Binding Of Probe Research Articles

AAp-MSMD: Amino Acid Preference Mapping on Protein-Protein Interaction Surfaces Using Mixed-Solvent Molecular Dynamics.

Peptides have attracted much attention recently owing to their well-balanced properties as drugs against protein-protein interaction (PPI) surfaces. Molecular simulation-based predictions of binding sites and amino acid residues with high affinity to PPI surfaces are expected to accelerate the design of peptide drugs. Mixed-solvent molecular dynamics (MSMD), which adds probe molecules or fragments of functional groups as solutes to the hydration model, detects the binding hotspots and cryptic sites induced by small molecules. The detection results vary depending on the type of probe molecule; thus, they provide important information for drug design. For rational peptide drug design using MSMD, we proposed MSMD with amino acid residue probes, named amino acid probe-based MSMD (AAp-MSMD), to detect hotspots and identify favorable amino acid types on protein surfaces to which peptide drugs bind. We assessed our method in terms of hotspot detection at the amino acid probe level and binding free energy prediction with amino acid probes at the PPI site for the complex structure that formed the PPI. In hotspot detection, the max-spatial probability distribution map (max-PMAP) obtained from AAp-MSMD detected the PPI site, to which each type of amino acid can bind favorably. In the binding free energy prediction using amino acid probes, ΔGFE obtained from AAp-MSMD roughly estimated the experimental binding affinities from the structure-activity relationship. AAp-MSMD, with amino acid probes, provides estimated binding sites and favorable amino acid types at the PPI site of a target protein.

Brain‐Penetrable Antibody Probes for in vivo Tau Imaging

AbstractBackgroundAntibody‐based PET ligands are desirable due to their high specificity; however, their brain uptakes are limited by the blood‐brain barrier (BBB). We previously demonstrated that transport of antibody across BBB can be facilitated through interaction with the transferrin receptor (TfR)1. We report here the first in vivo PET imaging studies using BBB permeable F‐18 radiolabeled bispecific antibodies specific to tau protein in mice.MethodWe performed multiple PET scans using each of the radio‐probes synthesized via conjugation of [18F]SFB with one of the four bispecific antibody constructs: 1) 6B2G12‐ScFv8D3 (full‐size Tau antibody conjugated with TfR fragment (ScFv8D3); abbreviated as TAUb), 2) ScFv235‐ScFv8D3 (small Tau‐TfR; TAUs), 3) 3D6‐ScFv8D3 (full‐size Aβ‐TfR; Aβb) or 4) ScFv3D6‐ScFv3D6 (small Aβ‐TfR; Aβs). We used a matched control design to compare a WT with one of the two types of transgenic (tg) tauopathy mice [PS19 (P301S) or JNPL3 (P301)] in each scan. Subjects were scanned on a MOLECUBES PET/CT scanner. An initial low‐dose (244 ± 54 μCi) dynamic scan (30 min) was performed, followed by two scans at 8hr and 12hr (20 min each static scans) after injection of a high dose (1832 ± 447 μCi) of the same probe. PMOD and FireVoxel were used for imaging processing. After co‐registration of PET/CT/atlas, data were quantified as SUV and SUVR [CB as a reference], and clearance was estimated as %change/hr.ResultTAUs probe displayed highest brain uptake with more specific binding retained in the brain, as compared to TAUb and Aβs. TAUs probe showed faster brain penetration in tau‐tg mice than in WT, with differences in amygdala, basal forebrain, and hypothalamus of 66%, 62% and 85%, respectively, at 8hr post‐injection (Fig. 1). Further, higher brain uptakes (TAUs > Aβs) in the same tau‐tg mouse (Fig. 2) suggest the specific binding of TAUs probe, while Aβs probe can serve as a control due to its lack of specific binding in tau‐tg mice.ConclusionWe have successfully synthesized and evaluated four novel F‐18 bispecific antibodies via in vivo PET. TAUs probe showed most promising BBB permeability and binding specificity to tau antigen with a reasonable clearance.

