binding to unintended targets, impairing assay specificity. Within this work, citrate-stabilized and silica-coated AuNPs (GNPs and SiGNPs, respectively) have been coated using N,N-dimethylacrylamide-based copolymers to increase their stability and enable their functionalization with biomolecules. AuNP stability after modification has been assessed by a combination of techniques including spectrophotometric characterization, nanoparticle tracking analysis, transmission electron microscopy and functional microarray tests. Two different copolymers were identified to provide a stable coating of AuNPs while enabling further modification through click chemistry reactions, due to the presence of azide groups in the polymers. Following this experimental design, AuNPs decorated with ssDNA and streptavidin were synthesized and successfully used in a biological assay. In conclusion, a functionalization scheme for AuNPs has been developed that offers ease of modification, often requiring single steps and short incubation time. The obtained functionalized AuNPs offer considerable flexibility, as the functionalization protocol can be personalized to match requirements of multiple assays.

Super-resolution Multispectral Imaging Nanoimmunosensor for Simultaneous Detection of Diverse Early Cancer Biomarkers.

In medical diagnosis, relying on only one type of biomarker is insufficient to accurately identify cancer. Blood-based multicancer early detection can help identify more than one type of cancer from a single blood sample. In this study, a super-resolution multispectral imaging nanoimmunosensor (srMINI) based on three quantum dots (QDs) of different color conjugated with streptavidin was developed for the simultaneous screening of various cancer biomarkers in blood at the single-molecule level. In the experiment, the srMINI chip was used to simultaneously detect three key cancer biomarkers: carcinoembryonic antigen (CEA), C-reactive protein (CRP), and alpha-fetoprotein (AFP). The srMINI chip exhibited 108 times higher detection sensitivity of 0.18-0.5 ag/mL (1.1-2.6 zM) for these cancer biomarkers than commercial enzyme-linked immunosorbent assay kits because of the absence of interfering signals from the substrate, establishing considerable potential for multiplex detection of cancer biomarkers in blood. Therefore, the simultaneous detection of various cancer biomarkers using the developed srMINI chip with high diagnostic precision and accuracy is expected to play a decisive role in early diagnosis or community screening as a single-molecule biosensor.

Concanavalin A Delivers a Photoactive Protein to the Bacterial Wall.

Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli. Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.

Polymers of functionalized diaminopropionic acid are efficient mediators of active exogenous enzyme delivery into cells.

Delivery of active protein especially enzyme is one of the major therapeutic challenge. Replacing or substituted invalid/improper acting protein offer fast and effective treatment of disease. Herein, we describe the synthesis and properties of biotinylated peptidomimetics consisting of oxoacid-modified 2,3, L-diaminopropionic acid residues with guanidine groups on its side chains. Electrophoretic analysis showed that the obtained compounds interact with FITC-labeled streptavidin or a streptavidin-ß-galactosidase hybrid in an efficient manner. Complexes formed by the abovementioned molecules are able to cross the cell membranes of cancer or healthy cells and show promising compatibility with live cells. Analysis of ß-galactosidase activity inside the cells revealed surprisingly high levels of active enzyme in complex-treated cells compared to controls. This observation was confirmed by immunochemical studies in which the presence of ß-galactosidase was detected in the membrane and vesicles of the cells.

Binding Site Programmable Self-Assembly of 3D Hierarchical DNA Origami Nanostructures.

DNA nanotechnology has broad applications in biomedical drug delivery and programmable materials. Characterization of the self-assembly of DNA origami and quantum dots (QDs) is necessary for the development of new DNA-based nanostructures. We use computation and experiment to show that the self-assembly of 3D hierarchical nanostructures can be controlled by programming the binding site number and their positions on DNA origami. Using biotinylated pentagonal pyramid wireframe DNA origamis and streptavidin capped QDs, we demonstrate that DNA origami with 1 binding site at the outer vertex can assemble multimeric origamis with up to 6 DNA origamis on 1 QD, and DNA origami with 1 binding site at the inner center can only assemble monomeric and dimeric origamis. Meanwhile, the yield percentages of different multimeric origamis are controlled by the QD:DNA-origami stoichiometric mixing ratio. DNA origamis with 2 binding sites at the αγ positions (of the pentagon) make larger nanostructures than those with binding sites at the αß positions. In general, increasing the number of binding sites leads to increases in the nanostructure size. At high DNA origami concentration, the QD number in each cluster becomes the limiting factor for the growth of nanostructures. We find that reducing the QD size can also affect the self-assembly because of the reduced access to the binding sites from more densely packed origamis.

Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

Learning Protein-Ligand Unbinding Pathways via Single-Parameter Community Detection.

Understanding the dynamics of biomolecular complexes, e.g., of protein-ligand (un)binding, requires the comprehension of paths such systems take between metastable states. In MD simulations, paths are usually not observable per se, but they need to be inferred from simulation trajectories. Here, we present a novel approach to cluster trajectories based on a community detection algorithm that necessitates only the definition of a single parameter. The unbinding of the streptavidin-biotin complex is used as a benchmark system and the A2a adenosine receptor in complex with the inhibitor ZM241385 as an elaborate application. We demonstrate how such clusters of trajectories correspond to pathways and how the approach helps in the identification of reaction coordinates for a considered (un)binding process.

Evaluation of a Commercial TIMS-Q-TOF Platform for Native Mass Spectrometry.

Mass-spectrometry based assays in structural biology studies measure either intact or digested proteins. Typically, different mass spectrometers are dedicated for such measurements: those optimized for rapid analysis of peptides or those designed for high molecular weight analysis. A commercial trapped ion mobility-quadrupole-time-of-flight (TIMS-Q-TOF) platform is widely utilized for proteomics and metabolomics, with ion mobility providing a separation dimension in addition to liquid chromatography. The ability to perform high-quality native mass spectrometry of protein complexes, however, remains largely uninvestigated. Here, we evaluate a commercial TIMS-Q-TOF platform for analyzing noncovalent protein complexes by utilizing the instrument's full range of ion mobility, MS, and MS/MS (both in-source activation and collision cell CID) capabilities. The TIMS analyzer is able to be tuned gently to yield collision cross sections of native-like complexes comparable to those previously reported on various instrument platforms. In-source activation and collision cell CID were robust for both small and large complexes. TIMS-CID was performed on protein complexes streptavidin (53 kDa), avidin (68 kDa), and cholera toxin B (CTB, 58 kDa). Complexes pyruvate kinase (237 kDa) and GroEL (801 kDa) were beyond the trapping capabilities of the commercial TIMS analyzer, but TOF mass spectra could be acquired. The presented results indicate that the commercial TIMS-Q-TOF platform can be used for both omics and native mass spectrometry applications; however, modifications to the commercial RF drivers for both the TIMS analyzer and quadrupole (currently limited to m/z 3000) are necessary to mobility analyze protein complexes greater than about 60 kDa.

Impact of sulfur substitution on biotin binding affinity to streptavidin.

In this study, we investigated how the replacement of the tetrahydrothiophene ring of biotin with either an oxolane or (methyl)pyrrolidine moiety may affect its molecular interactions, in an effort to identify alternative affinity ligands suitable for in vitro and in vivo applications in synthetic biology. Initial molecular dynamics (MD) simulations suggested the potential formation of a hydrogen bond between either the oxygen or nitrogen atom of the envisaged tetrahydroheteryl analogues and the Thr90 residue of streptavidin, mirroring the sulfur-centered hydrogen bond detected by the crystallographic analysis of the biotin-streptavidin interaction. Therefore, oxy-, aza-, and N-methylazabiotin were readily synthesized starting from chiral five- or six-carbon sugar precursors. Based on fluorescence-based titration experiments using the corresponding fluorescein conjugates, oxybiotin showed a binding behavior similar to biotin with streptavidin, while both amino analogues displayed lower binding capacities. Notably, azabiotin exhibited a pH-dependent interaction profile, demonstrating enhanced binding under acidic conditions but weaker binding under basic pH, which could be exploited for various purposes.

Tumor Pre-Targeting System Using Streptavidin-Expressing Bacteria.

