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1.
Mikrochim Acta ; 191(7): 369, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38834823

ABSTRACT

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.


Subject(s)
Arachidonic Acid , Biosensing Techniques , COVID-19 , SARS-CoV-2 , Humans , Arachidonic Acid/blood , COVID-19/blood , COVID-19/diagnosis , COVID-19/immunology , Biosensing Techniques/methods , SARS-CoV-2/immunology , Horseradish Peroxidase/chemistry , Respiratory Syncytial Viruses/immunology , Immunoassay/methods , Streptavidin/chemistry , Biotin/chemistry , Limit of Detection
2.
Commun Biol ; 7(1): 554, 2024 May 09.
Article in English | MEDLINE | ID: mdl-38724559

ABSTRACT

Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.


Subject(s)
Biotinylation , Humans , Biotin/chemistry , Biotin/metabolism , Proteomics/methods , Animals , Staining and Labeling/methods , Chromatography, Liquid/methods , Proteome/metabolism , Mass Spectrometry/methods
3.
Anal Chim Acta ; 1308: 342667, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38740453

ABSTRACT

BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.


Subject(s)
Biotin , DNA , MicroRNAs , Nucleic Acid Amplification Techniques , Streptavidin , MicroRNAs/blood , Humans , Streptavidin/chemistry , DNA/chemistry , DNA/blood , Biotin/chemistry , Biosensing Techniques/methods , Limit of Detection
4.
J Am Chem Soc ; 146(19): 13163-13175, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38698548

ABSTRACT

A pretargeted strategy that decouples targeting vectors from radionuclides has shown promise for nuclear imaging and/or therapy in vivo. However, the current pretargeted approach relies on the use of antibodies or nanoparticles as the targeting vectors, which may be compromised by poor tissue penetration and limited accumulation of targeting vectors in the tumor tissues. Herein, we present an orthogonal dual-pretargeted approach by combining stimuli-triggered in situ self-assembly strategy with fast inverse electron demand Diels-Alder (IEDDA) reaction and strong biotin-streptavidin (SA) interaction for near-infrared fluorescence (NIR FL) and magnetic resonance (MR) imaging of tumors. This approach uses a small-molecule probe (P-Cy-TCO&Bio) containing both biotin and trans-cyclooctene (TCO) as a tumor-targeting vector. P-Cy-TCO&Bio can efficiently penetrate subcutaneous HeLa tumors through biotin-assisted targeted delivery and undergo in situ self-assembly to form biotinylated TCO-bearing nanoparticles (Cy-TCO&Bio NPs) on tumor cell membranes. Cy-TCO&Bio NPs exhibited an "off-on" NIR FL and retained in the tumors, offering a high density of TCO and biotin groups for the concurrent capture of Gd-chelate-labeled tetrazine (Tz-Gd) and IR780-labeled SA (SA-780) via the orthogonal IEDDA reaction and SA-biotin interaction. Moreover, Cy-TCO&Bio NPs offered multiple-valent binding modes toward SA, which additionally regulated the cross-linking of Cy-Gd&Bio NPs into microparticles (Cy-Gd&Bio/SA MPs). This process could significantly (1) increase r1 relaxivity and (2) enhance the accumulation of Tz-Gd and SA-780 in the tumors, resulting in strong NIR FL, bright MR contrast, and an extended time window for the clear and precise imaging of HeLa tumors.


Subject(s)
Biotin , Cyclooctanes , Magnetic Resonance Imaging , Nanoparticles , Cyclooctanes/chemistry , Humans , Nanoparticles/chemistry , Magnetic Resonance Imaging/methods , HeLa Cells , Biotin/chemistry , Animals , Optical Imaging , Biotinylation , Mice , Streptavidin/chemistry , Cycloaddition Reaction , Fluorescence
5.
Mikrochim Acta ; 191(6): 321, 2024 05 10.
Article in English | MEDLINE | ID: mdl-38727732

ABSTRACT

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.


