Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

Combining avidin with CD63 improves basophil activation test accuracy in classifying peanut allergy.

BACKGROUND: Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS: Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS: Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS: Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Expanding the Hydrophobic Cavity Surface of Azocalix[4]arene to Enable Biotin/Avidin Affinity with Controlled Release.

The development of artificial receptors that combine ultrahigh-affinity binding and controllable release for active guests holds significant importance in biomedical applications. On one hand, a complex with an exceedingly high binding affinity can resist unwanted dissociation induced by dilution effect and complex interferents within physiological environments. On the other hand, stimulus-responsive release of the guest is essential for precisely activating its function. In this context, we expanded hydrophobic cavity surface of a hypoxia-responsive azocalix[4]arene, affording Naph-SAC4A. This modification significantly enhanced its aqueous binding affinity to 1013 M-1, akin to the naturally occurring strongest recognition pair, biotin/(strept-)avidin. Consequently, Naph-SAC4A emerges as the first artificial receptor to simultaneously integrate ultrahigh recognition affinity and actively controllable release. The markedly enhanced affinity not only improved Naph-SAC4A's sensitivity in detecting rocuronium bromide in serum, but also refined the precision of hypoxia-responsive doxorubicin delivery at the cellular level, demonstrating its immense potential for diverse practical applications.

The avidin-theophylline complex: A structural and computational study.

The interaction between avidin and its counterpart biotin is one of central importance in biology and has been reproposed and studied at length. However, the binding pocket of avidin is prone to promiscuous binding, able to accommodate even non-biotinylated ligands. Comprehending the factors that distinguish the extremely strong interaction with biotin to other ligands is an important step to fully picture the thermodynamics of these low-affinity complexes. Here, we present the complex between chicken white egg avidin and theophylline (TEP), the xanthine derivative used in the therapy of asthma. In the crystal structure, TEP lies in the biotin-binding pocket with the same orientation and planarity of the aromatic ring of 8-oxodeoxyguanosine. Indeed, its affinity for avidin measured by isothermal titration calorimetry is in the same µM range as those obtained for the previously characterized nucleoside derivatives. By the use of molecular dynamic simulations, we have investigated the most important intermolecular interactions occurring in the avidin-TEP binding pocket and compared them with those obtained for the avidin 8-oxodeoxyguanosine and avidin-biotin complexes. These results testify the capability of avidin to complex purely aromatic molecules.

Intra-articular kinetics of a cartilage targeting cationic PEGylated protein for applications in drug delivery.

OBJECTIVES: Cartilage targeting cationic glycoprotein Avidin was PEGylated to synthesize a multi-arm Avidin (mAv) nano-construct with high drug loading content. Here we investigate mAv biodistribution and kinetics over a 7-day period following intra-articular (IA) administration in rat knee joints. METHODS: Labeled mAv was injected into healthy rat knees, and joint tissues (articular cartilage, menisci, ligaments, tendons, fat pad) were harvested following sacrifice at 6 h, 1, 4 and 7 days. Its IA biodistribution and retention were measured using fluorescence microscopy. Tissue localization was compared in young vs old rats by immunohistochemistry. mAv chondrotoxicity and immune response were evaluated to determine safe carrier dose limits. RESULTS: mAv penetrated through the full thickness of rat cartilage and other joint tissues within 6 h, remaining detectable within most joint tissues over 7 days. Intra-tissue uptake correlated strongly with tissue GAG concentration, confirming the dominant role of electrostatic interactions between positively charged mAv and the negatively charged aggrecan proteoglycans. mAv was uptaken by chondrocytes and also penetrated the osteocyte lacuno-canalicular system of peri-articular bone in both young and old rats. mAv did not cause cytotoxicity at concentrations up to 300 µM but elicited a dose dependent immunogenic response. CONCLUSIONS: mAv's ability to target a variety of joint tissues, chondrocytes, and peri-articular osteocytes without sequestration in synovial fluid makes it a versatile carrier for delivering a wide range of drugs for treating a broad class of musculoskeletal diseases. Drugs can be conjugated using simple aqueous based avidin-biotin reaction, supporting its clinical prospects.

Sustained intra-cartilage delivery of interleukin-1 receptor antagonist using cationic peptide and protein-based carriers.

