Nonspecific Binding Events Research Articles

Sensitive electrochemical immunosensor for matrix metalloproteinase-3 based on single-wall carbon nanotubes.

A novel electrochemical immunosensor for the detection of matrix metalloproteinase-3 (MMP-3), a cancer biomarker protein, based on vertically aligned single-wall carbon nanotube (SWCNT) arrays is presented. Detection was based on a sandwich immunoassay consisting of horseradish peroxidase (14-16 labels) conjugated to a secondary antibody and/or a polymer bead loaded with multi-enzyme labels. Performance was optimized by effective minimization of non-specific binding (NSB) events using Bovine serum albumin (BSA), Tween-20 and optimization of the primary antibody and secondary antibody concentrations. Results provided a detection limit of 0.4 ng mL(-1) (7.7 pM) for the 14-16 label sensor protocol and 4 pg mL(-1) (77 fM) using a multiply enzyme labeled polymeric bead amplification strategy in 10 microL of calf serum. This immunosensor based on SWCNT arrays offers great promise for a rapid, simple, cost-effective method for clinical screening of cancer biomarkers for point-of-care diagnosis.

Specificity of a High-Sensitivity Cardiac Troponin I Assay Using Single-Molecule–Counting Technology

Cardiac troponin is the standard for diagnosis or exclusion of acute myocardial infarction. Recent guidelines recommend a cutoff value at the upper 99th percentile (99th%)1 of values for a reference population of healthy individuals and assay imprecision (CV) ≤10% at this cutoff (1). Many commercial assays cannot meet these criteria, and values below the 99th% cutoff appear to supply diagnostic and prognostic information (2)(3). Recently the Erenna™ cardiac troponin I (cTnI) immunoassay (Singulex) has been shown to provide sensitivity and precision that meet these goals (4), with a preliminary 99th% cutoff value at 8 ng/L and 10% CV of 1.8 ng/L (5). This novel assay uses single-molecule counting technology and has been previously described. However, we could not fully exclude the possibility that, with such sensitive limits of detection, low-level nonspecific binding (NSB) events might contribute to cTnI measurements. We explored this issue by testing potential sources of NSB events. To test for NSB of serum or plasma constituents, cTnI was quantified in 4 specimen types from 20 healthy individuals, serum, EDTA, lithium heparin, and sodium-citrated plasma. The protocol was approved by the University of California Committee on Human Research. All 20 individuals were free of cardiac disease or symptoms (13 female, 7 male, mean age 43 years, …

Analysis of P Element Transposase Protein-DNA Interactions during the Early Stages of Transposition

P elements are a family of transposable elements found in Drosophila that move by using a cut-and-paste mechanism and that encode a transposase protein that uses GTP as a cofactor for transposition. Here we used atomic force microscopy to visualize the initial interaction of transposase protein with P element DNA. The transposase first binds to one of the two P element ends, in the presence or absence of GTP, prior to synapsis. In the absence of GTP, these complexes remain stable but do not proceed to synapsis. In the presence of GTP or nonhydrolyzable GTP analogs, synapsis happens rapidly, whereas DNA cleavage is slow. Both atomic force microscopy and standard biochemical methods have been used to show that the P element transposase exists as a pre-formed tetramer that initially binds to either one of the two P element ends in the absence of GTP prior to synapsis. This initial single end binding may explain some of the aberrant P element-induced rearrangements observed in vivo, such as hybrid end insertion. The allosteric effect of GTP in promoting synapsis by P element transposase may be to orient a second site-specific DNA binding domain in the tetramer allowing recognition of a second high affinity transposase-binding site at the other transposon end.

Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory Syncytial Virus Infection in the Upper and Lower Respiratory Tract

Respiratory syncytial virus (RSV) is the leading cause of viral bronchiolitis and pneumonia in infants and children. Currently, palivizumab is the only approved monoclonal antibody (mAb) for prophylaxis of RSV. However, a small percentage of patients are not protected by palivizumab; in addition, palivizumab does not inhibit RSV replication effectively in the upper respiratory tract. We report here the development and characterization of motavizumab, an ultra-potent, affinity-matured, humanized mAb derived from palivizumab. Several palivizumab variants that enhanced the neutralization of RSV in vitro by up to 44-fold were generated; however, in vivo prophylaxis of cotton rats with these antibodies conferred only about a twofold improvement in potency over palivizumab. This unexpected small increase of in vivo potency was caused by poor serum pharmacokinetics and lung bio-availability that resulted from unexpectedly broad tissue binding. Subsequent analyses revealed that changes at three amino acids arising from the affinity maturation markedly increased the non-specific binding to various tissues. Our results suggested that kon-driven mutations are more likely to initiate non-specific binding events than koff-driven mutations. Reversion of these three residues to the original sequences greatly diminished the tissue binding. The resulting mAb, motavizumab, binds to RSV F protein 70-fold better than palivizumab, and exhibits about a 20-fold improvement in neutralization of RSV in vitro. In cotton rats, at equivalent concentrations, motavizumab reduced pulmonary RSV titers to up to 100-fold lower levels than did palivizumab and, unlike palivizumab, motavizumab very potently inhibited viral replication in the upper respiratory tract. This affinity-enhanced mAb is being investigated in pivotal clinical trials. Importantly, our engineering process offers precious insights into the improvement of other therapeutic mAbs.

Probing Orientations of Single Fluorescent Labels on a Peptide Reversibly Binding to the Human Delta-Opioid Receptor

We report the first in-depth study of single-molecule polarization behavior of a species that is undergoing reversible binding with its biological receptor. We examine the utility of the information in single-molecule polarization measurements for investigations of binding. The human delta-opioid receptor, which is a G protein-coupled receptor, was incorporated into a supported lipid bilayer. A Cy3 label was covalently attached by a hydrophilic linker to a peptide agonist, Deltorphin II (5,6 Ile-Ile). The fluorescence excitation was alternated between s- and p-polarization using a microscope having the capability of total internal reflectance fluorescence (TIRF) excitation. The polarization behavior reveals that nonspecific binding events for this system give emission that is mostly s-polarized, while binding to the receptor gives emission that has a strong component of p-polarization. The results show that a high signal-to-noise ratio is achievable with single-molecule polarization measurements. The experiment detected 37 binding events of short duration (<30 s) and 35 binding events of long duration (from 30 s to 500 s). The polarization studies indicate that the receptors in the bilayer do not freely rotationally diffuse in the plane of the bilayer when the peptide is bound. The system exhibits two types of polarization behavior. One type has the dye label with fixed orientation, which sometimes abruptly switches. The other type has the dye orientation continuously fluctuating over time, typically exhibiting occasional periods of fixed orientation. For a long binding event of fixed orientation, it is established through analysis of the variance that the orientation actually is fluctuating through a range of angles on the order of 6 degrees. It is shown that precise measurements of reorientation are achievable, with a detection limit of 1.3 degrees for a typical single-molecule signal.

Specificity of receptor–ligand interactions and their effect on dimerisation as observed by electrospray mass spectrometry: bile acids form stable adducts to the RXRα

Electrospray (ES) mass spectrometry data is presented showing that agonist binding to the nuclear receptor (NR), retinoid X receptor alpha (RXRalpha), is competitive. The competitive nature of agonist binding can be used to discriminate between the specific and non-specific binding of small lipophilic molecules to NRs. Further, data is presented which show that high-affinity ligand binding to the RXRalpha ligand-binding domain (LBD) stabilises the domain homodimer. The results indicate that homodimerisation, a functional property of the receptor associated with the binding of agonist ligands, could be used to discriminate between specific and non-specific binding events. Additionally, we report on the remarkable stability of the gas-phase complex between the RXRalpha LBD protein and endogenous bile acids. Protein-bile acid interactions in the gas phase were found to be surprisingly strong, withstanding 'in-source' fragmentation in the ES interface, and, in the case of taurocholic acid (TCA) and lithocholic acid-3-sulphate (LCA-3-sulphate), collision-induced dissociation within the collision cell of a tandem mass spectrometer. Bile acids were found to be inactive towards RXRalpha in transfection assays, and have not been reported to be ligands for the RXRalpha, although lithocholic acid (LCA) has been found to be a competitor in the photoaffinity labelling of RXRbeta with 9-cis-retinoic acid (9-cis-RA). The observation of strong RXRalpha-bile acid non-covalent complexes in ES mass spectrometry highlight the danger of extrapolating gas-phase binding data to the solution phase and further to a possible biological activity, particularly when surface-active compounds such as bile acids are involved. The introduction of a competitive ligand-binding experiment can alleviate this problem and allow the differentiation between specific and non-specific binding.