Subject(s)
Biotin , Gold , Limit of Detection , Metal Nanoparticles , MicroRNAs , Spectrum Analysis, Raman , MicroRNAs/blood , MicroRNAs/analysis , Metal Nanoparticles/chemistry , Gold/chemistry , Spectrum Analysis, Raman/methods , Biotin/chemistry , Humans , Catalysis , Streptavidin/chemistry
6.
Sensors (Basel) ; 24(9)2024 Apr 27.
Article in English | MEDLINE | ID: mdl-38732912

ABSTRACT

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.


Subject(s)
Aptamers, Nucleotide , Biotin , Nitrilotriacetic Acid , Streptavidin , Surface Plasmon Resonance , Streptavidin/chemistry , Biotin/chemistry , Aptamers, Nucleotide/chemistry , Nitrilotriacetic Acid/chemistry , Nitrilotriacetic Acid/analogs & derivatives , Biosensing Techniques/methods , Thrombin/chemistry , Organometallic Compounds
7.
J Med Chem ; 67(10): 8361-8371, 2024 May 23.
Article in English | MEDLINE | ID: mdl-38726551

ABSTRACT

Due to the complex heterogeneity in different cancer types, the heterodimeric strategy has been intensively practiced to improve the effectiveness of tumor diagnostics. In this study, we developed a series of novel 18F-labeled biotin/FAPI-conjugated heterobivalent radioligands ([18F]AlF-NSFB, [18F]AlF-NSFBP2, and [18F]AlF-NSFBP4), synergistically targeting both fibroblast activation protein (FAP) and biotin receptor (BR), to enhance specific tumor uptake and retention. The in vitro and in vivo biological properties of these dual-targeting tracers were evaluated, with a particular focus on positron emission tomography imaging in A549 and HT1080-FAP tumor-bearing mice. Notably, in comparison to the corresponding FAP-targeted monomer [18F]AlF-NSF, biotin/FAPI-conjugated heterodimers exhibited a high uptake in tumor and prolong retention. In conclusion, as a proof-of-concept study, the findings validated the superiority of biotin/FAPI-conjugated heterodimers and the positive influence of biotin and linker on pharmacokinetics of radioligands. Within them, the bispecific [18F]AlF-NSFBP4 holds significant promise as a candidate for further clinical translational studies.


Subject(s)
Biotin , Fluorine Radioisotopes , Animals , Humans , Fluorine Radioisotopes/chemistry , Biotin/chemistry , Biotin/pharmacokinetics , Mice , Drug Design , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacology , Positron-Emission Tomography , Mice, Nude , Tissue Distribution , Dimerization , Cell Line, Tumor , Mice, Inbred BALB C
8.
Anal Chem ; 96(21): 8791-8799, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38742926

ABSTRACT

MicroRNAs (miRNAs) are novel tumor biomarkers owing to their important physiological functions in cell communication and the progression of multiple diseases. Due to the small molecular weight, short sequence length, and low concentration levels of miRNA, miRNA detection presents substantial challenges, requiring the advancement of more refined and sensitive techniques. There is an urgent demand for the development of a rapid, user-friendly, and sensitive miRNA analysis method. Here, we developed an enhanced biotin-streptavidin dual-mode phase imaging surface plasmon resonance (PI-SPR) aptasensor for sensitive and rapid detection of miRNA. Initially, we evaluated the linear sensing range for miRNA detection across two distinct sensing modalities and investigated the physical factors that influence the sensing signal in the aptamer-miRNA interaction within the PI-SPR aptasensor. Then, an enhanced biotin-streptavidin amplification strategy was introduced in the PI-SPR aptasensor, which effectively reduced the nonspecific adsorption by 20% and improved the limit of detection by 548 times. Furthermore, we have produced three types of tumor marker chips, which utilize the rapid sensing mode (less than 2 min) of PI-SPR aptasensor to achieve simultaneous detection of multiple miRNA markers in the serum from clinical cancer patients. This work not only developed a new approach to detect miRNA in different application scenarios but also provided a new reference for the application of the biotin-streptavidin amplification system in the detection of other small biomolecules.


