Hybridization Affinity Research Articles

Design and Validation of Specific Oligonucleotide Probes on Planar Magnetic Biosensors.

Planar DNA biosensors employ surface-tethered oligonucleotide probes to capture target molecules for diagnostic applications. To improve the sensitivity and specificity of biosensing, hybridization affinities should be enhanced, and cross-hybridization with off-targets must be minimized. To this end, assays can be designed using the thermodynamic properties of hybridization between probes and on-targets or off-targets based on Gibbs free energies and melting temperatures. However, the nature of heterogeneous hybridization between the probes on the surface and the targets in a solution imposes challenges in predicting precise hybridization affinities and the degree of cross-hybridization due to indeterminable thermodynamic penalties induced by the solid surface and its status. Herein, we suggest practical and convenient guidelines for designing oligonucleotide probes based on data obtained from planar magnetic biosensors and thermodynamic properties calculated by using easily accessible solution-phase prediction. The suggested requirements comprised Gibbs free energy ≥ -7.5 kcal mol-1 and melting temperature ≤10 °C below the hybridization temperature, and we validated for the absence of cross-hybridization. Additionally, the effects of secondary structures such as hairpins and homodimers were investigated for better oligonucleotide probe designs. We believe that these practical guidelines will assist researchers in developing planar magnetic biosensors with high sensitivity and specificity for the detection of new targets.

Modulating of d-band center in Cu single-atom anchored Co3O4 nanozyme for sensitive immunochromatographic test strip

Immunochromatographic test strip (ITS) has been widely employed for point-of-care diagnoses. However, how to significantly enhance the analytical sensitivity of device-free ITS remains a challenge. Here, we synthesized a Cu single-atom anchored Co3O4 (CuSA/Co3O4) nanozyme that possesses ultrahigh peroxidase-mimicking catalytic activity owing to d-band center upshift mediated by Cu single-atom. Molecular orbital analysis demonstrated that d orbitals in CuSA/Co3O4 have stronger orbital hybridization affinity than Co3O4 to H2O2′s π* orbitals. This reinforces hydroxyl radical (OH) production and provides a stronger catalytic colorimetric signal, which is beneficial for improving the sensitivity of ITS. Moreover, we applied the CuSA/Co3O4 to ITS as a signal reporter for monitoring Xanthomonas oryzae pv. oryzicola (Xoc), the pathogen of rice bacterial leaf streak. The visual limit of detection (LOD) of the developed CuSA/Co3O4-based ITS was as low as 6.4 × 102 CFU mL−1, which is 25-fold more sensitive than the traditional Au nanoparticles (AuNP)-based ITS. Taken together, this work demonstrates the great potential of CuSA/Co3O4 nanocomposite as a signal reporter to improve the sensitivity of ITS for on-site Xoc detection.

New Benzylidene‐Triazole Tethered Coumarin Molecular Hybrids: Synthesis, SAR, Molecular Modelling Simulations Targeting Tubulin α/β Dimer and its Antimicrobial Evaluation

AbstractA series of new 7‐{3‐[3‐(Benzylidene‐amino)‐[1,2,4]triazole‐1‐yl]‐propoxy}‐4‐methyl‐chromen‐2‐one molecular hybrids 7(a–k) were synthesized using molecular hybridization approach. The synthesized new benzylidene‐triazole tethered coumarin molecular hybrids were characterized by spectroscopic studies and elemental analysis. The coumarin molecular hybrids have been explored for their antifungal efficacy in terms of tubulin inhibitors using molecular modelling studies against tubulin alphabeta hetero dimer protein. The results clearly demonstrated strong binding affinity of coumarin molecular hybrids with indole substituted and 2,4‐dihydroxy substituted as antagonists with respective binding scores of −10.5 and −10.7 kcal/mol towards tubulin alphabeta hetero dimer protein which was higher in comparison to standards griseofulvin (−8.2 kcal/mol) and fluconazole (−8.1 kcal/mol). In addition, broth dilution method was used to conduct in‐vitro antifungal studies against three fungal strains namely Candida albicans, Aspergillus niger and Aspergillus fumigatus. The results obtained clearly illustrate the antifungal activity of indole and 2,4‐dihydroxy substituted compounds which are consistent with docking simulations. Out of all the synthesized molecular hybrids, indole and 2,4‐dihydroxy substituted compounds depict outstanding antifungal activity with Minimum Inhibitory Concentration (MIC) value of 31.25 μg/ml and 62.5 μg/ml towards C.albicans and with MIC value of 31.25 for both these molecules towards A.niger in each case which is quite higher than griseofulvin and fluconazole respectively. Present work clearly demonstrates the potential efficacy of synthesized indole and 2,4‐dihydroxy substituted coumarin molecular hybrids to be considered as lead compounds for the development of new agents against resistant strains for the treatment of fungal infection.