Label-Free Electrochemical Hybridization Genosensor for the Detection of Hepatitis B Virus Genotype on the Development of Lamivudine Resistance

The resistance analysis related to the hepatitis B virus (HBV) genotyping and treatment procured key information for the study of infected patients. The aim of this study was to develop a novel assay for the voltammetric detection of DNA sequences related to the HBV genotype on the development of lamuvidine resistance by monitoring the oxidation signal of guanine. This new technique not only provides a rapid, cost-effective, simple analysis but also gives information concerning both genotyping and lamivudine resistance. Synthetic single-stranded oligonucleotides ("probe") including YMDD (HBV wild type) YVDD, or YIDD (mutations in the YMDD) variants have been immobilized onto pencil graphite electrodes with the adsorption at a controlled potential. The probes were hybridized with different concentrations of their complementary ("target") sequences such as synthetic complementary sequences, clonned PCR products, or real PCR samples. The formed synthetic hybrids on the electrode surface were evaluated by a differential pulse voltammetry technique using a label-free detection method. The oxidation signal of guanine was observed as a result of the specific hybridization between the probes and their synthetic targets and specific PCR products. The response of the hybridization of the probes with their single-base mismatch oligonucleotides at PGE was also detected. Control experiments using the noncomplementary oligonucleotides were performed to determine whether the DNA genosensor responds selectively. Numerous factors, affecting the probe immobilization, target hybridization, and nonspecific binding events, were optimized to maximize the sensitivity and reduce the assay time. Under the optimum conditions, 457 fmol/mL was found as the detection limit for target DNA. With the help of the appearance of the guanine signal, the new protocol is based on the electrochemical detection of HBV genotype for the development of lamuvidine resistance for the first time. Features of this protocol are discussed and optimized.

A Quantitative Description of the Binding States and In Vitro Function of Antitermination Protein N of Bacteriophage λ

The N protein of bacteriophage lambda activates transcription of genes that lie downstream of termination sequences by suppressing transcription termination. N binds to specific (boxB) and non-specific sites on the transcript RNA and contacts RNA polymerase via cis-RNA looping, resulting in "antitermination" of transcription. To find the effect of N-boxB binding on antitermination, we quantitatively relate binding measurements made in isolation to in vitro antitermination activity. We measure binding of N to boxB RNA, non-specific single-stranded RNA, and non-specific double-stranded DNA fluorimetrically, and use an equilibrium model to describe quantitatively the binding of N to nucleic acids of Escherichia coli transcription elongation complexes. We then test the model by comparison with in vitro N antitermination activity measured in reactions containing these same elongation complexes. We find that binding of N protein to the nucleic acid components of transcription elongation complexes can quantitatively predict antitermination activity, suggesting that antitermination in vitro is determined by a nucleic acid binding equilibrium with one molecule of N protein per RNA transcript being sufficient for antitermination. Elongation complexes contain numerous overlapping non-specific RNA and DNA-binding sites for N; the large number of sites compensates for the low N binding affinity, so multiple N proteins are expected to bind to elongation complexes. The occupancy/activity of these proteins is described by a binomial distribution of proteins on transcripts containing multiple non-specific sites. The contribution of specific (boxB) binding to activity also depends on this distribution. Specificity is not measured accurately by measurements made in the presence and in the absence of boxB. We find that antitermination is inhibited by non-productive binding of N to non-specific sites on template DNA, and that NusA protein covers RNA sites on the transcript, limiting N access and activity. The activity and specificity of regulatory proteins that loop from high-affinity binding sites are likely modulated by multiple non-specific binding events; in vivo activity may also be regulated by the modulation of non-specific binding.

