Specific Molecular Binding Research Articles

Lattice-based mesoscale simulations and mean-field theory of cell membrane adhesion.

Adhesion of cell membranes involves multi-scale phenomena, ranging from specific molecular binding at Angstrom scale all the way up to membrane deformations and phase separation at micrometer scale. Consequently, theory and simulations of cell membrane adhesion require multi-scale modeling and suitable approximations that capture the essential physics of these phenomena. Here, we present a mesoscale model for membrane adhesion which we have employed in a series of our recent studies. This model quantifies, in particular, how nanoscale lipid clusters physically affect and respond to the intercellular receptor-ligand binding that mediates membrane adhesion. The goal of this Chapter is to present all details and subtleties of the mean-field theory and Monte Carlo simulations of this mesoscale model, which can be used to further explore physical phenomena related to cell membrane adhesion.

Domestication and microbiome succession may drive pathogen spillover.

Emerging infectious diseases have posed growing medical, social and economic threats to humanity. The biological background of pathogen spillover or host switch, however, still has to be clarified. Disease ecology finds pathogen spillovers frequently but struggles to explain at the molecular level. Contrarily, molecular biological traits of host-pathogen relationships with specific molecular binding mechanisms predict few spillovers. Here we aim to provide a synthetic explanation by arguing that domestication, horizontal gene transfer even between superkingdoms as well as gradual exchange of microbiome (microbiome succession) are essential in the whole scenario. We present a new perspective at the molecular level which can explain the observations of frequent pathogen spillover events at the ecological level. This proposed rationale is described in detail, along with supporting evidence from the peer-reviewed literature and suggestions for testing hypothesis validity. We also highlight the importance of systematic monitoring of virulence genes across taxonomical categories and in the whole biosphere as it helps prevent future epidemics and pandemics. We conclude that that the processes of domestication, horizontal gene transfer and microbial succession might be important mechanisms behind the many spillover events driven and accelerated by climate change, biodiversity loss and globalization.

In vitro selection of a single-strand DNA aptamer targeting the receptor-binding domain of SARS-CoV-2 spike protein

Nucleic acid aptamers are developed from a pool of random oligonucleotide libraries with an in vitro selection through systematic evolution of ligands via exponential enrichment (SELEX) process, which are capable of specific and high-affinity molecular binding against targets. The receptor-binding domain (RBD) of spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is involved in the early stages of viral infection, is a promising target for aptamer selection. Currently, there are no effective approaches to prevent virus from spreading. In this study, a new ssDNA aptamer RBD/S-A1 binding to the RBD of spike protein from SARS-CoV-2 with high affinity (Kd=1.74 ± 0.2 nM) and low cross-binding activity was selected and evaluated. Although aptamers targeting the RBD of spike protein from SARS-CoV-2 have been described in a handful of previous studies, the RBD/S-A1 aptamer identified in this work may be considered as a potential supplementation for the current diagnosis and research of coronavirus SARS-CoV-2.

Nonspecific Binding-Fundamental Concepts and Consequences for Biosensing Applications.

Nature achieves differentiation of specific and nonspecific binding in molecular interactions through precise control of biomolecules in space and time. Artificial systems such as biosensors that rely on distinguishing specific molecular binding events in a sea of nonspecific interactions have struggled to overcome this issue. Despite the numerous technological advancements in biosensor technologies, nonspecific binding has remained a critical bottleneck due to the lack of a fundamental understanding of the phenomenon. To date, the identity, cause, and influence of nonspecific binding remain topics of debate within the scientific community. In this review, we discuss the evolution of the concept of nonspecific binding over the past five decades based upon the thermodynamic, intermolecular, and structural perspectives to provide classification frameworks for biomolecular interactions. Further, we introduce various theoretical models that predict the expected behavior of biosensors in physiologically relevant environments to calculate the theoretical detection limit and to optimize sensor performance. We conclude by discussing existing practical approaches to tackle the nonspecific binding challenge in vitro for biosensing platforms and how we can both address and harness nonspecific interactions for in vivo systems.

Low-Noise Solid-State Nanopore Enhancing Direct Label-Free Analysis for Small Dimensional Assemblies Induced by Specific Molecular Binding.

Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.

High contrast cleavage detection for enhancing porous silicon sensor sensitivity.