OBJECTIVE: Blocking the interleukin-1 (IL-1) catabolic cascade following joint trauma can be achieved using its receptor antagonist, IL-1Ra. However, its clinical translation for osteoarthritis therapy has been unsuccessful due to its rapid joint clearance and lack of targeting and penetration into deep cartilage layers at therapeutic concentrations. Here, we target the high negative charge of cartilage aggrecan-glycosaminoglycans (GAGs) by attaching cationic carriers to IL-1Ra. IL-1Ra was conjugated to the cartilage targeting glycoprotein, Avidin, and a short length optimally charged cationic peptide carrier (CPC+14). It is hypothesized that electro-diffusive transport and binding properties of IL-1Ra-Avidin and IL-1Ra-CPC+14 will create intra-cartilage depots of IL-1Ra, resulting in long-term suppression of IL-1 catabolism with only a single administration. DESIGN: IL-1Ra was conjugated to Avidin or CPC+14 using site specific maleimide linkers, and confirmed using gel electrophoresis, high-performance liquid chromatography (HPLC), and mass spectrometry. Intra-cartilage transport and retention of conjugates was compared with native IL-1Ra. Attenuation of IL-1 catabolic signaling with one-time dose of IL-1Ra-CPC+14 and IL-1Ra-Avidin was assessed over 16 days using IL-1α challenged bovine cartilage and compared with unmodified IL-1Ra. RESULTS: Positively charged IL-1Ra penetrated through the full-thickness of cartilage, creating a drug depot. A single dose of unmodified IL-1Ra was not sufficient to attenuate IL-1-induced cartilage deterioration over 16 days. However, when delivered using Avidin, and to a greater extent CPC+14, IL-1Ra significantly suppressed cytokine induced GAG loss and nitrite release while improving cell metabolism and viability. CONCLUSION: Charge-based cartilage targeting drug delivery systems hold promise as they can enable long-term therapeutic benefit with only a single dose.

DNA building blocks for AFM tip functionalization: An easy, fast and stable strategy.

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.

pH-Sensitive Twin Liposomes Containing Quercetin and Laccase for Tumor Therapy.

In this study, functional twin liposomes (TLs) were designed by linking avidin-anchored single liposomes and biotin-anchored single liposomes via avidin-biotin interactions. Here, we first punched a hole on the liposome surface using the liposome magnetoporation method to prepare functional single liposomes, which were used for safely encapsulating quercetin (QER, as a model prodrug) or laccase (LAC, as a bioactive enzyme) inside the liposomes without the use of organic solvents; the pores were then plugged by pH-sensitive glycol chitosan grafted with 3-diethylaminopropylamine (GDEAP) and avidin (or biotin). As a result, single liposomes with QER and biotin-GDEAP were efficiently coupled with other liposomes with LAC and avidin-GDEAP. We demonstrated that the TLs could accelerate QER and LAC release at acidic pH (6.8), improving the LAC-mediated oxidization of QER and significantly elevating tumor cell death, suggesting that this strategy can be used as an efficient method for the programmed action of prodrugs.

BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING).

Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.

Characterization of the kinetics and activation thermodynamics of intra- and inter-organism hybrid tetramers of pyruvate carboxylase.

In sedimentation velocity experiments, we have been able to detect hybrid Rhizobium etli pyruvate carboxylase tetramers formed between subunits that contain covalently bound biotin and mutant subunits that do not. This was performed by forming complexes of the tetramers with the biotin-binding protein avidin. In addition, we have shown that it is possible to form hybrid tetramers of pyruvate carboxylase subunits from two different organisms (bacteria - Rhizobium etli and fungi - Aspergillus nidulans). In hybrid tetramers containing mutant subunits that are not fully catalytically active and fully catalytically active subunits, the catalytic and regulatory properties of these hybrid tetramers are modified compared to homotetramers of the fully active pyruvate carboxylase subunits. Our data indicates that the model of catalysis involving half-of-the-sites activity in which there is obligatory alternation of pyruvate carboxylating activity between pairs of subunits either face of the tetramer, does not occur in the hybrid tetramers. Our results are also discussed in relation to recent findings that there are multiple pathways of biotin carboxylation and decarboxylation between subunits in pyruvate carboxylase tetramers.