Influence of Surfactants and Antibody Immobilization Strategy on Reducing Nonspecific Protein Interactions for Molecular Recognition Force Microscopy

Specific and nonspecific interactions between antibody-modified probes and substrate-immobilized proteins were monitored by atomic force microscopy (AFM). Probes were modified with anti-ovalbumin IgG antibodies immobilized in either an oriented or a random manner. The oriented immobilization of whole IgG was accomplished through the use of Protein A, and random immobilization was carried out with glutaraldehyde. Nonspecific interactions may lead to false detection of antibody-antigen binding events even when the antigen binding sites are properly positioned by an oriented immobilization strategy. Thus, nonionic and zwitterionic surfactants, including Tween 20, Tween 80, Triton X-100, and CHAPS, were evaluated to determine if nonspecific binding events could be reduced without compromising the desired specific antibody-antigen binding. Enzyme-linked immunosorbent assay and surface plasmon resonance assays were also employed to study antibody-antigen binding as a function of immobilization strategy and surfactant concentration. The data from these studies indicate that Protein A can be used to immobilize whole IgG onto AFM probes for force measurement experiments and that a surfactant is useful for improving the selectivity for such measurements.

Force Spectroscopy of the Double-Tethered Concanavalin-A Mannose Bond

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 ± 9 pN at a bond loading rate of ∼10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.

Detection of achondroplasia G380R mutation from PCR amplicons by using inosine modified carbon electrodes based on electrochemical DNA chip technology

The detection of Achondroplasia G380R mutation from real samples was performed by monitoring the oxidation signal of guanine. Inosine-substituted 12-mer capture probes related to the homozygous or heterozygous alleles of Achondroplasia G380R mutation were adsorbed onto carbon paste electrode (CPE) surface. No guanine signal was obtained from the capture probes, since the inosine bases were electroinactive. Then, these probes were hybridized with the denatured PCR samples on the CPE surface. The hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The oxidation signal of guanine was observed as a result of the specific hybridization between the probe and PCR sample. The changes in the peak height of the guanine signal provided the information whether the PCR sample contained heterozygous allele or homozygous allele. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 41.24 fmol/ml target DNA. The electrochemical detection of Achondroplasia G380R mutation from PCR samples greatly shortened and simplified the diagnosis for the homozygous mutant type, which is lethal for the newborn.

Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes.

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at approximately +1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5' end using N-(3-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated l-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5' phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base single-stranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications.

Allele-Specific Genotype Detection of Factor V Leiden Mutation from Polymerase Chain Reaction Amplicons Based on Label-Free Electrochemical Genosensor

An electrochemical genosensor for the genotype detection of allele-specific factor V Leiden mutation from PCR amplicons using the intrinsic guanine signal is described. The biosensor relies on the immobilization of the 21-mer inosine-substituted oligonucleotide capture probes related to the wild-type or mutant-type amplicons, and these probes are hybridized with their complementary DNA sequences at a carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The guanine signal was monitored as a result of the specific hybridization between the probe and amplicon at the CPE surface. No label-binding step was necessary, and the appearance of the guanine signal shortened the assay time and simplified the detection of the factor V Leiden mutation from polymerase chain reaction (PCR)-amplified amplicons. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the guanine signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 51.14 fmol/mL target DNA. With the help of the appearance of the guanine signal, the yes/no system is established for the electrochemical detection of allele-specific mutation on factor V for the first time. Features of this protocol are discussed and optimized.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription.

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

The MotA transcriptional activator of bacteriophage T4 binds to its specific DNA site as a monomer.

During bacteriophage T4 middle mode gene expression, the MotA transcription factor binds to T4 middle promoters at a -30 mot box consensus sequence to allow activation. Previous binding studies showed that MotA forms multiple gel-shifted complexes with DNA, and structural evidence suggested that MotA dimerizes upon DNA binding. We have shown that a short (13 bp) mot box DNA substrate binds MotA protein but fails to form slower migrating complexes. Therefore, the slower migrating complexes in gel shift assays are caused by DNA-mediated binding events. Competition experiments indicate that the slower migrating complexes are formed by nonspecific binding events, while the first-shifted complex is caused by specific binding to the mot box. Saturation binding experiments revealed that the stoichiometry of MotA binding to DNA is 1:1 in the first-shifted complex, while the slower complexes apparently contain MotA multimers. Gel shift assays using mixtures of MotA and a GST-MotA fusion protein supported the conclusion that the first-shifted complex contains one protein molecule bound to DNA. Furthermore, MotA monomers were cross-linked by glutaraldehyde under conditions where slower complexes exist, but not under conditions that lead to only the first-shifted complex. We conclude that MotA binds specifically to the mot box as a monomer and that additional nonspecific binding events require flanking DNA.