Development of a neuroimaging biomarker of the pro‐coagulant state in Alzheimer’s disease: The BioClotAD Project

AbstractBackgroundAlzheimer’s disease (AD) is the most common form of dementia1. Its multifactorial profile includes a strong vascular factor, partially driven by an early haemostatic dysregulation. Accordingly, fibrin, the main protein component of blood clots, is significantly increased in AD‐patients’ brains2. Furthermore, amyloid‐β contributes to this process by interacting with fibrin and producing degradation‐resistant clots3. Consequently, this pro‐thrombotic milieu intensifies hypoperfusion, neurodegeneration and blood‐brain barrier (BBB) disruption4, but it does not affect all patients. Therefore, early detection of these phenomena would be invaluable to identify patients likely to benefit from anticoagulant therapies5. Therefore, the BioClotAD Project aims to develop an imaging biomarker based on a fibrin binding probe (FBP)6 to non‐invasively identify AD’s pro‐coagulant state.MethodBioClotAD involves four partners in three European countries with complementary expertise. To optimize and test FBP we use extensive in vitro, ex vivo and in vivo assays, including AD animal models and human AD samples. Our project is comprised of three blocks: 1) Testing the FBP to in vivo detect the cerebral occlusions by nuclear imaging. 2) Detecting fibrin deposits inside the brain parenchyma with FBP coupled to a transferrin receptor antibody (FBP‐TfR) to facilitate BBB crossing7. FBP‐TfR will be labelled for optical and nuclear imaging. 3) Validating the most promising FBP probes in frozen brain samples of AD patients by autoradiography or fluorescence microscopy.ResultBioClotAD provides feasible neuroimaging strategies to in vivo detect the fibrin cerebral accumulation of AD models and identify the specific regional distribution in the brain. Our project will set the basis for future clinical trials on neuroimaging of the cerebral fibrin accumulation in AD.ConclusionBioClotAD neuroimaging biomarkers will allow the early detection of the pro‐thrombotic state in AD, opening a window of opportunity to delay the disease progression by personalized anticoagulant therapies. Funding: EU Joint Programme – Neurodegenerative Disease Research (JPND)

Plasma Proteome Profiling of Sickle Cell Patients Reveals Activation of Inflammatory Pathways during Steady State and Vaso-Occlusive Crisis

Background The pathophysiology of Sickle Cell Disease (SCD) is characterized by chronic intravascular hemolysis and ischemia-reperfusion, resulting in oxidative stress and chronic inflammation. Although inflammation seems to play an important role in the development of tissue damage in SCD, little is known about the various cellular and cytokine pathways involved in the pathophysiology of SCD. This study aims to further elucidate the inflammatory pathways in SCD using targeted plasma protein profiling both in steady state and during vaso-occlusive crisis (VOC). Methods In this prospective study, SCD patients with the genotypes HbSS and HbSβ 0-thalassemia and healthy ethnicity matched controls were included. Plasma levels of 92 proteins involved in various inflammatory pathways were measured in EDTA plasma using Olink Proximity Extension Assay (PEA) technology. In brief, pairs of oligonucleotide-labeled antibody probes were added to 20 μL of plasma, resulting in the binding of these antibody probes to their targeted protein. Subsequently DNA polymerase was added, leading to a proximity-dependent DNA polymerization event, generating a unique PCR target sequence. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, which is an arbitrary unit on a log2-scale with an NPX difference (estimate) of 1 meaning a doubling of protein concentration. RStudio version 4.2.1 was used for data analysis and visualization. Differences between subgroups were tested using an unpaired or paired t-test as appropriate. Results were corrected for multiple testing using the Benjamini-Hochberg method. Results with p < 0.05 were considered significant. Furthermore Over-representation Analysis (ORA) was performed to investigate the association of the differentially expressed proteins with specific biological processes. Results Plasma samples were collected from 43 SCD patients in steady state (median age: 25 years (IQR: 20 - 32)) and 13 healthy ethnicity matched controls (median age: 29 (IQR: 28-39)). From 15 SCD patients plasma samples were collected during both steady state and VOC (median age: 27 (IQR: 19 - 34)). Comparison of protein levels between healthy controls and SCD patients in steady state identified 38 proteins (IL-18, ANGPT2, Gal-9, CD4, HGF, HO-1, CCL4, ARG1, ADA, GZMA, PD-L1, CCL23, LAG3, TNFRSF4, CCL3, CXCL11, CRTAM, IL-10, GZMB, CD27, FGF2, IL-8, IL-6, TNF, PGF, NCR1, TNFSF14, CASP-8, IL-7, FASLG, TNFRSF9, LAP TGF-beta-1, PD-L2, CCL19, CD83, GZMH, MCP-4, CD244, TIE2) that were significantly higher in SCD patients as shown in Figure 1. Furthermore soluble CD70 was significantly lower in SCD patients in steady state compared to healthy controls (estimate: -0.34, p < 0.05). Over-representation Analysis revealed a significant upregulation of proteins involved in the regulation of hematopoiesis, the IL-18 signaling pathway, T-cell activation, leukocyte differentiation and leukocyte adhesion (Figure 2). Pairwise comparison of protein levels between steady state and VOC in SCD patients revealed 6 proteins (TWEAK, VEGFR-2, CD244, PDCD1, GZMA, TNFRSF4) which were significantly lower and 12 proteins (IL-6, TNFSF14, ADA, IL-15, HGF, IL-10, IL-8, Gal-9, CSF-1, CCL20, MMP7, VEGFA) which were significantly higher during VOC as compared to steady state. During VOC, pathways involved in the regulation of cell death (IL-6, TNFSF14, ADA, HGF, IL-10, PDCD1, VEGFA) and tissue development (IL-6, ADA, HGF, IL-10, VEGFA) were significantly enriched. Summary/conclusion The results of these proteomic analyses provide new insights into the pathways that regulate inflammation in SCD, both during steady state and VOC. These findings might contribute to a better understanding of SCD pathophysiology and identifying potential new targets for therapeutic interventions in SCD patients. Our results warrant further in-depth investigation of the different activated pathways.