An Electrochemical Sense Array Based on Aptamer and Biotin-Avidin System for the Selective Detection of Glucagon-Like Peptide-1.

BACKGROUND: GLP-1 as an incretin, has the ability to decrease blood sugar levels in a glucose-dependent manner by enhancing the secretion of insulin. Besides the insulinotropic effects, GLP-1 has been associated with numerous regulatory and protective effects. Thus, the action of GLP-1 is preserved in patients with type 2 diabetes and substantial pharmaceutical research has therefore been directed towards the development of GLP-1-based treatment. METHODS: In this work, we reported an electrochemical sense array based on the aptamer and biotin-avidin system for the detection of glucagon-like peptide-1 (GLP-1). The sense array employed a "stem-loop" conformation ap-tamer which was immobilized on the electrode of the 16-unit gold array via pre-labeled thiol group (-SH). Pre-labeled biotin serves as an affinity tag for the binding of avidin-horseradish peroxidase (avidin-HRP). The stem-loop structure of the aptamer kept the biotin from being approached by a bulky avidin-HRP by means of the steric hindrance. After the interaction of the target (GLP-1) and the aptamer, the aptamer would undergo a significant conformational change to force biotin away from the electrode, giving the avidin-HRP easy access to the labeled biotin. The HRP in the substrate could sensitively transduce the concentration of GLP-1 into the electrical signals, which were then measured by electrochemical technology of cyclic voltammetry and amperometric i-t curve. RESULTS: Under the optimal experimental conditions, the proposed sense array for GLP-1 had a good linear relationship from 0.1 pmol/L to 20 pmol/L with a detection limit of 0.05 pmol/L and can be used to accurately detect the GLP-1 in serum. CONCLUSIONS: The experimental results show that GLP-1 could be selectively detected by the electrochemical sense array, indicating that the proposed sense array based on the biotin-avidin system and the stem-loop aptamer has great potential in the detection of GLP-1.

Protein Motion and Configurations in a Form-Fitting Nanopore: Avidin in ClyA.

We probe the molecular dynamics and states of an avidin protein as it is captured and trapped in a voltage-biased cytolysin A nanopore using time-resolved single-molecule electrical conductance signals. The data for very large numbers of single-molecule events are analyzed and presented by a new method that provides clear visual insight into the molecular scale processes. Avidin in cytolysin A has surprisingly rich conductance spectra that reveal transient and more permanently trapped protein configurations in the pore and how they evolve into one another. We identify a long-lasting, stable, and low-noise configuration of avidin in the nanopore into which avidin can be reliably trapped and released. This may prove useful for single-molecule studies of other proteins that can be biotinylated and then transported by avidin to the pore via their coupling to avidin with biotin-avidin linking. We demonstrate the sensitivity of this system with detection of biotin attached to avidin captured by the pore.

Metal-Ion-Responsive Bionanocomposite for Selective and Reversible Enzyme Inhibition.

A bionanocomposite with artificial binding pockets for a DNA repair enzyme has been developed by in situ assembly of an affinity protein with a surrounding contact surface of polydopamine on the surface of silica coated magnetic nanoparticles via molecular imprinting reactions. The obtained nanoparticles exhibited antibody-like binding behavior toward the target enzyme with highly specific and efficient inhibition effect. Moreover, the binding and inhibition could be flexibly tuned by the addition of metal ions such as Mn2+ and Mg2+, which provided a convenient tool to regulate enzyme activity with artificially engineered nanoinhibitors.

Dynamic Fingering in Adhered Lipid Membranes.

Artificial lipid membranes incorporating proteins have frequently been used as models for the dynamic organization of biological structures in living cells as well as in the development of biology-inspired technologies. We report here on the experimental demonstration and characterization of a pattern-forming process that occurs in a lipid bilayer membrane adhered via biotin-avidin binding to a second lipid membrane that is supported by a solid substrate. Adhesion regions are roughly circular with a diameter of about 25 µm. Using confocal fluorescence microscopy, we record time series of dynamic fingering patterns that grow in the upper lipid membrane and intermembrane biotin-avidin bonds. The fingers are micrometer-scale elongated pores that grow from the edge of an already-stabilized hole. Finger growth is saltatory on the scale of tens of seconds. We find that as the fingers grow and the density of adhesion proteins increases, the rate of finger growth decreases exponentially and the width of newly formed fingers decreases linearly. We show that these findings are consistent with a thermodynamic description of dynamic pore formation and stabilization.