Crossing the Halfway Point: Aptamer-Based, Highly Multiplexed Assay for the Assessment of the Proteome.

Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.

Novel coumarin-piperazine-2(5H)-furanone hybrids as potential anti-lung cancer agents: Synthesis, biological evaluation and molecular docking studies

Novel coumarin-piperazine-2(5H)-furanone hybrids 5a–l were efficiently synthesized by introducing a furanone scaffold into coumarin using piperazine as a linker. The cytotoxicity of all hybrids 5a–l were evaluated by MTT assay on human lung cancer A549 cells and normal human lung fibroblast WI-38 cells with cytarabine (CAR) as a positive control. Hybrid 5l (IC50 = 11.28 μM) was the most toxic to A549 cells, 18-fold more toxic than the reference CAR (IC50 = 202.57 μM). Moreover, hybrid 5l (IC50 = 411.93 μM) was less toxic to WI-38 cells, with a much higher selectivity (5l, SI ≈ 37, WI-38/A549) than CAR (SI ≈ 2). Structure–activity relationship analysis showed that both the cytotoxicity against A549 cells and selectivity (WI-38/A549) were greatly improved when the bornyl group was incorporated in the hybrids (5c, 5f, 5i and 5l). Further, hybrid 5l was more toxic and selective against four types of human lung cancer cells (A549, Calu-1, PC-9 and H460; IC50 = 5.72–45.46 μM; SI ≈ 9–72) than three other types of human cancer cells (SK-BR-3, 786-O and SK-OV-3, IC50 = 39.07–130.82 μM; SI ≈ 0–2), showing remarkable specificity. In particular, hybrid 5l (IC50 = 5.72 μM) showed the highest cytotoxicity against H460 cells with the highest selectivity of up to 72 (WI-38/H460). Flow cytometric analysis showed that hybrid 5l induced apoptosis in H460 cells in a concentration-dependent manner. Molecular docking studies revealed a high binding affinity of hybrid 5l with CDK2 protein. Hybrid 5l is expected to be a leading candidate for anti-lung cancer agents.

Adaptive Conditional Denoising Diffusion Model With Hybrid Affinity Regularizer for Generalized Zero-Shot Learning

Adaptive Conditional Denoising Diffusion Model With Hybrid Affinity Regularizer for Generalized Zero-Shot Learning

An Aptamer-Based Hydrogel Sensing Platform Enabling Low Micromolar Detection of Small Molecules Using Low Sample Volumes.

Poor fluorescence recovery at low analyte dosages and slow ligand binding kinetics are critical challenges currently limiting the use of aptamer-functionalized hydrogels for sensing small molecules. In this paper, we report an adenosine-responsive hydrogel sensor that integrates FRET-signaling aptamer switches into in situ-gelling thin-film hydrogels. The hydrogel sensor is able to entrap a high proportion of the sensing probes (>70% following vigorous washing), delay nucleolytic degradation, stabilize weak aptamer complexes to improve hybridization affinity and suppress fluorescence background, and provide high sensitivity in biological fluids (i.e., undiluted human serum). Furthermore, the developed hydrogel sensors were able to achieve low limits of detection (5.3 μM in buffer and 8.8 μM in serum) within 4 min of exposure to the sample, with signal generation requiring only 20 μL/well of analyte sample. The physical nature of the aptamer encapsulation allows this approach to accommodate virtually any small-molecule aptamer, avoiding the need for covalent anchoring and the complex modification of nucleic acid sequences typically required for effective aptamer-based molecular recognition.