Using porous silicon (PSi) interferometer sensors, we show the first experimental implementation of the high contrast cleavage detection (HCCD) mechanism. HCCD makes use of dramatic optical signal amplification caused by cleavage of high-contrast nanoparticle labeled reporters instead of the capture of low-index biological molecules. An approximately 2 nm reflectance peak shift was detected after cleavage of DNA-quantum dot reporters from the PSi surface via exposure to a 12.5 nM DNase enzyme solution. This signal change is 20 times greater than the resolution of the spectrometer used for the interferometric measurements, and the interferometric measurements agree with the response predicted by simulations and fluorescence measurements. These proof of principle experiments show a clear path to achieving a real-time, highly sensitive readout for a broad range of biological diagnostic assays that generate a signal via nucleic acid cleavage triggered by specific molecular binding events.

Point-of-care-ready nanoscale ISFET arrays for sub-picomolar detection of cytokines in cell cultures

Rapid and frequent screening of cytokines as immunomodulation agents is necessary for precise interventions in severe pathophysiological conditions. In addition to high-sensitivity detection of such analytes in complex biological fluids such as blood, saliva, and cell culture medium samples, it is also crucial to work out miniaturized bioanalytical platforms with potential for high-density integration enabling screening of multiple analytes. In this work, we show a compact, point-of-care-ready bioanalytical platform for screening of cytokines such as interleukin-4 (IL-4) and interleukin-2 (IL-2) based on one-dimensional ion-sensitive field-effect transistors arrays (nanoISFETs) of silicon fabricated at wafer-scale via nanoimprint lithography. The nanoISFETs biofunctionalized with receptor proteins alpha IL-4 and alpha IL-2 were deployed for screening cytokine secretion in mouse T helper cell differentiation culture media, respectively. Our nanoISFETs showed robust sensor signals for specific molecular binding and can be readily deployed for real-time screening of cytokines. Quantitative analyses of the nanoISFET-based bioanalytical platform was carried out for IL-4 concentrations ranging from 25 fg/mL (1.92 fM) to 2.5 μg/mL (192 nM), showing a limit of detection down to 3–5 fM, which was found to be in agreement with ELISA results in determining IL-4 concentrations directly in complex cell culture media.Graphical abstract

Structure-based identification of a potential non-catalytic binding site for rational drug design in the fructose 1,6-biphosphate aldolase from Giardia lamblia

Giardia lamblia is the causal agent of giardiasis, one of the most prevalent parasitosis in the world. Even though effective pharmacotherapies against this parasite are available, the disadvantages associated with its use call for the development of new antigiardial compounds. Based on the Giardia dependence on glycolysis as a main energy source, glycolytic enzymes appear to be attractive targets with antiparasitic potential. Among these, fructose 1,6-biphosphate aldolase (GlFBPA) has been highlighted as a promising target for drug design. Current efforts are based on the design of competitive inhibitors of GlFBPA; however, in the kinetic context of metabolic pathways, competitive inhibitors seem to have low potential as therapeutic agents. In this work, we performed an experimental and in silico structure-based approach to propose a non-catalytic binding site which could be used as a hot spot for antigardial drug design. The druggability of the selected binding site was experimentally tested; the alteration of the selected region by site directed mutagenesis disturbs the catalytic properties and the stability of the enzyme. A computational automated search of binding sites supported the potential of this region as functionally relevant. A preliminary docking study was performed, in order to explore the feasibility and type of molecules to be able to accommodate in the proposed binding region. Altogether, the results validate the proposed region as a specific molecular binding site with pharmacological potential.

A Fluidic Biosensor Based on a Phase-Sensitive Low-Coherence Spectral-Domain Interferometer.

A phase-sensitive fluidic biosensor based on a spectral-domain low-coherence interferometer is presented in this paper. With a fiber optic probe employing the common-path interferometric configuration, subnanometric changes in thickness of the molecular layers can be detected through phase analysis of the acquired interference signal from the sensor surface. Advantages of this biosensor include its picometer-scale thickness sensitivity, 13.9-ms time response, and tolerance to the fluctuation in concentration of the target solution. The capabilities of this biosensor in monitoring specific molecular binding and recognizing specific molecular was successfully demonstrated by using the reactions between the molecules of protein A and IgG. The calculated minimum detectable concentration of IgG is 0.11 µg/mL.

