Biotin-streptavidin Interaction Research Articles

Multifunctional self-assembled nanoparticles serving as the signal labels of fluorescent immunoassays

Nanolabels composed of multiple small molecules have shown great potential as the signal tags of immunoassays. However, their practical applications are limited by the complex preparation and modification procedures. This study proposed the design of multifunctional nanoparticles as the signal labels of fluorescent immunoassays, which were prepared by the self-assembly of biotinylated pyrene-phenylalanine (bio-PyF) monomers. The recognition elements of biotin moieties on the nanoparticles enabled their attachment on the sensor surface through streptavidin–biotin interactions. The tetrameric streptavidin proteins serving as the crosslinkers induced the in-situ assembly of bio-PyF nanoparticles (bio-PyFNPs) into network architectures on the sensor interface, thus achieving the second signal amplification. The captured nanoparticles were disrupted into a large number of signal reporters with pyrene tags under external surfactant stimulus. The inherent fluorescent property of pyrene allowed for the detection of targets with high simplicity and sensitivity. The analytical performances of the immunoassays were evaluated by determining alpha-fetoprotein (AFP) in buffer and serum samples, achieving a detection limit down to 0.5 pg/mL. This method offers a simple, sensitive, and selective alternative to conventional enzyme-linked immunosorbent assays for clinical diagnosis. The proposed strategy holds great promise for the development of novel signal labels and self-assembly-based biosensors.

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.

Magnetic Metalloimmunoassays: Influence of Particle Shape and Size on Galvanic Exchange-Based Electrochemical Detection

The electrooxidation of silver (Ag) offers improved signal sharpness and intensity, enabling precise measurements and enhanced resolution in electrochemical sensing. Leveraging silver nanoparticles (AgNPs) as indirect labels in electrochemical assays provides a distinct advantage for detecting various biomarkers at clinically relevant concentrations. In our recent work, we have introduced an electrochemical approach that involves a galvanic exchange (GE) reaction between electrodeposited gold and AgNPs, followed by anodic stripping voltammetry (ASV), with potential applications in immunosensing. In this assay protocol, specific monoclonal antibodies immobilize the biomarker of interest separately on AgNPs and magnetic beads. Subsequently, magnetic bead-AgNP conjugates are attracted to the electrode surface by a magnetic force. However, to achieve the expected analytical performance of the bioassay, it is imperative to characterize the composition, size, shape, and surface modifications of the AgNPs. Consequently, our research focuses on investigating the electrochemical properties of a model bioconjugate formed between magnetic microbeads (MB) and AgNPs through Streptavidin-biotin interactions under various assay conditions. Notably, our recent studies reveal that conducting the GE cycle for three repetitions significantly enhances the Ag signal, yielding an improvement of approximately two-fold or greater compared to previously reported methods. Previous studies have shown that altering the shape of AgNPs, transitioning from nanospheres to other forms like nanocubes and nanodisks enhances both the limit of detection and the range of analyte detection. However, despite these enhancements, spherical AgNPs remain the most stable and easily synthesized option, characterized by excellent homogeneity. Recently, we compared AgNPs with diameters ranging from 20 to 100 nm paired with magnetic microbeads with diameters spanning from 50 nm to 3 µm to identify the optimal combination. Preliminary results indicate that the Mb (3 µm)-AgNP (50 nm) conjugate exhibits the highest Ag charge and Ag collection efficiency. Nonetheless, critical knowledge gaps persist regarding the fundamental understanding of the electrogenerated GE process and we are continually working on refining the electrochemical and assay parameters to develop a robust universal platform for GE-based electrochemical immunosensing.

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.

Controlled In Situ Self-Assembly of Biotinylated Trans-Cyclooctene Nanoparticles for Orthogonal Dual-Pretargeted Near-Infrared Fluorescence and Magnetic Resonance Imaging.

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.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

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.