Wetting of a Hydrophobic Surface: Far-IR Action Spectroscopy and Dynamics of Microhydrated Naphthalene.

The interaction of water and polycyclic aromatic hydrocarbons is of fundamental importance in areas as diverse as materials science and atmospheric and interstellar chemistry. The interplay between hydrogen bonding and dipole-π interactions results in subtle dynamics that are challenging to describe from first principles. Here, we employ far-IR action vibrational spectroscopy with the infrared free-electron laser FELIX to investigate naphthalene with one to three water molecules. We observe diffuse bands associated with intermolecular vibrational modes that serve as direct probes of the loose binding of water to the naphthalene surface. These signatures are poorly reproduced by static DFT or Møller-Plesset computations. Instead, a rationalization is achieved through Born-Oppenheimer Molecular Dynamics simulations, revealing the active mobility of water over the surface, even at low temperatures. Therefore, our work provides direct insights into the wetting interactions associated with shallow potential energy surfaces while simultaneously demonstrating a solid experimental-computational framework for their investigation.

Selective quinizarin-linked bis-1,2,3-triazoles as probes for Fe(II)/(III): Characterization, metal ion binding, DFT, and antiallergy activity via docking studies

Herein, two new molecular probes of quinizarin-linked bis-1,2,3-triazoles have been synthesized via CuAAC reaction to detect transition metal(s), such as iron (Fe). FTIR, 1H & 13C-NMR, and HRMS spectroscopy fully characterized the synthesized probes. Absorption spectroscopy showed selective binding of probes with Fe(II) and Fe(III) ions. Job's plot suggested a 1:1 stoichiometric ratio of ligand and metal ion. Both probes 3a and 3b showed association constant of 4.42 × 103 M−1 and 4.35 × 103M−1 for Fe(II) and 4.35 × 103 M−1 and 4.59 × 103 M−1 for Fe(III), through Benesi-Hildebrand (B-H) plots. The DFT was performed for geometrical optimization of probes and their complexes, supported by Mulliken charges and Molecular electrostatic potential. Above and beyond, probes and their complexes were also docked with human histamine H1 receptor (PDB ID: 3RZE) protein, providing foretastes of its anti-allergic activity.