A New Analytical Device Based on Gated Ion Channels: A Peptide-Channel Biosensor

Binding of a protein at a ligand-modified ion channel can lead to closure or distortion of the channel, resulting in a digital on/off response of the ion flux. This principle could be applied as a strategy for sensor design using artificial membrane ligands or receptors. Important details of modeling and a technique of realization of the new molecular electronic sensor device are presented. The response of the sensor induced by analyte binding depends on the analyte's size and ability to close or distort the ion channel. Testing different ion channels in a typical single molecule-binding event modifies the membrane current by about 0.2 to 20 pA. The gated channel-based sensor is set up by using elements from different fields. It consists of a stable transmembrane channel (for example, a chemically engineered 6.3 helix peptide antibiotic) with a ligand covalently bound at the peptide channel entrance, an electrochemical sensor chip with a photostructurized hydrophobic polymer frame, a hydrophilic ion-conducting membrane support, a lipid membrane incorporating the engineered ion channel, and a sensitive membrane current amplifier or a sensitive fluorescence monitor. Detection of channel opening or closure can either be obtained directly by monitoring membrane conductivity or by monitoring a transient change in pH or ion concentration within the membrane compartment. This transient change can be induced by various means, and its decay is directly correlated to the ion permeability of the membrane. Nonspecific binding at a lipid membrane does not interfere with the ion flux through ion channels. This is a basic advantage of this principle compared to that of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or hybridization of labeled DNA/RNA probes. Furthermore, because nonspecific binding events are mainly transient, specific binding events directly at the ion channel can be separated from them by evaluating the current peak time scale. Theoretically, the sensitivity of such a device can approach one molecule. Under practical conditions, the sensitivity is limited by the diffusion process of the analyte to the receptor ligand but not by the molecular electronic device. The new type of biosensor combines sensor design and technology from immunoassays and biorecognition assays with automated analysis systems and knowledge from the field of nanotechnology. Based on the detection principle, miniaturized portable sensor chips with either electronic or optical detection are under construction. These may have capabilities exceeding those of existing analytical instruments (for example, ELISA tests, medical analyzer, and high-pressure liquid chromatography [HPLC].

Advances in Understanding the Pharmacological Properties of Antisense Oligonucleotides

A rapid progress in the field of antisense technology and in exploiting it for therapeutic purposes has become intense. This chapter provides a summary of progress, to assess the status of the technology, to place the technology in the pharmacological context in which it is best understood, and the interpretation of experiments. The ultimate biological effect of an oligonucleotide should be influenced by the local concentration of the oligonucleotide at the target RNA, the concentration of the RNA, the rates of synthesis and degradation of the RNA, the type of terminating mechanism, and the rates of the events that result in termination of the activity of the RNA. Binding of oligonucleotides to specific sequences may inhibit the interaction of the RNA with proteins, other nucleic acids, or other factors required for essential steps in the intermediary metabolism of the RNA or its utilization by the cell. RNA molecules regulate their own metabolism. A number of structural features of RNA are known to influence stability, various processing events, subcellular distribution, and transport. Phosphorothioate oligonucleotides bind to proteins. The interactions with proteins can be divided into nonspecific, sequence-specific, and structure-specific binding events, each of which may have different characteristics and effects. To study the pharmacokinetics of phosphorothioate oligonucleotides, a variety of labeling techniques have been used. In addition to local and regional effects of antisense oligonucleotides, a growing number of well-controlled studies have demonstrated systemic effects of phosphorothioate oligodeoxynucleotides that have a therapeutic index that supports their evaluation for a number of therapeutic indications. As several clinical trials are in progress with phosphorothioates and others should be initiated shortly, more definitive information must be available about the activities, toxicities, and value of this class of antisense drugs in human beings.