Improving CEA detection Sensitivity: Carboxyfluorescein-Loaded liposomes in aptamer sandwich assay

This study aimed to develop an effective method for highly sensitive detection of carcinoembryonic antigen (CEA), a tumor biomarker, using an aptasensor sandwich assay based on a liposome loaded with carboxyfluorescein. In this approach, liposomes loaded with carboxyfluorescein (CF) were anchored with a cholesterol-labeled anti-CEA aptamer (Apt1-Lip) using a post-insertion technique. Furthermore, the successful insertion of the aptamer into the liposome outer layer was confirmed through UV absorption, PAGE electrophoresis, dynamic light scattering (DLS), and zeta potential measurements. Additionally, a secondary aptamer was conjugated with magnetic beads via biotin-streptavidin interactions and used as a capture probe (MBs/Apt2). Aptamer-decorated liposomes successfully bound to the CEA antigen in the presence of the target, leading to the formation of a sandwich assay when combined with MBs/Apt2. The specific binding signal was further increased by dissolving the lipid nanospheres with Triton X-100. This liposome-based aptasensor demonstrated a detection limit of 35.48 pg/mL over a wide linear range from 0.1 ng/mL to 100 ng/mL, with high specificity and sensitivity at CEA. Additionally, the proposed approach exhibited notable promise for on-site testing in early clinical diagnosis, as it showed excellent selectivity, satisfactory reproducibility, and good recovery in CEA detection from human serum samples.

Abstract B026: Rotational magnetic drug targeting for leptomeningeal metastases

Abstract Background: Magnetic nanoparticles (MNPs) have potential for enhancing the delivery of therapeutic molecules to cancer cells and micronodules, including non-obstructing metastases within cerebrospinal fluid (CSF) pathways. Rotational magnetic drug targeting (rMDT) takes advantage of the surface-walking capability of clusters of MNPs, which can be activated remotely. Here, we present data related to 1) the creation, characterization, and in vitro testing of a variety of MNPs utilized with bound or unbound chemotherapeutic agents, and 2) the design and construction of four different magnetomotive systems used to propel MNPs at clinically-relevant distances. The goal was to create an in vitro model mimicking conditions found in patients with leptomeningeal metastases. Methods: Gold-iron alloy nanoparticles were combined with biotinylated etoposide via the streptavidin-biotin interaction. Unbound MNPs were tested with etoposide and doxorubicin. MNPs were characterized by techniques including electron microscopy, Zeta potential analysis, and dynamic light scattering. 3D-printed and acrylic trays were used as conduits for measuring translational motion over distances up to 30 cm through saline, culture media, and artifical CSF. MNP velocities were determined by videography. For propelling the MNPs, one electromagnetic system used a Helmholtz pair of coils on each side of an underlying perpendicular coil. The second electromagnetic system utilized 2 angled coils. Two rotating permanent magnet systems used neodymium-boron-iron magnets of different sizes and rotational speeds. Cultured cells included fibroblasts, lung cancer, glioblastoma, and medulloblastoma cell lines. Light microscopy and cytotoxicty assays (by MTT and trypan blue exclusion) were used to assess preservation of tumoricidal activity. Results: In unbound MNP studies, both etoposide and doxorubin could be dispersed over physiologic distances through saline and artificial CSF at velocities exceeding 1 cm/sec - much faster than by diffusion alone. Drugs conveyed in this manner could be transported at 37°C across cell surfaces and retain tumoricidal effect on cultured cells. With rotating magnets, MNPs could be moved against a saline flow rate of 1 cm/sec. In bound MNP studies, true rMDT was possible - all four magnetomotive systems could be shut off, depositing the MNP-drug conjugate at the desired location. Rotating permanent magnets were found to be advantageous for unilateral device design; electromagnetic devices, however, are more likely to be compatible with concurrent magnetic particle imaging (currently under development). Conclusions: These in vitro findings indicate that rMDT within CSF pathways is feasible and should advance to animal studies. Bound MNPs may prove to be more useful for intrathecal use (administered via lumbar puncture or Ommaya reservoir), considering dilutional effects and CSF flow. An improved ability to direct therapeutic agents within CSF would represent a major step forward in the treatment of patients with leptomeningeal metastases. Citation Format: Herbert Engelhard, Alexander Willis, Tolou Shokuhfar, Lamar Mair. Rotational magnetic drug targeting for leptomeningeal metastases [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B026.

Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 μM) underlines potential interest of this nanoplatform for biomedical applications.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

Unraveling blunt-end RNA binding and ATPase-driven translocation activities of the RIG-I family helicase LGP2.