Proof of concept: Pull down assay using bovine serum albumin and an immunomodulator small molecule

In the past decade, there has been increasing interest in use of small molecules for immunomodulation. The affinity-based pull-down purification is an essential tool for target identification of small molecules and drug discovery. This study presents our recent efforts to investigate the cellular target(s) of Compound A, a small molecule with demonstrated immunomodulatory properties in human peripheral blood mononuclear cells (PBMCs). While we have previously observed the immunomodulatory activity of Compound A in PBMCs, the specific molecular targets underlying its effects remains elusive. To address this challenge, we synthesized a trifluoromethyl phenyl diazirine (TPD)-bearing trifunctional Probe 1 based on the chemical structure of Compound A, which could be used in a pull-down assay to efficiently bind to putative cellular targets via photoaffinity labelling. In this report, we utilized bovine serum albumin (BSA) as a model protein to establish a proof-of-concept in order to assess the suitability of Probe 1 for binding to an endogenous target. By the successful synthesis of Probe 1 and demonstrating the efficient binding of Probe 1 to BSA, we propose that this method can be used as a tool for further identification of potential protein targets of small molecules in living cells. Our findings provide a valuable starting point for further investigations into the molecular mechanisms underlying the immunomodulatory effects of Compound A.

Heteromultivalency enables enhanced detection of nucleic acid mutations.

Detecting genetic mutations such as single nucleotide polymorphisms (SNPs) is necessary to prescribe effective cancer therapies, perform genetic analyses and distinguish similar viral strains. Traditionally, SNP sensing uses short oligonucleotide probes that differentially bind the SNP and wild-type targets. However, DNA hybridization-based techniques require precise tuning of the probe's binding affinity to manage the inherent trade-off between specificity and sensitivity. As conventional hybridization offers limited control over binding affinity, here we generate heteromultivalent DNA-functionalized particles and demonstrate optimized hybridization specificity for targets containing one or two mutations. By investigating the role of oligo lengths, spacer lengths and binding orientation, we reveal that heteromultivalent hybridization enables fine-tuned specificity for a single SNP and dramatic enhancements in specificity for two non-proximal SNPs empowered by highly cooperative binding. Capitalizing on these abilities, we demonstrate straightforward discrimination between heterozygous cis and trans mutations and between different strains of the SARS-CoV-2 virus. Our findings indicate that heteromultivalent hybridization offers substantial improvements over conventional monovalent hybridization-based methods.

Chemoproteomic capture of RNA binding activity in living cells

Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5500 cysteine sites across ~3000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.

Spectroscopic and theoretical studies on a novel bis(salamo)-like probe for highly effective fluorimetric-colorimetric identification of Fe3+ and Cu2+ in aquo-organic medium

A novel bis(salamo)-type sensor FT for fluorescence-colorimetric recognition of Fe3+/Cu2+ has been created, which revealed significant fluorescent performance and colorimetric sensing ability for Cu2+ and Fe3+ ions, superior to other related competitive metal ions. Interestingly, the binding of the FT probe with Cu2+ ions manifested an instant color change from colorless to red in sunlight, which is detectable by the naked-eye, and a fluorescence turn-off response under UV light for Fe3+ and Cu2+. The results demonstrated that the probe exhibits better sensitivity, greater affinity and lower limit of detection leading to quick response time in an aquo-organic medium. The excited state property of the FT probe and in the presence of Cu2+/Fe3+ was evaluated on the basis of DFT & TD-DFT results. Furthermore, test strips have been provided for convenient monitoring of Cu2+ and Fe3+ ions by naked eye and fluorescence method.

SaeR as a novel target for antivirulence therapy against Staphylococcus aureus

ABSTRACT Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilizing a GFP-Lux dual reporter system driven by saeP1 promoter. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and haemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR’s DNA-binding domain. When SaeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target’s specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure–activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four times more potent than HR3744 and demonstrated identical antivirulence properties and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence properties, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections.

FISHing for Rickettsia in tardigrades: additional evidence for tardigrade endosymbionts

Abstract Many ecdysozoans harbour endosymbiotic bacteria within their microbiota, and these endosymbionts can have a range of positive and negative effects on their hosts. Recent 16S rRNA gene amplicon sequencing studies have provided evidence for endosymbionts within the tardigrade microbiota. In a previous amplicon study, we determined that sequences corresponding to the endosymbiotic genus Rickettsia were significantly more associated with tardigrades than with the substrate from which they were isolated. Here, we performed fluorescence in situ hybridization (FISH) using a Rickettsia-specific probe, RickB1, to determine if Rickettsia could be found in tardigrades. RickB1 and a probe targeting most bacteria, EUB338, colocalized within tardigrade tissues, indicating the presence of Rickettsia. We also performed FISH using RickB1 and a nonsense probe, which allowed us to distinguish between false-positives and true positives. This method revealed RickB1 signals in tardigrades that were not due to erroneous probe binding, providing further evidence that Rickettsia is present in tardigrades. Future research will be necessary to determine the effects, if any, of these endosymbionts on their tardigrade hosts.

