Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions

<p>Abstract</p> <p>Background</p> <p>The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector.</p> <p>Results</p> <p>We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed.</p> <p>Conclusion</p> <p>Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins.</p

Nanopore Detector based analysis of single-molecule conformational kinetics and binding interactions

BACKGROUND: A Nanopore Detector provides a means to transduce single molecule events into observable channel current changes. Nanopore-based detection can report directly, or indirectly, on single molecule kinetics. The nanopore-based detector can directly measure molecular characteristics in terms of the blockade properties of individual molecules – this is possible due to the kinetic information that is embedded in the blockade measurements, where the adsorption-desorption history of the molecule to the surrounding channel, and the configurational changes in the molecule itself, imprint on the ionic flow through the channel. This rich source of information offers prospects for DNA sequencing and single nucleotide polymorphism (SNP) analysis. A nanopore-based detector can also measure molecular characteristics indirectly, by using a reporter molecule that binds to certain molecules, with subsequent distinctive blockade by the bound-molecule complex. RESULTS: It is hypothesized that reaction histories of individual molecules can be observed on model DNA/DNA, DNA/Protein, and Protein/Protein systems. Preliminary results are all consistent with this hypothesis. Nanopore detection capabilities are also described for highly discriminatory biosensing, binding strength characterization, and rapid immunological screening. CONCLUSION: In essence, the heart of chemistry is now accessible to a new, single-molecule, observation method that can track both external molecular binding states, and internal conformation states

Development of New Biological Nanopores and Their Application for Biosensing and Disease Detection

Nanopore technology has recently emerged as a new real-time single molecule sensing method. The current dominant technologies, such as mass spectrometry and immunoassay, for protein analysis is still slow and complex, which can’t meet the urgent need and fields of use. Development of a highly simple, portable and sensitive detection system for pathogen detection, disease diagnosis, and environmental monitoring is in great need. Membrane embedded Phi29 connector nanopore, the first protein nanopore coming from bacteriophage, was mainly focusing on DNA and RNA translocation in previous studies. Here, Phi29 connector nanopore was first time established for antibody detection by engineering Epithelial Cell Adhesion Molecule peptide as a probe. The results demonstrate that the specific antibody can be detected in presence of diluted serum or non-specific antibody. To enable detecting more different types of analytes with high sensitivity, developing new nanopore with various properties, such as size, charge, hydrophilic/hydrophobic and physical dimension, is needed. In this work, besides Phi29 nanopore, several new protein nanopores that derived from T3, T4, and SPP1 bacteriophages were developed. A shared property of three step conformational change among these portal channel has been discovered. Elucidating the sequence and oligomeric states of proteins and peptides is critical for understanding their biological functions. Here, SPP1 nanopore was used to characterize the translocation of TAT peptide with dimer and monomer forms. Translocation of the peptide was confirmed by optical single molecule imaging for the first time, and analyzed quantitatively. The dynamics of peptide oligomeric states were clearly differentiated based on their characteristic electronic signatures. Main challenge for probing protein structure, folding, detection and sequencing using nanopore is the ultra-fast translocation speed which normally beyond electronic detection limit. In this work, the peptides translocation was slowed in SPP1 nanopore by changing the charge shielding of the channel. A 500-fold reduction was observed for TAT peptide translocation. By using this method, arginine chain peptide as short as two arginine can be detected first time. Further improving the bandwidth may lead to single amino acid detection and has the potential for protein sequencing. Compared with protein nanopore, de novo designed nanopore can provide numerous advantages, such as tunable size and functionality, ease of construction, scale up and modification. In the final study, an RNA-based biomimetic nanopore was first time constructed. The insertion of RNA nanopore into lipid bilayer and cell membrane were characterized and translocation of short amino acids through RNA nanopore was detected. This new artificial nanopore has the potential to be used for sensing, disease diagnosis, and even protein sequencing

Pore forming protein assembly and the use in nanopore sensing: a study on E. coli proteins ClyA and OmpG

Pore forming proteins are typically the proteins that form channels in membranes. They have several roles ranging from molecule transport to triggering the death of a cell. This work focuses on two E. coli pore forming proteins that have vastly differing roles in nature. Outer membrane protein G (OmpG) is an innocuous β-barrel porin while Cytolysin A (ClyA) is an α-helical pore forming toxin. For OmpG we probed its potential to be a nanopore sensor for protein detection and quantification. A small high affinity ligand, biotin, was covalently attached to loop 6 of OmpG and used to capture biotin-binding proteins. OmpG specifically interacted with target proteins and with a high sensitivity. In addition, we found that OmpG could discriminate among eight target proteins, four avidin homologues and four antibody homologues. Our work was the first revelation that a “noisy” nanopore sensor could not only discriminate among homologous proteins but could maintain this sensitivity and specificity in a serum sample. Unlike OmpG which is a monomeric pore, the ClyA pore is an oligomer which allowed us to probe its assembly. We found an alternative mechanism of assembly of the ClyA pore from its soluble monomer. Our results revealed an off-pathway oligomerization of ClyA. These oligomers are stable in solution but are less efficient at converting into transmembrane pores in the membrane than monomeric ClyA