The RIG-I family helicases, comprising RIG-I, MDA5and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity. We show that LGP2, like RIG-I, prefers binding blunt-ended double-stranded (ds) RNAs over internal dsRNA regions or RNA overhangs and associates with blunt-ends faster than with overhangs. Unlike RIG-I, a 5'-triphosphate (5'ppp), Cap0, or Cap1 RNA-end does not influence LGP2's RNA binding affinity. LGP2 hydrolyzes ATP in the presence of RNA but at a 5-10 fold slower rate than RIG-I. Nevertheless, LGP2 uses its ATPase activity to translocate and displace biotin-streptavidin interactions. This activity is significantly hindered by a methylated RNA patch, particularly on the 3'-strand, suggesting a 3'-strand tracking mechanism like RIG-I. The preference of LGP2 for blunt-end RNA binding, its insensitivity to Cap0/Cap1 modification, and its translocation/protein displacement ability have substantial implications for how LGP2 regulates the RNA sensing process by MDA5/RIG-I.

Combining hybrid nanoflowers with hybridization chain reaction for highly sensitive detection of SARS-CoV-2 nucleocapsid protein

BackgroundCOVID-19 (coronavirus disease 2019) pandemic has had enormous social and economic impacts so far. The nucleocapsid protein (N protein) is highly conserved and is a key antigenic marker for the diagnosis of early SARS-CoV-2 infection. ResultsIn this study, the N protein was first captured by an aptamer (Aptamer 58) coupled to magnetic beads (MBs), which in turn were bound to another DNA sequence containing the aptamer (Aptamer 48-Initiator). After adding 5′-biotinylated hairpin DNA Amplifier 1 and Amplifier 2 with cohesive ends for complementary hybridization, the Initiator in the Aptamer 48-Initiator began to trigger the hybridization chain reaction (HCR), generating multiple biotin-labeled DNA concatamers. When incubated with synthetic streptavidin-invertase-Ca3(PO4)2 hybrid nanoflower (SICa), DNA concatamers could specifically bind to SICa through biotin-streptavidin interaction with high affinity. After adding sucrose, invertase in SICa hydrolyzed sucrose to glucose, whose concentration could be directly read with a portable glucometer, and its concentration was positively correlated with the amount of captured N protein. The method is highly sensitive with a detection limit as low as 1 pg/mL. SignificanceWe believe this study provided a practical solution for the early detection of SARS-CoV-2 infection, and offered a new method for detecting other viruses through different target proteins.

Peptide Bispecifics Inhibiting HIV-1 Infection by an Orthogonal Chemical and Supramolecular Strategy.

Viral infections pose a significant threat to human health, and effective antiviral strategies are urgently needed. Antiviral peptides have emerged as a promising class of therapeutic agents due to their unique properties and mechanisms of action. While effective on their own, combining antiviral peptides may allow us to enhance their potency and to prevent viral resistance. Here, we developed an orthogonal chemical strategy to prepare a heterodimeric peptide conjugate assembled on a protein-based nanoplatform. Specifically, we combined the optimized version of two peptides inhibiting HIV-1 by distinct mechanisms. Virus-inhibitory peptide (VIRIP) is a 20 amino acid fragment of α1-antitrypsin that inhibits HIV-1 by targeting the gp41 fusion peptide. Endogenous peptide inhibitor of CXCR4 (EPI-X4) is a 16-residue fragment of human serum albumin that prevents HIV-1 entry by binding to the viral CXCR4 co-receptor. Optimized forms of both peptides are assembled on supramolecular nanoplatforms through the streptavidin-biotin interaction. We show that the construct consisting of the two different peptides (SAv-VIR-102C9-EPI-X4 JM#173-C) shows increased activity against CCR5- and CXCR4-tropic HIV-1 variants. Our results are a proof of concept that peptides with different modes of action can be assembled on nanoplatforms to enhance their antiviral activity.

In Situ Capturing and Counting Device for the Specific Depletion and Purification of Cancer-Derived Exosomes.