Rapid Diagnostic Platform for Personalized Vitamin B6 Detection in Erythrocytes via PLP Cofactor Mimics.

Personalized assessment of vitamin levels in point-of-care (POC) devices is urgently needed to advance the recognition of diseases associated with malnutrition and unbalanced diets. We here introduce a diagnostic platform, which showcases an easy and rapid readout of vitamin B6 (pyridoxal phosphate, PLP) levels in erythrocytes as a first step toward a home-use POC. The technology is based on fluorescent probes, which bind to PLP-dependent enzymes (PLP-DEs) and thereby indirectly report their occupancy with endogenous B6. For example, low vitamin levels result in high probe binding, yielding a strong signal and vice versa. Antibodies against signature human PLP-DEs were immobilized on microarrays to capture probe labeled enzymes for fluorescent detection. Calibrating the system with defined B6 levels revealed a concentration-depended readout as well as sufficient sensitivity for its detection in erythrocytes. To account for individual differences in protein expression, a second antibody was used to normalize protein abundance. This sandwiched assay correctly reported relative B6 levels in human erythrocyte samples, as confirmed by classical laboratory diagnostics. In principle, the platform layout can be easily expanded to other crucial vitamins beyond B6 via an analogous probe strategy.

Fluorescent probe for the identification of potent inhibitors of the macrophage infectivity potentiator (Mip) protein of Burkholderia pseudomallei.

The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.

Wash-Free and High-Contrast Imaging of Single-Nucleotide Variation in Single Cells Using Transcription-Amplified Light-Up RNA Aptamer.

Single-nucleotide variation (SNV) imaging can indicate cellular heterogeneity and spatial pattern, but it remains challenging to produce high-gain signal while also yielding single-nucleotide resolution. Herein, we developed a light-up strategy for visualizing SNVs based on transcription amplification, enabling wash-free and high-contrast imaging of SNVs inside cells. The discrimination of SNVs is achieved by ligase-assisted transcription reaction. Employing a light-up RNA aptamer as a reporter eliminates nonspecific probe binding and the washing process and contributes to a 2-fold improvement of signal gain compared to that using the fluorescence in situ hybridization (FISH) method. The method allowed us to precisely quantify drug-resistant strains in the bacteria mixture and identify drug-resistant Salmonella enterica (S. enterica) isolated from poultry farm. Using this approach, we explored the colonization features of drug-resistant and drug-sensitive S. enterica in the mice intestinal tract and screened the prebiotics for Salmonella colonization inhibition. The SNV imaging method promises for the interrogation of genotypes in physiological and pathological states at the single-cell level.

Free Energy Perturbation Calculations of Mutation Effects on SARS-CoV-2 RBD::ACE2 Binding Affinity

The strength of binding between human angiotensin converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of viral spike protein plays a role in the transmissibility of the SARS-CoV-2 virus. In this study we focus on a subset of RBD mutations that have been frequently observed in infected individuals and probe binding affinity changes to ACE2 using surface plasmon resonance (SPR) measurements and free energy perturbation (FEP) calculations. Our SPR results are largely in accord with previous studies but discrepancies do arise due to differences in experimental methods and to protocol differences even when a single method is used. Overall, we find that FEP performance is superior to that of other computational approaches examined as determined by agreement with experiment and, in particular, by its ability to identify stabilizing mutations. Moreover, the calculations successfully predict the observed cooperative stabilization of binding by the Q498R N501Y double mutant present in Omicron variants and offer a physical explanation for the underlying mechanism. Overall, our results suggest that despite the significant computational cost, FEP calculations may offer an effective strategy to understand the effects of interfacial mutations on protein–protein binding affinities and, hence, in a variety of practical applications such as the optimization of neutralizing antibodies.