Growth inhibition of human ovarian carcinoma by a novel AvidinOX-anchored biotinylated camptothecin derivative.

Oxidized form of avidin, named AvidinOX, provides stable fixation of biotinylated molecules in tissues thus representing a breakthrough in topical treatment of cancer. AvidinOX proved to be a stable receptor for radiolabeled biotin, biotinylated antibodies and cells. In order to expand applicability of the AvidinOX-based delivery platform, in the present study we investigated the possibility to hold biotinylated chemotherapeutics in AvidinOX-treated sites. A novel biotinylated gimatecan-derived camptothecin, coded ST8161AA1, was injected at suboptimal doses into human tumors xenografted in mice alone or pre-complexed to AvidinOX. Significantly higher growth inhibition was observed when the drug was anchored to AvidinOX suggesting the potential utility of this delivery modality for the local treatment of inoperable tumors.

A dipeptide with enhanced anion binding affinity enables cell uptake and protein delivery.

Herein, we report a rather simple strategy to enhance the anion binding ability of a dipeptide to achieve cell uptake and also protein delivery. Peptide 1, composed of only two synthetic amino acids with an artificial anion binding site in the side chains, has an overall molecular weight of only 630 Da and demonstrated strong binding affinity (107 M-1) and clustering ability with heparin as a model for cell surface sugars. Furthermore, peptide 1 is also efficiently taken up by cells most likely via endocytosis. The uptake efficiency is dependent on the amount of glycosaminoglycans on the cell surface. Cells with reduced amounts of surface bound glycosaminoglycans show significantly less uptake of peptide 1. Moreover, 1 induced the uptake of a model protein (avidin, around 67 kDa) into cells, which makes 1 a highly attractive candidate for drug and protein delivery, especially as 1 has negligible cytotoxicity.

Autodisplay of an avidin with biotin-binding activity on the surface of Escherichia coli.

OBJECTIVES: To display a recombinant avidin fused to the autotransporter ShdA to bind biotinylated molecules on the surface of Escherichia coli. RESULTS: Two chimeric protein constructs containing avidin fused to the autotransporter ShdA were expressed on the surface of Escherichia coli DH5α. One fusion protein contained 476 amino acids of the ShdA α and ß domains, whereas the second consisted of a 314 amino acid from α and truncated ß domains. Protein production was verified by SDS-PAGE using an antibody to the molecular FLAG-tag. The surface display of the avidin-shdA fusion protein was confirmed by confocal microscopy and flow cytometry analysis, and the biotin-binding activity was evaluated by fluorescence microscopy and flow cytometry using biotin-4-fluorescein and biotinylated-ovalbumin (OVA). CONCLUSIONS: Expression of a recombinant avidin with biotin-binding activity on the surface of E. coli was achieved using the autotransporter ShdA. This system is an alternative to bind biotinylated molecules to E. coli.

Development of a peptide-modified siRNA nanocomplex for hepatic stellate cells.

Insulin-like growth factor 2 receptor (IGF2R) is overexpressed in activated hepatic stellate cells (HSCs) and therefore can be utilized for HSC-specific drug delivery. We recently discovered an IGF2R-specific peptide using a novel biopanning. Here, we adopted biotin-conjugated IGF2R-specific peptide, cholesterol, and vitamin A as the targeting ligands for the neutravidin-based siRNA nanocomplex to deliver PCBP2 siRNA, a potentially antifibrotic agent, to HSCs. Compared to vitamin A and cholesterol, the IGF2R-specific peptide exhibited the highest targeting effect to human LX-2 HSC, rat HSC-T6 cell line, and activated primary rat HSCs. Accordingly, the IGF2R-specific peptide coupled nanocomplex demonstrated higher silencing activity of PCBP2 and better inhibition on the migration of activated HSCs. Compared to free siRNA and the nanocomplexes coupled with vitamin A and cholesterol, the IGF2R-specific peptide coupled nanocomplex showed the highest uptake in the liver and lowest uptake in the lung and kidney of the rats with CCl4-induced liver fibrosis.