PURPOSE: A major obstacle to targeted cancer therapy is identifying suitable targets that are specifically and abundantly expressed by solid tumors. Certain bacterial strains selectively colonize solid tumors and can deliver genetically encoded cargo molecules to the tumor cells. Here, we engineered bacteria to express monomeric streptavidin (mSA) in tumors, and developed a novel tumor pre-targeting system by visualizing the presence of tumor-associated mSA using a biotinylated imaging probe. PROCEDURES: We constructed a plasmid expressing mSA fused to maltose-binding protein and optimized the ribosome binding site sequence to increase solubility and expression levels. E. coli MG1655 was transformed with the recombinant plasmid, expression of which is driven by the pBAD promotor. Expression of mSA was induced by L-arabinose 4 days after injection of bacteria into mice bearing CT26 mouse colon carcinoma cells. Selective accumulation of mSA in tumor tissues was visualized by optical imaging after administration of a biotinylated fluorescent dye. Counting of viable bacterial cells was also performed. RESULTS: Compared with a conventional system, the novel expression system resulted in significantly higher expression of mSA and sustained binding to biotin. Imaging signals in tumor tissues were significantly stronger in the mSA-expressing group than in non-expressing group (P = 0.0005). Furthermore, the fluorescent signal in tumor tissues became detectable again after multiple inductions with L-arabinose. The bacterial counts in tumor tissues showed no significant differences between conditions with and without L-arabinose (P = 0.45). Western blot analysis of tumor tissues confirmed expression and binding of mSA to biotin. CONCLUSIONS: We successfully engineered tumor-targeting bacteria carrying a recombinant plasmid expressing mSA, which was targeted to, and expressed in, tumor tissues. These data demonstrate the potential of this novel tumor pre-targeting system when combined with biotinylated imaging probes or therapeutic agents.

Construction of a streptavidin-based dual-localized DNAzyme walker for disease biomarker detection.

A dual-localized DNAzyme walker (dlDW) was constructed by utilizing multiple split DNAzymes with probes, and their substrates are separately localized on streptavidin and AuNPs, serving as walking pedals and tracks, respectively. Based on dlDW, biosensing platform was successfully constructed and showed great potential application in clinical disease diagnosis.

Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1ß, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Interface-constrained catalytic hairpin assembly permits highly sensitive SERS signaling of miRNA.

The rapid and precise monitoring of peripheral blood miRNA levels holds paramount importance for disease diagnosis and treatment monitoring. In this study, we propose an innovative research strategy that combines the catalytic hairpin assembly reaction with SERS signal congregation and enhancement. This combination can significantly enhance the stability of SERS detection, enabling stable and efficient detection of miRNA. Specifically, our paper-based SERS detection platform incorporates a streptavidin-modified substrate, biotin-labeled catalytic hairpin assembly reaction probes, 4-ATP, and primer-co-modified gold nanoparticles. In the presence of miRNA, the 4-ATP and primer-co-modified gold nanoparticles can specifically recognize the miRNA and interact with the biotin-labeled CHA probes to initiate an interfacial catalytic hairpin assembly reaction. This enzyme-free high-efficiency catalytic process can accumulate a large amount of biotin on the gold nanoparticles, which then bind to the streptavidin on the substrate with the assistance of the driving liquid, forming red gold nanoparticle stripes. These provide a multitude of hotspots for SERS, enabling enhanced signal detection. This innovative design achieves a low detection limit of 3.47 fM while maintaining excellent stability and repeatability. This conceptually innovative detection platform offers new technological possibilities and solutions for clinical miRNA detection.

Label-free OIRD detection of protein microarrays on high dielectric constant substrate with enhanced intrinsic sensitivity.

Oblique-incidence reflectivity difference (OIRD) is a dielectric constant-sensitive technique and exhibits intriguing applications in label-free and high-throughput detection of protein microarrays. With the outstanding advantage of being compatible with arbitrary substrates, however, the effect of the substrate, particularly its dielectric constant on the OIRD sensitivity has not been fully disclosed. In this paper, for the first time we investigated the dependence of OIRD sensitivity on the dielectric constant of the substrate under top-incident OIRD configuration by combining theoretical modeling and experimental evaluation. Optical modeling suggested that the higher dielectric constant substrate exhibits a higher intrinsic sensitivity. Experimentally, three substrates including glass, fluorine-doped tin oxide (FTO) and silicon (Si) with different dielectric constants were selected as microarray substrates and their detection performances were evaluated. In good agreement with the modeling, high dielectric constant Si-based microarray exhibited the highest sensitivity among three chips, reaching a detection limit of as low as 5 ng mL-1 with streptavidin as the model target. Quantification of captured targets on three chips with on-chip enzyme-linked immunosorbent assay (ELISA) further confirmed that the enhanced performance originates from the high dielectric constant enhanced intrinsic OIRD sensitivity. This work thus provides a new way to OIRD-based label-free microarrays with improved sensitivity.