Label-free, ultra-low detection limit DNA biosensor using high quality optical microcavity functionalized by DNA tetrahedral nanostructure probes

Abstract This study proposes and demonstrates a novel label-free DNA biosensor using high quality optical microcavity functionalized by 3D DNA nanostructure probes. To achieve ultra-low limit DNA biosensing, optical sensing interface of the hollow-core, thin wall-thickness microcavity was functionalized by self-assembled DNA tetrahedral nanostructure (DTN) probes with size of 17 bp and length of ∼5.8 nm. High efficiency binding of the DTN probes with the optical sensor interface and the target ssDNA are achieved. Whispering gallery mode (WGM) spectra with high-Q factor of >107 are excited and traced for DNA detection inside the microfluidic channel of the microcavity, with a small sample volume of nL. Incorporation of nanoscale DTN probes onto surface of the optical microcavity makes it an effective way for increasing efficient probe density and eliminating entanglement between DNA probes, thus ∼1000 times lower detection limit is achieved as compared to using 1D ssDNA probes. Due to its desirable merits of label-free, ultra-low LoD, real time and compact size, the proposed DNA biosensor has broad application prospects in bioengineering and medical diagnosis.

Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes

ω-Alkynyl-fatty acids can be used as probes for covalent binding to intracellular macromolecules. To inform future in vivo studies, we determined the rates of reaction of ω-alkynyl-labeled linoleate with recombinant enzymes of the skin 12R-lipoxygenase (12R-LOX) pathway involved in epidermal barrier formation (12R-LOX, epidermal lipoxygenase-3 (eLOX3), and SDR9C7). We also examined the reactivity of ω-alkynyl-arachidonic acid with representative lipoxygenase enzymes employing either “carboxyl end-first” substrate binding (5S-LOX) or “tail-first” (platelet-type 12S-LOX). ω-Alkynyl-linoleic acid was oxygenated by 12R-LOX at 62 ± 9 % of the rate compared to linoleic acid, the alkynyl-9R-HPODE product was isomerized by eLOX3 at only 43 ± 1 % of the natural substrate, whereas its epoxy alcohol product was converted to epoxy ketone linoleic by an NADH-dependent dehydrogenase (SDR9C7) with 91 ± 1 % efficiency. The results suggest the optimal approach will be application of the 12R-LOX/eLOX3-derived epoxyalcohol, which should be most efficiently incorporated into the pathway and allow subsequent analysis of covalent binding to epidermal proteins. Regarding the orientation of substrate binding in LOX catalysis, our results and previous reports suggest the ω-alkynyl group has a stronger inhibitory effect on tail-first binding, as might be expected. Beyond slowing the reaction, however, we found that the tail-first binding and transformation of ω-alkynyl-arachidonic acid by platelet-type 12S-LOX results in almost complete enzyme inactivation, possibly due to reactive intermediates blocking the enzyme active site. Overall, the results reinforce the conclusion that ω-alkynyl-fatty acids are suitable for selected applications after appropriate reactivity is established.

Exploring magnetic capture to improve the detection of human norovirus in strawberries

AbstractHuman norovirus is a leading cause of foodborne illness worldwide, and its detection in foods remains a challenge due to the difficulty of eluting and enriching trace amounts of viral particles. In this study, we developed porcine gastric mucin–labeled magnetic capture probes and optimized the conditions for their binding with norovirus, elution of viral particles, and RNA isolation based on the previous research. We evaluated the magnetic capture‐based pretreatment method in artificially contaminated strawberry samples and compared it with the conventional polyethylene glycol precipitation–based method using reverse transcriptional‐quantitative PCR (RT‐qPCR). The results revealed that Tris·HCl (pH 9.5) was the optimal buffer for the binding of magnetic probes with viral particles, and RNA recovery rate obtained by heating‐based release was significantly higher than that obtained by a commercial kit, with only active virus being captured. This buffer was similar to the RT‐qPCR buffer, which facilitated the sensitivity of RT‐qPCR. Furthermore, the buffer was an effective solution for eluting viral particles from foods in previous research and was optimized to ensure maximum elution and no inhibitory effect on viral capture. We explored multiple factors or steps to ensure that the sensitivity of our method was three orders of magnitude higher than that of the conventional method. Our findings suggest that magnetic capture‐based detection has great potential for the sensitive detection of human norovirus.

Nanoparticle-Coupled Single-Molecule Kinetic Fingerprinting for Enzymatic Activity Detection.

The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.