Subject(s)
Aptamers, Nucleotide , Biotin , MicroRNAs , Streptavidin , Surface Plasmon Resonance , MicroRNAs/analysis , MicroRNAs/blood , Biotin/chemistry , Surface Plasmon Resonance/methods , Streptavidin/chemistry , Humans , Aptamers, Nucleotide/chemistry , Limit of Detection , Biomarkers, Tumor/blood , Biomarkers, Tumor/analysis , Biosensing Techniques/methods
9.
Bioorg Med Chem Lett ; 108: 129803, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38777280

ABSTRACT

Targeted delivery of radionuclides to tumors is significant in theranostics applications for precision medicine. Pre-targeting, in which a tumor-targeting vehicle and a radionuclide-loaded effector small molecule are administered separately, holds promise since it can reduce unnecessary internal radiation exposure of healthy cells and can minimize radiation decay. The success of the pre-targeting delivery requires an in vivo-stable tumor-targeting vehicle selectively binding to tumor antigens and an in vivo-stable small molecule effector selectively binding to the vehicle accumulated on the tumor. We previously reported a drug delivery system composed of a low-immunogenic streptavidin with weakened affinity to endogenous biotin and a bis-iminobiotin with high affinity to the engineered streptavidin. It was, however, unknown whether the bis-iminobiotin is stable in vivo when administered alone for the pre-targeting applications. Here we report a new in vivo-stable bis-iminobiotin derivative. The keys to success were the identification of the degradation site of the original bis-iminobiotin treated with mouse plasma and the structural modification of the degradation site. We disclosed the successful pre-targeting delivery of astatine-211 (211At), α-particle emitter, to the CEACAM5-positive tumor in xenograft mouse models.


Subject(s)
Biotin , Streptavidin , Animals , Streptavidin/chemistry , Mice , Biotin/chemistry , Humans , Drug Delivery Systems , Cell Line, Tumor , Mutation , Molecular Structure
10.
Proc Natl Acad Sci U S A ; 121(22): e2403013121, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38781207

ABSTRACT

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.


Subject(s)
Avidin , Biomolecular Condensates , Biotin , Poloxamer , Biomolecular Condensates/chemistry , Biomolecular Condensates/metabolism , Poloxamer/chemistry , Biotin/chemistry , Biotin/metabolism , Avidin/chemistry , Avidin/metabolism , Fluorescence Recovery After Photobleaching/methods , Surface Properties , Surface-Active Agents/chemistry , Surface-Active Agents/metabolism , Single Molecule Imaging/methods
11.
Chem Commun (Camb) ; 60(47): 6059-6062, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38780054

ABSTRACT

We developed a system to detect multiple target biomolecules through sensing motif-tethered oligodeoxynucleotides. DNA-based molecular probes gave the primary amine motif upon reaction with the target biomolecules, glutathione (GSH) and H2O2. After labelling with biotin, the product DNAs were selectively collected to be quantified by qPCR.


Subject(s)
Biotin , Glutathione , Hydrogen Peroxide , Oligodeoxyribonucleotides , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/analysis , Glutathione/chemistry , Glutathione/analysis , Biotin/chemistry , DNA/chemistry , Biosensing Techniques/methods
12.
J Phys Chem B ; 128(22): 5327-5335, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38771940

ABSTRACT

Carboxy-biotin serves as a coenzyme in certain carboxylases, exhibiting the remarkable capability to transfer a carboxy group to specific substrates. This process is made possible by the presence of biotin, a unique molecule that consists of a sulfur-containing tetrahydrothiophene ring fused to a ureido group. It is covalently attached to the enzyme via a flexible linker, allowing for its functionality. Biotin-dependent carboxylases consist of two distinct domains. The first domain (BC) facilitates biotin carboxylation by utilizing ATP, while the second domain (CT) transfers CO2 to the substrate. The process of ATP-dependent carboxylation using bicarbonate in the biotin carboxylase domain (BC) is well-known. However, the precise mechanism by which CO2 is released in the carboxyltransferase domain (CT) is still not fully understood. We employed advanced computational chemistry methods to investigate the decarboxylation process of carboxy-biotin in various molecular environments and different protonation states. Regardless of the polarity of the molecular surroundings, decarboxylation only occurs spontaneously in the protonated form. To determine the protonation state of biotin in different environments, we established an accurate computational chemistry method for calculating the pKa value of carboxy-biotin, reaching sub-kcal/mol accuracy. Based on our findings, nonpolar environments, such as the active site of the carboxyltransferase domain, have the ability to cause the spontaneous release of CO2 from carboxy-biotin. The CO2 release takes place spontaneously from protonated carboxy-biotin, promoting the carboxylation of substrates.


Subject(s)
Biotin , Carbon Dioxide , Biotin/chemistry , Biotin/metabolism , Carbon Dioxide/chemistry , Carbon Dioxide/metabolism
13.
ACS Appl Mater Interfaces ; 16(17): 21534-21545, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38634566

ABSTRACT

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.


Subject(s)
Click Chemistry , DNA , DNA/chemistry , Biosensing Techniques/methods , Surface Properties , Animals , Mice , Azides/chemistry , Biotin/chemistry , Nanostructures/chemistry , Polyethylene Glycols/chemistry , Ligands , Avidin/chemistry
14.
Angew Chem Int Ed Engl ; 63(23): e202402139, 2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38563765

ABSTRACT

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.


Subject(s)
Avidin , Biotin , Calixarenes , Hydrophobic and Hydrophilic Interactions , Calixarenes/chemistry , Biotin/chemistry , Avidin/chemistry , Avidin/metabolism , Humans , Surface Properties , Doxorubicin/chemistry , Doxorubicin/pharmacology , Doxorubicin/metabolism , Delayed-Action Preparations/chemistry , Phenols/chemistry
15.
J Control Release ; 369: 325-334, 2024 May.
Article in English | MEDLINE | ID: mdl-38565395

ABSTRACT

Challenges for glioma treatment with nanomedicines include physio-anatomical barriers (the blood-brain barrier and blood-brain tumor barrier), low drug loading capacity, and limited circulation time. Here, a red blood cell membrane-coated docetaxel drug nanocrystal (pV-RBCm-NC(DTX)), modified with pHA-VAP (pV) for all-stage targeting of glioma, was designed. The NC(DTX) core exhibited a high drug loading capacity but low in vivo stability, and the RBCm coating significantly enhanced the stability and prolonged in vivo circulation. Moreover, the Y-shaped targeting ligand pV was modified by a mild avidin-biotin interaction, which endowed RBCm-NC(DTX) with superior barrier-crossing ability and therapeutic efficacy. The integration of nanocrystal technology, cell membrane coating, and the avidin-biotin insertion method into this active targeting biomimetic formulation represents a promising drug delivery strategy for glioma.


Subject(s)
Antineoplastic Agents , Brain Neoplasms , Docetaxel , Erythrocyte Membrane , Glioma , Nanoparticles , Docetaxel/administration & dosage , Docetaxel/pharmacokinetics , Docetaxel/chemistry , Glioma/drug therapy , Animals , Nanoparticles/chemistry , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Erythrocyte Membrane/drug effects , Erythrocyte Membrane/chemistry , Cell Line, Tumor , Brain Neoplasms/drug therapy , Male , Drug Delivery Systems , Avidin/administration & dosage , Avidin/chemistry , Humans , Biotin/chemistry , Biotin/administration & dosage , Rats, Sprague-Dawley , Blood-Brain Barrier/metabolism , Mice, Inbred BALB C , Mice, Nude
16.
Spectrochim Acta A Mol Biomol Spectrosc ; 315: 124274, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38640627

ABSTRACT

γ-Glutamyl transpeptidase (GGT), a cell-surface enzyme, is strongly implicated in mammalian malignancy growth and migration processes including human hepatocarcinogens. However, simply and conveniently detect of GGT on the cell membrane remains highly challenging. In this study, a biotin-tagged fluorescent probe Nap-biotin-glu was developed using glutamic acid, naphthalimide, and biotin as the reaction site, fluorescent reporter, and membrane-targeting group, which required only three steps. Colocalization fluorescence imaging and immunofluorescence analysis indicated that probe Nap-biotin-glu was successfully realized in situ visualizing of GGT on the cell membrane.Owing to the significant over-expressed GGT level in tumor, the probe was successfully applied to distinguish cancer tissues from adjacent normal tissues.


Subject(s)
Biotin , Fluorescent Dyes , gamma-Glutamyltransferase , gamma-Glutamyltransferase/metabolism , gamma-Glutamyltransferase/analysis , Fluorescent Dyes/chemistry , Humans , Biotin/chemistry , Neoplasms , Naphthalimides/chemistry , Cell Line, Tumor , Glutamic Acid/analysis , Glutamic Acid/metabolism
17.
ACS Appl Mater Interfaces ; 16(17): 22334-22343, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38635042

ABSTRACT

The number of applications of self-assembled deoxyribonucleic acid (DNA) origami nanoparticles (DNA NPs) has increased drastically, following the development of a variety of single-stranded template DNA (ssDNA) that can serve as the scaffold strand. In addition to viral genomes, such as M13 bacteriophage and lambda DNAs, enzymatically produced ssDNA from various template sources is rapidly gaining traction and being applied as the scaffold for DNA NP preparation. However, separating fully formed DNA NPs that have custom scaffolds from crude assembly mixes is often a multistep process of first separating the ssDNA scaffold from its enzymatic amplification process and then isolating the assembled DNA NPs from excess precursor strands. Only then is the DNA NP sample ready for downstream characterization and application. In this work, we highlight a single-step purification of custom sequence- or M13-derived scaffold-based DNA NPs using photocleavable biotin tethers. The process only requires an inexpensive ultraviolet (UV) lamp, and DNA NPs with up to 90% yield and high purity are obtained. We show the versatility of the process in separating two multihelix bundle structures and a wireframe polyhedral architecture.


Subject(s)
Biotin , DNA, Single-Stranded , Nanoparticles , Biotin/chemistry , Nanoparticles/chemistry , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/isolation & purification , Bacteriophage M13/chemistry , Bacteriophage M13/genetics , DNA/chemistry , DNA/isolation & purification , Ultraviolet Rays
18.
Anal Biochem ; 691: 115543, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38636731

ABSTRACT

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.


Subject(s)
Biotin , Melanoma , Oxidation-Reduction , Proteomics , Proteomics/methods , Melanoma/metabolism , Melanoma/pathology , Humans , Cell Line, Tumor , Biotin/chemistry , Biotin/metabolism , Sulfhydryl Compounds/chemistry , Sulfhydryl Compounds/metabolism
19.
ACS Biomater Sci Eng ; 10(5): 3017-3028, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38655791

ABSTRACT

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.


Subject(s)
Cryogels , Oxidation-Reduction , Polyethylene Glycols , Cryogels/chemistry , Polyethylene Glycols/chemistry , Animals , Concanavalin A/chemistry , Concanavalin A/metabolism , Methacrylates/chemistry , Mice , Mannose/chemistry , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Sulfhydryl Compounds/chemistry , Streptavidin/chemistry , Streptavidin/metabolism , Proteins/chemistry , Proteins/metabolism , Biotin/chemistry , Biotin/metabolism , Biotin/analogs & derivatives , Porosity
20.
Methods Mol Biol ; 2797: 351-362, 2024.
Article in English | MEDLINE | ID: mdl-38570472

ABSTRACT

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.


Subject(s)
Genes, ras , Proto-Oncogene Proteins p21(ras) , Animals , Mice , Proto-Oncogene Proteins p21(ras)/genetics , Alleles , Biotin/chemistry , Streptavidin , Mutation
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