New Ciprofloxacin–Pyrazolopyrimidine Hybrids as Topoisomerase IV and Gyrase Inhibitors against MRSA AUMC 261

AbstractEleven novel ciprofloxacin–pyrazolopyrimidine hybrids 4 a–k were synthesized and structurally elucidated using NMR, elemental analysis, and mass spectrometry. The antibacterial screening showed that the newly synthesized compounds revealed a remarkable shifting of antibacterial activity from Gram–negative toward Gram–positive bacteria. Most of the synthesized hybrids displayed good to excellent activity against Staph aureus ATCC 6538 especially hybrids 4 a and 4 g which revealed MICs of 12.5 and 6.25 μg/ml, respectively, compared to the ciprofloxacin MIC, 25 μg/ml as a reference drug. In addition, both hybrids displayed potent activity toward MRSA AUMC 261 with MICs, 71 and 36 μg/ml respectively, compared to the ciprofloxacin MIC, 190 μg/ml as a reference drug. Additionally, hybrids, 4 a and 4 g exhibited effective inhibitory activity against DNA gyrase and topoisomerase IV enzymes with IC50 values, 0.404, 1.626 μM and 3.647 and 3.791 μM, respectively, compared to ciprofloxacin IC50 values 0.661 and 8.159. A molecular modeling investigation showed a high fitting affinity of hybrids 4 a and 4 g toward gyrase and topoisomerase IV active sites compared to ciprofloxacin.

A Canvas of Spatially Arranged DNA Strands that Can Produce 24-bit Color Depth.

Nucleic acid microarray photolithography combines density, throughput, and positional control in DNA synthesis. These surface-bound sequence libraries are conventionally used in large-scale hybridization assays against fluorescently labeled, perfect-match DNA strands. Here, we introduce another layer of control for in situ microarray synthesis─hybridization affinity─to precisely modulate fluorescence intensity upon duplex formation. Using a combination of Cy3-, Cy5-, and fluorescein-labeled targets and an ensemble of truncated DNA probes, we organize 256 shades of red, green, and blue intensities that can be superimposed and merged. In so doing, hybridization alone is able to produce a large palette of 16 million colors or 24-bit color depth. Digital images can be reproduced with high fidelity at the micrometer scale by using a simple process that assigns sequence to any RGB value. Largely automated, this approach can be seen as miniaturized DNA-based painting.

Transition of average drug-to-antibody ratio of trastuzumab deruxtecan in systemic circulation in monkeys using a hybrid affinity capture liquid chromatography-tandem mass spectrometry.

Trastuzumab deruxtecan (T-DXd, DS-8201a) is an antibody-drug conjugate, comprising an anti-HER2 antibody at a drug-to-antibody ratio of 7-8 with the topoisomerase I inhibitor DXd. In this study, the concentrations of antibody-conjugated DXd and total antibody were determined and observed to decrease over time following intravenous administration of T-DXd to monkeys. The drug-to-antibody ratio of T-DXd also decreased in a time-dependent manner, which reached approximately 2.5 in 21days after administration. It was suggested that antibody-conjugated DXd of T-DXd was relatively stable in vivo compared with that of other reported antibody-drug conjugates.

Global Stability of a Fourth-Order Hybrid Affine System

Global Stability of a Fourth-Order Hybrid Affine System

STUDY OF HYBRIDIZATION OF COMPLEMENTARY SINGLE-STRANDED POLYNUCLEOTIDES POLY(rA) AND POLY(rU)

The study of the interaction of intercalators ethidium bromide (EtBr), methylene blue (MB) and groove binding compound Hoechst 33258 (H33258) with single-stranded synthetic polyribonucleotides poly(rA) and poly(rU) has been carried out by the method of UV-melting, at ionic strengths of the solution 0.04 M and 0.1 M. Stirring of poly(rA) and poly(rU) by equal-molar concentrations was shown to result in hybridization with formation of double-stranded (ds-) structure poly(rA)-poly(rU). It was revealed that EtBr, with higher degree in comparison with MB, stimulates hybridization and stabilizes the formed ds-structure poly(rA)-poly(rU). It was also found out that the hybridization process and affinity of EtBr and MB depend on the ionic strength of the solution and these processes occur much more effectively at the ionic strength 0.04 M. On the other hand, it was shown that the groove binding ligand H33258 practically does not affect the stabilization of the formed ds-structure poly(rA)-poly(rU).

Quantitative [123]I-Ioflupane DaTSCAN single-photon computed tomography-computed tomography in Parkinsonism.