Nanostructured Microcantilevers for the Sensing of Volatile Organic Compounds for Environmental and Food Safety Applications

Biomolecular detection has applications in many fields, including environmental monitoring, food quality monitoring, hazardous gas detection, and medical diagnostics. Three volatile analytes are of particular interest; trimethylamine (TMA) is associated with seafood spoilage and Trimethylaminuria [1], acetic acid (AA) is a biomarker for asthma [2], and ammonia (NH3) is a biomarker for protein degradation [3]. Detection of these molecules requires a highly sensitive and highly specific sensing platform. For the purpose of detecting these molecules, a microcantilever is designed with a nanostructured surface and is functionalized with odorant-binding peptides. The hammerhead microcantilever shown in Figure 1 was designed and fabricated at the Georgia Institute of Technology. The hammerhead shape provides greater surface area and causes the cantilever to resonant in-plane. Nanostructure is fabricated on the microcantilever surface in order to increase the sensitivity of the device. Silicon dioxide nanostructure is created using glancing angle deposition (GLAD) and increases the overall surface area, allowing more molecules to be captured. Images of the nanostructure taken using a high-resolution scanning electron microscope are shown in Figure 2 and 3. The odorant-binding peptides are attached to the SiO2 surface using aminosilane. Validation of the surface chemistry was performed by attaching biotin-streptavadin with a fluorescence tag and viewing under a Fluorescein Isothiocyanate (FITC) light microscope. Further validation is performed by attaching a fluorescence tag to the ammonia-binding peptide and introducing it to a SiO2 surface. Additionally, Raman spectroscopy was used to show the attachment of the silane. The peptides are modeled using the PEP-FOLD servers [4], and binding interactions are modeled using the AutoDock routines in the PyRx software, Figure 4. A scanning Kelvin probe (SKP) is used to demonstrate a charge differential between the flat surface and nanostructure-coated surface. In addition, a charge differential is shown between a surface that is functionalized with peptides and one that is not. This charge differential can be used as a numerical indication of the success of the nanostructure and surface chemistry strategies. The improvement in sensor surface area with added 250 nm nanostructure is estimated to be on the order of a 12-fold increase. BET with Krypton analysis is being used to confirm the amount of nanostructured surface area for both the 250 and 600 nm nanostructure additions. Peptide-functionalized nanostructured cantilevers are being tested for specific VOC molecular binding characteristics. The relevant parameter is the change (decrease) in resonance frequency with added molecular mass. Acknowledgements Research supported by NSF Award #1445488 to KMH. Additional support for RJM provided by the University of Dayton Graduate School.

Coexistence of p210BCR-ABL and CBFβ-MYH11 fusion genes in myeloid leukemia: A report of 4 cases.

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17–25% of patients with acute lymphocytic leukemia and 0.9–3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-β-myosin heavy chain 11 (CBFβ-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFβ-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFβ-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFβ-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFβ-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFβ-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFβ-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

Catch-and-Release of Target Cells Using Aptamer-Conjugated Electroactive Zwitterionic Oligopeptide SAM

Nucleic acid aptamers possess attractive features such as specific molecular recognition, high-affinity binding, and rapid acquisition and replication, which could be feasible components for separating specific cells from other cell types. This study demonstrates that aptamers conjugated to an oligopeptide self-assembled monolayer (SAM) can be used to selectively trap human hepatic cancer cells from cell mixtures containing normal human hepatocytes or human fibroblasts. Molecular dynamics calculations have been performed to understand how the configurations of the aptamers are related to the experimental results of selective cell capture. We further demonstrate that the captured hepatic cancer cells can be detached and collected along with electrochemical desorption of the oligopeptide SAM, and by repeating these catch-and-release processes, target cells can be enriched. This combination of capture with aptamers and detachment with electrochemical reactions is a promising tool in various research fields ranging from basic cancer research to tissue engineering applications.