From metabolic waste to biological mediators, exosomes have emerged as the key player in a variety of pathological processes, particularly in oncogenesis. The exosome-mediated communication network involves nearly every step of cancer progression, promoting the proliferation and immune escape of cancer cells. Therefore, the removal of cancer-derived exosomes has profound clinical significance. Current methods for exosome separation and enrichment are either for large-scale samples or require complex pretreatment processes, lacking effective methods for trace-volume exosome capture in situ. Herein, we have developed an in situ exosome capturing and counting device based on the antibody-functionalized capillary. Specific antibodies targeting exosome biomarkers were immobilized to the inner wall of the capillary via biotin-streptavidin interaction for direct cancer exosome capturing. Subsequent exosome staining enabled imaging and enumeration. Acceptable linearity and reproducibility were achieved with our device, with the capturing and detective range between 3.3 × 104 and 3.3 × 108 particles, surpassing the nanoparticle tracking analysis by 2 orders of magnitude while requiring merely 30 μL sample. We demonstrated that MCF-7-derived exosomes induced epithelial-mesenchymal transition of epithelial cells MCF-10A, and our method was able to completely or partially reverse the transition by complete depletion or specific depletion of cancer exosomes without any preprocessing. Moreover, both whole exosomes and cancer-specific exosomes alone from mimic blood samples were successfully captured and counted, without obvious non-specific adsorption. In all, our approach realized the in situ depletion and number-counting of cancer-derived exosomes directly from the complex humoral environment, having the potential to provide a comprehensive tumor therapeutic and prognosis evaluation tool by targeted hemodialysis and counting of tumor-derived exosomes.

Linkage Pathways of DNA–Nanoparticle Conjugates and Biological Applications

DNA–nanoparticle conjugates have extraordinary optical and catalytic properties that have attracted great interest in biosensing and biomedical applications. Combining these special qualities has made it possible to create extremely sensitive and selective biomolecule detection methods, as well as effective nanopharmaceutical carriers and therapy medications. In particular, inorganic nanoparticles, such as metal nanoparticles, metal–organic framework nanoparticles, or upconversion nanoparticles with relatively inert surfaces can easily bind to DNA through covalent bonds, ligand bonds, electrostatic adsorption, biotin–streptavidin interactions and click chemistry to form DNA–nanoparticle conjugates for a broad range of applications in biosensing and biomedicine due to their exceptional surface modifiability. In this review, we summarize the recent advances in the assembly mechanism of DNA–nanoparticle conjugates and their biological applications. The challenges of designing DNA–nanoparticle conjugates and their further applications are also discussed.

Heterodimers with various bonding positions: Synthesis, plasmon coupling and surface enhanced Raman scattering

The novel plasmonic heterodimer with an adjustable self-assembled hot spot is synthesized via the bonding of single core-shell Au@Ag nanocube (Au@Ag NC) and varisized Au nanosphere (Au NS) based on the biotin-streptavidin interaction between single and ensemble assemblies. Due to the various bonding positions, three modes of heterodimers nanostructures (Middle, Vicinity, and Vertice modes) are prepared, and an obvious hot spot generates between the nanosphere and nanocube, showing different characteristics for surface plasmonic resonance effect. The obtained surface enhanced Raman scattering enhancement factors for the three modes of nanostructures have a narrow distribution and can be controlled in order of magnitude via adjusting the dimension of Au NS on single Au@Ag NC. Specifically, the heterodimer with Vertice mode can significant enhance the electric field in the bonding position, which reflects the synergistic action for the inter-particle plasmon coupling effect and the lightning rod effect. This microstructure and corresponding optical characteristic of the heterodimers are of great significance for research in related fields, including plasmonic device, biological detection, and in situ tracking in cells.

Construction of a Highly Selective Enrichment, Ionization, and Detection Platform Based on a Broad-Spectrum Antibody.

Ambient mass spectrometry (AMS) allows direct analysis of various raw food samples with minimal or no sample pretreatment, but the trace analytes in complex food samples still have problems with limitations. In this work, we developed a platform based on coated stainless steel sheet spray mass spectrometry for fast, in situ, high-throughput, and high selectivity multiresidue analysis of fluoroquinolone drugs (FQs). The sensitivity of the platform was enhanced via coupling broad-spectrum antibodies against FQs to graphene oxide coated blade spray (CBS)-MS through a streptavidin-biotin (SA-biotin) interaction. The prepared platform had sufficient loading capacity for SA (1.37 mg/piece) and the antibody (84.8 μg/piece), which is greater than that of physical mixing and the EDC/NHS covalent coupling strategy. With simplified sample pretreatment, this platform demonstrated comparable sensitivity to high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) (0.08-0.16 ng/mL in phosphate-buffered saline and 0.21-0.32 ng/mL in diluted milk). Meanwhile, compared with HPLC-MS/MS, the method is rapid (enrichment: 10 min, detection: <1 min) and acceptable recoveries (81.94-102.08%) can be obtained. The presence of analytes can be monitored by MS/MS spectra, and multiple analytes can be measured simultaneously in a single assay. This study is expected to provide a powerful and portable tool for rapid laboratory analysis and reliable screening in the field.