[123]I-Ioflupane (DaTSCAN) binds to the presynaptic dopamine transporter (DAT) and with a lower affinity to the serotonin transporter (SERT). We aimed to develop a novel method to quantify absolute uptake in the striatal (predominantly DAT binding) and extra-striatal regions (mainly SERT binding) using single-photon computed tomography-computed tomography (SPECT-CT) DaTSCAN and to improve DaTSCAN image quality. Twenty-six patients with Parkinsonism underwent DaTSCAN SPECT-CT prospectively. The scans were visually analyzed independently by two experienced reporters. Specific binding ratios (SBRs) from Chang attenuation corrected SPECT were obtained using GE DaTQuant. Normalized concentrations and specific uptakes (NSU) from measured attenuation and modelled scatter-corrected SPECT-CT were obtained using HERMES Hybrid Recon and Affinity and modified EARL volumes of interest. Striatal NSU and SBR positively correlate ( R = 0.65-0.88, P = 0.00). SBR, normalized concentrations, and NSU box plots differentiated between scans without evidence of dopaminergic deficit and abnormal scans. Interestingly, body weight inversely correlated with normalized concentrations values in extra-striatal regions [frontal ( R = 0.81, P = 0.00); thalamus ( R = 0.58, P = 0.00); occipital ( R = 0.69, P = 0.00)] and both caudate nuclei [ R = 0.42, P = 0.03 (Right), R = 0.52, P = 0.01 (Left)]. Both reporters noted improved visual quality of SPECT-CT versus SPECT images for all scans. DaTSCAN SPECT-CT resulted in more accurate quantification, improved image quality, and enabled absolute quantification of extra-striatal regions. More extensive studies are required to establish the full value of absolute quantification for diagnosis and monitoring the progression of neurodegenerative disease, to assess an interplay between DAT and SERT, and to verify whether serotonin and DATs are potentially dysfunctional in obesity.

Label-Free Quantification of DNA Hybridization Kinetics and Affinity Enabled by an Electro-Optical Modulation on Single Nanoparticles.

Since the formation of a DNA duplex plays a vital role in gene expression and regulation across all kinds of organisms, quantifying the interaction during DNA hybridization is essential for understanding various biological processes in living systems on the molecular scale of nucleic acids. Here, we developed a label-free method to measure the binding kinetics and affinity of DNA hybridization with an electro-optical modulation on the individual gold nanorods based on a total internal reflection dark-field imaging microscopy. Under the electrochemical modulation, a Fourier transform filter was utilized to extract the optical scattering of the nanorods, which varies with the DNA binding due to their impedance change. We validated the imaging principle and established an analytical model to monitor the optical response during the DNA hybridization. Using the presented platform, we measured the binding kinetics and affinities of different DNA pairs and demonstrated its capability to distinguish the DNA hybridization with only single-base mismatch, which may provide guidance to explore the etiology and pathogenesis of diseases associated with single point gene mutations. Furthermore, the method allows for simultaneous imaging of multiple nanoparticles, thus enabling a high-throughput detection and opening possibilities for the study of the interfacial heterogeneity.

Variable Step Size Methods of the Hybrid Affine Projection Adaptive Filtering Algorithm under Symmetrical Non-Gaussian Noise

The idea of variable step-size was introduced into the Hybrid Affine Projection Algorithm (H-APA) and we propose two variable step size algorithms based on H-APA, which are called the Variable Step-Size Hybrid Affine Projection Algorithm (VSS-H-APA) and the Modified Variable Step-Size Hybrid Affine Projection Algorithm (MVSS-H-APA). These are two variable-step algorithms aim to further improve the robust performance and convergence speed of H-APA under non-Gaussian noise. This allows for faster convergence while maintaining stability. The MVSS-H-APA goes further than VSS-H-APA to estimate the noise in order to achieve better convergence performance. The proposed algorithm performs better than the existing algorithms in system identification under symmetric non-Gaussian noise.

Designing and fabrication of electrochemical nano-biosensor for the fast detection of SARS-CoV-2-RNA

SARS-CoV-2 caused a global panic among populations. Rapid diagnostic procedures for the virus are crucial for disease control. Thus, the designed signature probe from a highly conserved region of the virus was chemically immobilized onto the nanostructured-AuNPs/WO3-screen printed electrodes. Different concentrations of the matched oligonucleotides were spiked to test the specificity of the hybridization affinity whereas the electrochemical impedance spectroscopy was used for tracking the electrochemical performance. After a full assay optimization, limits of detection and quantification were calculated based on linear regression and were valued at 298 and 994 fM, respectively. Further, the high performance of the fabricated RNA-sensor chips was confirmed after testing the interference status in the presence of the mismatched oligos in one nucleotide and completely one. Worthy to mention that the single-stranded matched oligos can be hybridized to the immobilized probe in 5 min at room temperature. The designed disposable sensor chips are capable of detecting the virus genome directly. Therefore, the chips are a rapid tool for SARS-CoV-2 detection.