The NTD Nanoscope: potential applications and implementations

<p>Abstract</p> <p>Background</p> <p>Nanopore transduction detection (NTD) offers prospects for a number of highly sensitive and discriminative applications, including: (i) single nucleotide polymorphism (SNP) detection; (ii) targeted DNA re-sequencing; (iii) protein isoform assaying; and (iv) biosensing via antibody or aptamer coupled molecules. Nanopore event transduction involves single-molecule biophysics, engineered information flows, and nanopore cheminformatics. The NTD Nanoscope has seen limited use in the scientific community, however, due to lack of information about potential applications, and lack of availability for the device itself. Meta Logos Inc. is developing both pre-packaged device platforms and component-level (unassembled) kit platforms (the latter described here). In both cases a lipid bi-layer workstation is first established, then augmentations and operational protocols are provided to have a nanopore transduction detector. In this paper we provide an overview of the NTD Nanoscope applications and implementations. The NTD Nanoscope Kit, in particular, is a component-level reproduction of the standard NTD device used in previous research papers.</p> <p>Results</p> <p>The NTD Nanoscope method is shown to functionalize a single nanopore with a channel current modulator that is designed to transduce events, such as binding to a specific target. To expedite set-up in new lab settings, the calibration and troubleshooting for the NTD Nanoscope kit components and signal processing software, the NTD Nanoscope Kit, is designed to include a set of test buffers and control molecules based on experiments described in previous NTD papers (the model systems briefly described in what follows). The description of the Server-interfacing for advanced signal processing support is also briefly mentioned.</p> <p>Conclusions</p> <p>SNP assaying, SNP discovery, DNA sequencing and RNA-seq methods are typically limited by the accuracy of the error rate of the enzymes involved, such as methods involving the polymerase chain reaction (PCR) enzyme. The NTD Nanoscope offers a means to obtain higher accuracy as it is a single-molecule method that does not inherently involve use of enzymes, using a functionalized nanopore instead.</p

Nanopores with Fluid Walls for Characterizing Proteins and Peptides.

Nanopore-based, resistive-pulse sensing is a simple single-molecule technique, is label free, and employs basic electronic recording equipment. This technique shows promise for rapid, multi-parameter characterization of single proteins; however, it is limited by transit times of proteins through a nanopore that are too fast to be resolved, non-specific interactions of proteins with the nanopore walls, and poor specificity of nanopores for particular proteins. This dissertation introduces the concept of nanopores with fluid walls and their applications in sensing and characterization of proteins, disease-relevant aggregates of amyloid-beta peptides, and activity of membrane-active enzymes. Inspired by lipid-coated nanostructures found in the olfactory sensilla of insect antennae, this work demonstrates that coating nanopores with a fluid lipid bilayer confers unprecedented capabilities to a nanopore such as precise control and dynamic actuation of nanopore diameters with sub-nanometer precision, well-defined control of protein transit times, simultaneous multi-parameter characterization of proteins, and an ability to monitor phospholipase D. Using these bilayer-coated nanopores with lipids presenting a ligand, proteins binding to the ligand were captured, concentrated on the surface, and selectively transported to the nanopore, thereby, conferring specificity to a nanopore. These assays enabled the first combined determination of a protein’s volume, shape, charge, and affinity for the ligand using a single molecule technique. For non-spherical proteins, the dipole moment and rotational diffusion coefficient could be determined from a single protein. Additionally, the fluid, biomimetic surface of a bilayer-coated nanopore was non-fouling and enabled characterization of Alzheimer’s disease-related amyloid-beta aggregates. The presented method and analysis fulfills a previously unmet need in the amyloid research field: a method capable of determining the size distributions and concentrations of amyloid-beta aggregates in solution. The experiments presented here demonstrate that the concept of a nanopore with fluid walls enables new nanopore-based assays. In particular, it demonstrates the benefits of this concept for simultaneous, multi-parameter characterization of proteins with a single-molecule method; this technique may, therefore, be well-suited for identification of proteins directly in complex biological fluids. Based on these findings, the addition of fluid walls to nanopores holds great promise as a tool for simple, portable single-molecule assays and protein characterization.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96049/1/ecyus_1.pd

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications

Channel-Forming Bacterial Toxins in Biosensing and Macromolecule Delivery

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications