Competitive SPR Chaser Assay to Study Biomolecular Interactions with Very Slow Binding Dissociation Rate Constant.

Binding kinetics of drug and its target protein is crucial for the efficacy and safety of the drug. Using surface plasmon resonance (SPR) technology, we performed a competitive SPR chaser assay, a method to study biomolecular interactions with very slow dissociation rate constants (kd < 1E-4 s-1). This report described the principle and the experimental setup of the chaser assay, which involves using a competitive probe (chaser) to detect changes in target occupancy by a test molecule over time. We demonstrated the applicability of the chaser assay for both small and large molecules and compared the results with conventional SPR kinetic analysis and other methods. We suggest that the chaser assay is a useful and robust technique to characterize very tight biomolecular interactions, and that it can also be used to study cooperativity in ternary complex formation.

Characterization of PARP1 binding to c-KIT1 G-quadruplex DNA: Insights into domain-specific interactions.

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme involved in catalyzing Poly-(ADP-ribosyl)ation. PARP1 binds to different forms of DNA and DNA breaks and thus plays important roles in several cellular processes, including DNA damage repair, cell cycle regulation, chromatin remodeling, and maintaining genomic stability. In this study, we conducted biochemical and biophysical characterization of PARP1 binding to G-quadruplex DNA (G4-DNA). Our investigation identified ZnF1, ZnF3, and WGR as the critical domains to mediate PARP1 binding to G4-c-KIT1. Also, our results show that these domains together show cooperativity for G4-c-KIT1 recognition. Further, we establish that the presence of an oxidized (5-carboxylcytosine) base in the loop region of G4-c-KIT1 (G4-5caC-cKIT1) does not affect its recognition by PARP1. Both G4-c-KIT1 and G4-5caC-cKIT1 are potent stimulators of PARP1's catalytic activity. Our study advances the understanding of PARP1's versatile DNA binding capabilities for G4-c-KIT1 DNA irrespective of the oxidation/ modification in the DNA base. These insights into PARP1's domain-specific contributions to G4-c-KIT1 DNA recognition and catalysis expand our knowledge of its multifaceted roles in DNA repair and genome maintenance.

Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab'-DNA ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

Competitive Binding Kinetics of Methylmercury-Cysteine with Dissolved Organic Matter: Critical Impacts of Thiols on Adsorption and Uptake of Methylmercury by Algae.

Complexes with low-molecular-weight thiols are crucial species of methylmercury (MeHg) excreted by anaerobic Hg-methylating microbes, notably, MeHg-cysteine (MeHg-Cys). As MeHg-Cys diffuses into surface water, it would undergo a ligand exchange process with dissolved organic matter (DOM) under nonsulfidic conditions, inevitably altering MeHg speciation and bioavailability to phytoplankton. In this study, we investigated the competitive binding kinetics between MeHg-Cys and Suwannee River natural organic matter, and their influence on the adsorption and uptake of MeHg by the cyanobacterium, Synechocystis sp. PCC6803. Liquid chromatography-inductively coupled plasma mass spectrometry was employed to monitor the kinetics processes involving competition of DOM with Cys for MeHg binding, which revealed that competitive binding kinetics were dictated by the abundance of thiol moieties in DOM. Thiol concentrations of 0.97 and 49.34 µmol of thiol (g C)-1 resulted in competitive binding rate constant (k values) of 0.30 and 3.47 h-1, respectively. Furthermore, the time-dependent competitive binding of DOM toward MeHg-Cys significantly inhibited MeHg adsorption and uptake by cyanobacteria, an effect that was amplified by an increased thiol abundance in DOM. These findings offer valuable insights into the kinetic characteristics of MeHg's fate and transport, as well as their impact on bioconcentration in aquatic organisms within natural aquatic ecosystems.

Protein Interaction Kinetics Delimit the Performance of Phosphorylation-Driven Protein Switches.

Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo-designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between "on" and "off" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.

TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization.

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode.

Insight into the avidity-affinity relationship of the bivalent, pH-dependent interaction between IgG and FcRn.

Monoclonal antibodies (mAbs) as therapeutics necessitate favorable pharmacokinetic properties, including extended serum half-life, achieved through pH-dependent binding to the neonatal Fc receptor (FcRn). While prior research has mainly investigated IgG-FcRn binding kinetics with a focus on single affinity values, it has been shown that each IgG molecule can engage two FcRn molecules throughout an endosomal pH gradient. As such, we present here a more comprehensive analysis of these interactions with an emphasis on both affinity and avidity by taking advantage of switchSENSE technology, a surface-based biosensor where recombinant FcRn was immobilized via short DNA nanolevers, mimicking the membranous orientation of the receptor. The results revealed insight into the avidity-to-affinity relationship, where assessing binding through a pH gradient ranging from pH 5.8 to 7.4 showed that the half-life extended IgG1-YTE has an affinity inflection point at pH 7.2, reflecting its engineering for improved FcRn binding compared with the wild-type counterpart. Furthermore, IgG1-YTE displayed a pH switch for the avidity enhancement factor at pH 6.2, reflecting strong receptor binding to both sides of the YTE-containing Fc, while avidity was abolished at pH 7.4. When compared with classical surface plasmon resonance (SPR) technology and complementary methods, the use of switchSENSE demonstrated superior capabilities in differentiating affinity from avidity within a single measurement. Thus, the methodology provides reliable kinetic rate parameters for both binding modes and their direct relationship as a function of pH. Also, it deciphers the potential effect of the variable Fab arms on FcRn binding, in which SPR has limitations. Our study offers guidance for how FcRn binding properties can be studied for IgG engineering strategies.

Time-dependent ligand-receptor binding kinetics and functionality in a heterodimeric receptor model.

GPCRs heteromerize both in CNS and non-CNS regions. The cell uses receptor heteromerization to modulate receptor functionality and to provide fine tuning of receptor signaling. In order for pharmacologists to explore these mechanisms for therapeutic purposes, quantitative receptor models are needed. We have developed a time-dependent model of the binding kinetics and functionality of a preformed heterodimeric receptor involving two drugs. Two cases were considered: both or only one of the drugs are in excess with respect to the total concentration of the receptor. The latter case can be applied to those situations in which a drug causes unwanted side effects that need to be reduced by decreasing its concentration. The required efficacy can be maintained by the allosteric effects mutually exerted by the two drugs in the two-drug combination system. We discuss this concept assuming that the drug causing unwanted side effects is an opioid and that analgesia is the therapeutic effect. As additional points, allosteric modulation by endogenous compounds and synthetic bivalent ligands was included in the study. Receptor heteromerization offers a mechanistic understanding and quantification of the pharmacological effects elicited by combinations of two drugs at different doses and with different efficacies and cooperativity effects, thus providing a conceptual framework for drug combination therapy.

Ligand Gaussian accelerated Molecular Dynamics 3 (LiGaMD3): Improved Calculations of Binding Thermodynamics and Kinetics of Both Small Molecules and Flexible Peptides.

Binding thermodynamics and kinetics play critical roles in drug design. However, it has proven challenging to efficiently predict ligand binding thermodynamics and kinetics of small molecules and flexible peptides using conventional Molecular Dynamics (cMD), due to limited simulation timescales. Based on our previously developed Ligand Gaussian accelerated Molecular Dynamics (LiGaMD) method, we present a new approach, termed "LiGaMD3", in which we introduce triple boosts into three individual energy terms that play important roles in small-molecule/peptide dissociation, rebinding and system conformational changes to improve the sampling efficiency of small-molecule/peptide interactions with target proteins. To validate the performance of LiGaMD3, MDM2 bound by a small molecule (Nutlin 3) and two highly flexible peptides (PMI and P53) were chosen as model systems. LiGaMD3 could efficiently capture repetitive small-molecule/peptide dissociation and binding events within 2 microsecond simulations. The predicted binding kinetic constant rates and free energies from LiGaMD3 agreed with available experimental values and previous simulation results. Therefore, LiGaMD3 provides a more general and efficient approach to capture dissociation and binding of both small-molecule ligand and flexible peptides, allowing for accurate prediction of their binding thermodynamics and kinetics.

Non-linear blood-brain barrier transport and dosing strategies influence receptor occupancy ratios of morphine and its metabolites in pain matrix.

BACKGROUND AND PURPOSE: Morphine is important for treatment of acute and chronic pain. However, there is high interpatient variability and often inadequate pain relief and adverse effects. To better understand variability in the dose-effect relationships of morphine, we investigated the effects of its non-linear blood-brain barrier (BBB) transport on µ-receptor occupancy in different CNS locations, in conjunction with its main metabolites that bind to the same receptor. EXPERIMENTAL APPROACH: CNS exposure profiles for morphine, M3G and M6G for clinically relevant dosing regimens based on intravenous, oral immediate- and extended-release formulations were generated using a physiology-based pharmacokinetic model of the CNS, with non-linear BBB transport of morphine. The simulated CNS exposure profiles were then used to derive corresponding µ-receptor occupancies at multiple CNS pain matrix locations. KEY RESULTS: Simulated CNS exposure profiles for morphine, M3G and M6G, associated with non-linear BBB transport of morphine resulted in varying µ-receptor occupancies between different dose regimens, formulations and CNS locations. At lower doses, the µ-receptor occupancy of morphine was relatively higher than at higher doses of morphine, due to the relative contribution of M3G and M6G. At such higher doses, M6G showed higher occupancy than morphine, whereas M3G occupancy was low throughout the dose ranges. CONCLUSION AND IMPLICATIONS: Non-linear BBB transport of morphine affects the µ-receptor occupancy ratios of morphine with its metabolites, depending on dose and route of administration, and CNS location. These predictions need validation in animal or clinical experiments, to understand the clinical implications.

Binding and unbinding of potent melatonin receptor ligands: Mechanistic simulations and experimental evidence.

The labeled ligand commonly employed in competition binding studies for melatonin receptor ligands, 2-[125I]iodomelatonin, showed slow dissociation with different half-lives at the two receptor subtypes. This may affect the operational measures of affinity constants, which at short incubation times could not be obtained in equilibrium conditions, and structure-activity relationships, as the Ki values of tested ligands could depend on either interaction at the binding site or the dissociation path. To address these issues, the kinetic and saturation binding parameters of 2-[125I]iodomelatonin as well as the competition constants for a series of representative ligands were measured at a short (2 h) and a long (20 h) incubation time. Concurrently, we simulated by molecular modeling the dissociation path of 2-iodomelatonin from MT1 and MT2 receptors and investigated the role of interactions at the binding site on the stereoselectivity observed for the enantiomers of the subtype-selective ligand UCM1014. We found that equilibrium conditions for 2-[125I]iodomelatonin binding can be reached only with long incubation times, particularly for the MT2 receptor subtype, for which a time of 20 h approximates this condition. On the other hand, measured Ki values for a set of ligands including agonists, antagonists, nonselective, and subtype-selective compounds were not significantly affected by the length of incubation, suggesting that structure-activity relationships based on data collected at shorter time reflect different interactions at the binding site. Molecular modeling simulations evidenced that the slower dissociation of 2-iodomelatonin from the MT2 receptor can be related to the restricted mobility of a gatekeeper tyrosine along a lipophilic path from the binding site to the membrane bilayer. The enantiomers of the potent, MT2-selective agonist UCM1014 were separately synthesized and tested. Molecular dynamics simulations of the receptor-ligand complexes provided an explanation for their stereoselectivity as due to the preference shown by the eutomer at the binding site for the most abundant axial conformation adopted by the ligand in solution. These results suggest that, despite the slow-binding kinetics occurring for the labeled ligand, affinity measures at shorter incubation times give robust results consistent with known structure-activity relationships and with interactions taken at the receptor binding site.

BiSim Tool: a binding simulation tool to aid and simplify ligand-binding assay design and development.

Ligand-binding assays (LBAs) rely on the reversible, noncovalent binding between the analyte of interest and the assay reagents, and understanding their dynamic equilibrium is key to building robust LBA methods. Although the dynamic interplay of free and bound fractions can be calculated using mathematical models, these are not routinely applied. This approach is costly in terms of both assay development time and reagents, and can result in an under-exploration of the possible parameter combinations. Therefore, we have created a user-friendly simulation tool to facilitate LBA development (the BiSim Tool). We describe the models driving the mathematical simulations and the main features of our software solution by means of case studies, illustrating the tool's value in drug development. To support drug development for all patients worldwide, the BiSim Tool is now available as an open-source code project and as a free web-based tool at https://proteinbindingsimulation.shinyapps.io/BiSim-ProteinBindingSimulation [1].

[Box: see text].

Interaction Between SARS-CoV-2 Spike Protein S1 Subunit and Oyster Heat Shock Protein 70.

There is growing evidence that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminates the marine environment and is bioaccumulated in filter-feeding shellfish. Previous study shows the Pacific oyster tissues can bioaccumulate the SARS-CoV-2, and the oyster heat shock protein 70 (oHSP70) may play as the primary attachment receptor to bind SARS-CoV-2's recombinant spike protein S1 subunit (rS1). However, detailed information about the interaction between rS1 and oHSP70 is still unknown. In this study, we confirmed that the affinity of recombinant oHSP70 (roHSP70) for rS1 (KD = 20.4 nM) is comparable to the receptor-binding affinity of rACE2 for rS1 (KD = 16.7 nM) by surface plasmon resonance (SPR)-based Biacore and further validated by enzyme-linked immunosorbent assay (ELISA). Three truncated proteins (roHSP70-N/C/M) and five mutated proteins (p.I229del, p.D457del, p.V491_K495del, p.K556I, and p.ΣroHSP70) were constructed according to the molecular docking results. All three truncated proteins have significantly lower affinity for rS1 than the full-length roHSP70, indicating that all three segments of roHSP70 are involved in binding to rS1. Further, the results of SPR and ELISA showed that all five mutant proteins had significantly lower affinity for rS1 than roHSP70, suggesting that amino acids at these sites are involved in binding to rS1. This study provides a preliminary theoretical basis for the bioaccumulation of SARS-CoV-2 in oyster tissues or using roHSP70 as the capture unit to selectively enrich virus particles for detection.

Differential Roles of Kinetic On- and Off-Rates in T-Cell Receptor Signal Integration Revealed with a Modified Fab'-DNA Ligand.

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly-binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide-MHC (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRß H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rate of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells (LAT), which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from synthetic ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

Ligand-induced protein transition state stabilization switches the binding pathway from conformational selection to induced fit.

Protein-ligand complex formation is fundamental to biological function. A central question is whether proteins spontaneously adopt binding-competent conformations to which ligands bind conformational selection (CS) or whether ligands induce the binding-competent conformation induced fit (IF). Here, we resolve the CS and IF binding pathways by characterizing protein conformational dynamics over a wide range of ligand concentrations using NMR relaxation dispersion. We determined the relative flux through the two pathways using a four-state binding model that includes both CS and IF. Experiments conducted without ligand show that galectin-3 exchanges between the ground-state conformation and a high-energy conformation similar to the ligand-bound conformation, demonstrating that CS is a plausible pathway. Near-identical crystal structures of the apo and ligand-bound states suggest that the high-energy conformation in solution corresponds to the apo crystal structure. Stepwise additions of the ligand lactose induce progressive changes in the relaxation dispersions that we fit collectively to the four-state model, yielding all microscopic rate constants and binding affinities. The ligand affinity is higher for the bound-like conformation than for the ground state, as expected for CS. Nonetheless, the IF pathway contributes greater than 70% of the total flux even at low ligand concentrations. The higher flux through the IF pathway is explained by considerably higher rates of exchange between the two protein conformations in the ligand-associated state. Thus, the ligand acts to decrease the activation barrier between protein conformations in a manner reciprocal to enzymatic transition-state stabilization of reactions involving ligand transformation.

A Sensitive Technique Unravels the Kinetics of Activation and Trans-Cleavage of CRISPR-Cas Systems.

Activation of the CRISPR-Cas13a system requires the formation of a crRNA-Cas13a ribonucleoprotein (RNP) complex and the binding of an RNA activator to the RNP. These two binding processes play a crucial role in the performance of the CRISPR-Cas13a system. However, the binding kinetics remain poorly understood, and a main challenge is the lack of a sensitive method for real-time measurements of the dynamically formed active CRISPR-Cas13a enzyme. We describe here a new method to study the binding kinetics and report the rate constants (kon and koff) and dissociation constant (Kd) for the binding between Cas13a and its activator. The method is able to unravel and quantify the kinetics of binding and cleavage separately, on the basis of measuring the real-time trans-cleavage rates of the CRISPR-Cas system and obtaining the real-time concentrations of the active CRISPR-Cas ternary complex. We further discovered that once activated, the Cas13a system operates at a wide range of temperatures (7-37 °C) with fast trans-cleavage kinetics. The new method and findings are important for diverse applications of the Cas13a system, such as the demonstrated quantification of microRNA at ambient temperatures (e.g., 25 °C).

Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

Reconstitution of cytolinker-mediated crosstalk between actin and vimentin.

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called 'ACTIF' that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.

High-Precision Viral Detection Using Electrochemical Kinetic Profiling of Aptamer-Antigen Recognition in Clinical Samples and Machine Learning.

High-precision viral detection at point of need with clinical samples plays a pivotal role in the diagnosis of infectious diseases and the control of a global pandemic. However, the complexity of clinical samples that often contain very low viral concentrations makes it a huge challenge to develop simple diagnostic devices that do not require any sample processing and yet are capable of meeting performance metrics such as very high sensitivity and specificity. Herein we describe a new single-pot and single-step electrochemical method that uses real-time kinetic profiling of the interaction between a high-affinity aptamer and an antigen on a viral surface. This method generates many data points per sample, which when combined with machine learning, can deliver highly accurate test results in a short testing time. We demonstrate this concept using both SARS-CoV-2 and Influenza A viruses as model viruses with specifically engineered high-affinity aptamers. Utilizing this technique to diagnose COVID-19 with 37 real human saliva samples results in a sensitivity and specificity of both 100 % (27 true negatives and 10 true positives, with 0 false negative and 0 false positive), which showcases the superb diagnostic precision of this method.

Transcription factor exchange enables prolonged transcriptional bursts.

Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding is related to transcription activation. Here, we devised a microscopy method to simultaneously measure transient TF binding at a single locus and the effect of these binding events on transcription. We show that DNA binding of the yeast TF Gal4 activates transcription of a target gene within a few seconds, with at least ∼20% efficiency and with a high initiation rate of ∼1 RNA/s. Gal4 DNA dissociation decreases transcription rapidly. Moreover, at a gene with multiple binding sites, individual Gal4 molecules only rarely stay bound throughout the entire burst but instead frequently exchange during a burst to increase the transcriptional burst duration. Our results suggest a mechanism for enhancer regulation in more complex eukaryotes, where TF cooperativity and exchange enable robust and responsive transcription regulation.