Streptavidin-Conjugated DNA for the Boronate Affinity-Based Detection of Poly(ADP-Ribose) Polymerase-1 with Improved Sensitivity.

This work reports the development of a fluorescence method for the detection of poly(ADP-ribose) polymerase-1 (PARP1), in which a phenylboronic acid-modified fluorescein isothiocyanate dye (FITC-PBA) was used to recognize the formed poly(ADP-ribose) (PAR) polymer. The detection system was designed by conjugating recombinant streptavidin (rSA) with PARP1-specific double-stranded DNA (dsDNA) through streptavidin-biotin interaction. Capture of PARP1 via rSA-biotin-dsDNA allowed for the poly-ADP-ribosylation (PARylation) of both rSA and PARP1 in a homogeneous solution. The resulting rSA-biotin-dsDNA/PAR conjugates were then captured and separated via the commercialized nitrilotriacetic acid-nickel ion-modified magnetic bead (MB-NTA-Ni) through the interaction between NTA-Ni on MB surface and oligohistidine (His6) tag in rSA. The PAR polymer could capture the dye of FITC-PBA through the borate ester interaction between the boronic acid moiety in PBA and the cis-diol group in ribose, thus causing a decrease in fluorescence signal. The PARylation of streptavidin and the influence of steric hindrance on PARylation efficiency were confirmed using reasonable detection strategies. The method showed a wide linear range (0.01~20 U) and a low detection limit (0.01 U). This work should be valuable for the development of novel biosensors for the detection of poly(ADP-ribose) polymerases and diol-containing species.

Direct Comparison of Lysine versus Site-Specific Protein Surface Immobilization in Single-Molecule Mechanical Assays.

Single-molecule force spectroscopy (SMFS) is powerful for studying folding states and mechanical properties of proteins, however, it requires protein immobilization onto force-transducing probes such as cantilevers or microbeads. A common immobilization method relies on coupling lysine residues to carboxylated surfaces using 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS). Because proteins typically contain many lysine groups, this strategy results in a heterogeneous distribution of tether positions. Genetically encoded peptide tags (e.g., ybbR) provide alternative chemistries for achieving site-specific immobilization, but thus far a direct comparison of site-specific vs. lysine-based immobilization strategies to assess effects on the observed mechanical properties was lacking. Here, we compared lysine- vs. ybbR-based protein immobilization in SMFS assays using several model polyprotein systems. Our results show that lysine-based immobilization results in significant signal deterioration for monomeric streptavidin-biotin interactions, and loss of the ability to correctly classify unfolding pathways in a multipathway Cohesin-Dockerin system. We developed a mixed immobilization approach where a site-specifically tethered ligand was used to probe surface-bound proteins immobilized through lysine groups, and found partial recovery of specific signals. The mixed immobilization approach represents a viable alternative for mechanical assays on in vivo-derived samples or other proteins of interest where genetically encoded tags are not feasible.

Magnetically Assisted Immobilization-Free Detection of microRNAs Based on the Signal Amplification of Duplex-Specific Nuclease.

The double specific nuclease (DSN)-based methods for microRNAs (miRNAs) detection usually require the immobilization of DNA probes on a solid surface. However, such strategies have the drawbacks of low hybridization and cleavage efficiency caused by steric hindrance effect and high salt concentration on the solid surface. Herein, we proposed an immobilization-free method for miRNA detection on the basic of DSN-assisted signal amplification. The biotin- and fluorophore-labeled probes were captured by streptavidin-modified magnetic beads through streptavidin-biotin interactions, thus producing a poor fluorescence signal. Once the DNA probes were hybridized with target miRNA in solution to form DNA-miRNA duplexes, DNA stands in the duplexes would be selectively digested by DSN. The released target miRNA could initiate the next hybridization/cleavage recycling in the homogeneous solution, finally resulting in the release of numerous fluorophore-labeled fragments. The released fluorophores remained in solution and emitted strong fluorescence after treatment by the streptavidin-modified magnetic beads. The immobilization-free method achieved the assays of miRNA-21 with a detection limit down to 0.01 pM. It was employed to evaluate the expression levels of miRNA-21 in different cancer cells with satisfactory results.