Controlling size and uniformity of molecularly imprinted nanoparticles using auxiliary template

Molecularly imprinted nanomaterials are gaining substantial importance. As a simple and efficient synthetic method, precipitation polymerization has been used to prepare uniform molecularly imprinted microspheres for numerous template compounds. Despite of its general applicability, the difficulty of obtaining uniform particles for some difficult templates by precipitation polymerization has been reported. In this work, we attempted to produce uniform atrazine-imprinted nanoparticles using propranolol as an auxiliary template under standard precipitation polymerization condition. When propranolol was added in the prepolymerization mixture for atrazine imprinting, it displayed a significant effect on particle size and size distribution of atrazine-imprinted polymers. The molecular binding characteristics of the molecularly imprinted polymer (MIP) nanoparticles were found to be dependent on the relative ratios of the two templates. Under an optimal template propranolol-atrazine ratio of 1:3 mol/mol, very uniform imprinted nanoparticles (d(H) = 106 nm) with a polydispersity index of 0.07 were obtained. The loading of the auxiliary template (propranolol) could be reduced to as low as 5% without sacrificing the uniformity of the MIP nanoparticles. The uniform MIP nanoparticles could be easily encapsulated into polyethylene terephthalate nanofibers using a simple electrospinning technique. The composite nanofibers containing the MIP nanoparticles maintained specific molecular binding capability for both atrazine and propranolol.

Effects of functionally graded materials on dynamics of molecular bond clusters

Unlike nonspecific adhesion of conventional hard materials in engineering commonly described by JKR and DMT type models, the molecular adhesion via specific receptor-ligand bonds is stochastic by nature and has the feature that its strength strongly depends on the medium stiffness surrounding the adhesion. In this paper, we demonstrate in a stochastic-elasticity framework that a type of materials with linearly graded elastic modulus can be designed to achieve “equal load sharing” and enhanced cooperative rebinding among interfacial molecular bonds. Upon modulus gradation, multiple molecular bonds can be elastically decoupled but statistically cooperative. In general, uniform molecular adhesion can be accomplished by two strategies: homogeneous materials with sufficient stiffness higher than a threshold or heterogeneous materials satisfying the criterion on modulus gradation. These results not only provide a theoretical principle for possible applications of functionally graded materials in quantitatively controlling cell-matrix adhesion, but also have general implications on adhesion between soft materials mediated by specific molecular binding.

Modeling nanoparticle uptake and intracellular distribution using stochastic process algebras

Computational modeling is increasingly important to help understand the interaction and movement of nanoparticles (NPs) within living cells, and to come to terms with the wealth of data that microscopy imaging yields. A quantitative description of the spatio-temporal distribution of NPs inside cells; however, it is challenging due to the complexity of multiple compartments such as endosomes and nuclei, which themselves are dynamic and can undergo fusion and fission and exchange their content. Here, we show that stochastic pi calculus, a widely-used process algebra, is well suited for mapping surface and intracellular NP interactions and distributions. In stochastic pi calculus, each NP is represented as a process, which can adopt various states such as bound or aggregated, as well as be passed between processes representing location, as a function of predefined stochastic channels. We created a pi calculus model of gold NP uptake and intracellular movement and compared the evolution of surface-bound, cytosolic, endosomal, and nuclear NP densities with electron microscopy data. We demonstrate that the computational approach can be extended to include specific molecular binding and potential interaction with signaling cascades as characteristic for NP-cell interactions in a wide range of applications such as nanotoxicity, viral infection, and drug delivery.

Applications de l’imagerie pour l’étude de la biodistribution et du métabolisme de nouvelles molécules

Due to numerous technical developments, in vivo imaging is suitable for pharmacokinetic and metabolism studies of new chemical entities as well as for evaluating their pharmacological or biological effects. MRI, nuclear medicine, X-Ray, ultrasound and optical imaging are available for both clinical and experimental imaging with even higher performance. For all these imaging modalities, diagnostic agents are useful to improve contrast and specificity. Specific targeting of biological events is addressed by molecular imaging. From a pharmacodynamic perspective, radiolabeling of a new chemical entity allows in vivo visualization quantitative measure of its biodistribution, its elimination and its specific molecular binding. Non-invasive imaging methods are useful for longitudinal investigations of biological changes. Based on nanotechnologies, specificity of drug delivery can be monitored by imaging. New developments in hybrid imaging technologies as well as multimodal contrast agents reinforce in vivo experimental and clinical proof of mechanism of new chemical entities.

The detection of specific biomolecular interactions with micro-Hall magnetic sensors

The detection of reagent-free specific biomolecular interactions through sensing ofnanoscopic magnetic labels provides one of the most promising routes to biosensing withsolid-state devices. In particular, Hall sensors based on semiconductor heterostructureshave shown exceptional magnetic moment sensitivity over a large dynamic field rangesuitable for magnetic biosensing using superparamagnetic labels. Here we demonstrate thecapability of such micro-Hall sensors to detect specific molecular binding usingbiotin–streptavidin as a model system. We apply dip-pen nanolithography to selectivelybiotinylate the active areas of InAs micro-Hall devices with nanoscale precision. Specificbinding of complementarily functionalized streptavidin-coated superparamagnetic beads tothe Hall crosses occurs via molecular recognition, and magnetic detection of theassembled beads is achieved at room temperature using phase sensitive micro-Hallmagnetometry. The experiment constitutes the first unambiguous demonstrationof magnetic detection of specific biomolecular interactions with semiconductormicro-Hall sensors, and the selective molecular functionalization and resultinglocalized bead assembly demonstrate the possibility of multiplexed sensing ofmultiple target molecules using a single device with an array of micro-Hall sensors.

Key Role of Local Regulation in Chemosensing Revealed by a New Molecular Interaction-Based Modeling Method

The signaling network underlying eukaryotic chemosensing is a complex combination of receptor-mediated transmembrane signals, lipid modifications, protein translocations, and differential activation/deactivation of membrane-bound and cytosolic components. As such, it provides particularly interesting challenges for a combined computational and experimental analysis. We developed a novel detailed molecular signaling model that, when used to simulate the response to the attractant cyclic adenosine monophosphate (cAMP), made nontrivial predictions about Dictyostelium chemosensing. These predictions, including the unexpected existence of spatially asymmetrical, multiphasic, cyclic adenosine monophosphate–induced PTEN translocation and phosphatidylinositol-(3,4,5)P3 generation, were experimentally verified by quantitative single-cell microscopy leading us to propose significant modifications to the current standard model for chemoattractant-induced biochemical polarization in this organism. Key to this successful modeling effort was the use of “Simmune,” a new software package that supports the facile development and testing of detailed computational representations of cellular behavior. An intuitive interface allows user definition of complex signaling networks based on the definition of specific molecular binding site interactions and the subcellular localization of molecules. It automatically translates such inputs into spatially resolved simulations and dynamic graphical representations of the resulting signaling network that can be explored in a manner that closely parallels wet lab experimental procedures. These features of Simmune were critical to the model development and analysis presented here and are likely to be useful in the computational investigation of many aspects of cell biology.

Nanoscale adhesion, friction and wear studies of biomolecules on silicon based surfaces

Protein layers are deployed over the surfaces of microdevices such as biological microelectromechanical systems (bioMEMS) and bioimplants as functional layers that confer specific molecular recognition or binding properties or to facilitate biocompatibility with biological tissue. When a microdevice comes in contact with any exterior environment, like tissues and/or fluids with a variable pH, the biomolecules on its surface may get abraded. Silicon based bioMEMS are an important class of devices. Adhesion, friction and wear properties of biomolecules (e.g., proteins) on silicon based surfaces are therefore important. Adhesion was studied between streptavidin and a thermally grown silica substrate in a phosphate buffered saline (PBS) solution with various pH values as a function of the concentration of biomolecules in the solution. Friction and wear properties of streptavidin (protein) biomolecules coated on silica by direct physical adsorption and a chemical linker method were studied in PBS using the tapping mode atomic force microscopy at a range of free amplitude voltages. Fluorescence microscopy was used to study the detailed wear mechanism of the biomolecules. Based on this study, adhesion, friction and wear mechanisms of biomolecules on silicon based surfaces are discussed.

RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach

Developing a readily available biosensor with excellent performances is the main focus of many research groups. Recently, major breakthroughs in miniaturization of molecular analysis have produced DNA and protein microarrays. The aim of our group is to develop a sensitive technique for analyzing signals on protein microarray by applying the surface plasmon resonance (SPR) method. This new detection technique for specific molecular binding utilizes rolling circles amplification (RCA) post-signal processing method [Nat. Genet. 19 (1998) 225–232] and optical visualization by nanogold particle-labeled molecules on a micro-structured chip surface. By covalent bonding of the RCA primer to the detection antibody guarantees that the linkage between the analyte and the amplified RCA product is maintained during the assay. Experimental results show that RCA has significantly enhanced sensitivity compared to conventional methods. This combination of an easily detectable signal with chip technology should have the potential to become a successful commercial application.