Rapid Visible Detection of African Swine Fever Virus Using Hybridization Chain Reaction-Sensitized Magnetic Nanoclusters and Affinity Chromatography.

African swine fever virus (ASFV) is a severe and persistent threat to the global swine industry. As there are no vaccines against ASFV, there is an immense need to develop easy-to-use, cost-effective, and rapid point-of-care (POC) diagnostic platforms to detect and prevent ASFV outbreaks. Here, a novel POC diagnostic system based on affinity column chromatography for the optical detection of ASFV is presented. This system employs an on-particle hairpin chain reaction to sensitize magnetic nanoclusters with long DNA strands in a target-selective manner, which is subsequently fed into a column chromatography device to produce quantitatively readable and colorimetric signals. The detection approach does not require expensive analytical apparatus or immobile instrumentation. The system can detect five genes constituting the ASFV whole genome with a detection limit of ≈19.8 pm in swine serum within 30min at laboratory room temperature. With an additional pre-amplification step using polymerase chain reaction (PCR), the assay is successfully applied to detect the presence of ASFV in 30 suspected swine samples with 100% sensitivity and specificity, similar to quantitative PCR. Thus, this simple, inexpensive, portable, robust, and customizable platform for the early detection of ASFV can facilitate the timely surveillance and implementation of control measures.

Robust hybrid affine projection filtering algorithm under [formula omitted]-stable environment

A novel hybrid affine projection algorithm (H-APA) is proposed. This algorithm is considered to be a derivation of the affine projection sign algorithm (APSA). The APSA can be greatly affected by impulsive noise. To solve this problem, the proposed algorithm is applied with the robust mixed-norm algorithm (RMNA). The simulated results show that the proposed algorithm is insensitive to impulsive noise with fast convergence rate under the colored input condition. In addition, a simplified H-APA (SH-APA) is derived based on the proposed H-APA to reduce the complexity of the algorithm. Numerical simulations show that compared with the existing AP-type algorithms, the proposed algorithms have faster convergence speed.

A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples

Therapeutic oligonucleotides, such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA), are a new class of therapeutics rapidly growing in drug discovery and development. A sensitive and reliable method to quantify oligonucleotides in biological samples is critical to study their pharmacokinetic and pharmacodynamic properties. Hybridization LC-MS/MS was recently established as a highly sensitive and specific methodology for the quantification of single-stranded oligonucleotides, e.g., ASOs, in various biological matrices. However, there is no report of this methodology for the bioanalysis of double-stranded oligonucleotides (e.g., siRNA). In this work, we investigated hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides in biological samples using an siRNA compound, siRNA-01, as the test compound. The commonly used DNA capture probe and a new peptide nucleic acid (PNA) probe were compared for the hybridization extraction of siRNA-01 under different conditions. The PNA probe achieved better extraction recovery than the DNA probe, especially for high concentration samples, which may be due to its stronger hybridization affinity. The optimized hybridization method using the PNA probe was successfully qualified for the quantitation of siRNA-01 in monkey plasma, cerebrospinal fluid (CSF), and tissue homogenates over the range of 2.00-1000 ng/mL. This work is the first report of the hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides. The developed methodology will be applied to pharmacokinetic and toxicokinetic studies of siRNA-01. This novel methodology can also be used for the quantitative bioanalysis of other double-stranded oligonucleotides.

Multivalent forms of the ribonuclease H1 hybrid binding domain are high-affinity binders of RNA-DNA hybrids.

The hybrid binding domain (HBD) is a conserved fold present in ribonucleases H1 that selectively recognizes RNA-DNA hybrids, which are structures present in cellular R-loops and participate in diverse biological processes. We engineered multivalent HBD proteins to create high-affinity hybrid binders. Using EMSA- and SPR-based analyses, we showed that the triple-HBD protein exhibits a ~ 22 000-fold increase in hybrid affinity (KD 370 pm) relative to the single HBD (KD 8.29 μm), with the length and sequence of the linkers enabling optimal function. These findings provide a framework for testing models that correlate multivalency and affinity to understand how multivalent proteins function and also can serve to guide applications that exploit multivalency as a strategy to enhance binding affinity.