Nanopore Analysis as a Tool for Studying Rapid Holliday Junction Dynamics and Analyte Binding.

Holliday junctions (HJs) are an important class of nucleic acid structure utilized in DNA break repair processes. As such, these structures have great importance as therapeutic targets and for understanding the onset and development of various diseases. Single-molecule fluorescence resonance energy transfer (smFRET) has been used to study HJ structure-fluctuation kinetics, but given the rapid time scales associated with these kinetics (approximately sub-milliseconds) and the limited bandwidth of smFRET, these studies typically require one to slow down the structure fluctuations using divalent ions (e.g., Mg2+). This modification limits the ability to understand and model the underlying kinetics associated with HJ fluctuations. We address this here by utilizing nanopore sensing in a gating configuration to monitor DNA structure fluctuations without divalent ions. A nanopore analysis shows that HJ fluctuations occur on the order of 0.1-10 ms and that the HJ remains locked in a single conformation with short-lived transitions to a second conformation. It is not clear what role the nanopore plays in affecting these kinetics, but the time scales observed indicate that HJs are capable of undergoing rapid transitions that are not detectable with lower bandwidth measurement techniques. In addition to monitoring rapid HJ fluctuations, we also report on the use of nanopore sensing to develop a highly selective sensor capable of clear and rapid detection of short oligo DNA strands that bind to various HJ targets.

Biological nanopores elucidate the differences between isomers of mercaptobenzoic-capped gold clusters.

Identification of isomers using traditional mass spectroscopy methods has proven an interesting challenge due to their identical mass to charge ratios. This proves particularly consequential for gold clusters, as subtle variations in the ligand and cluster structure can have drastic effects on the cluster functionalization, solubility, and chemical properties. Biological nanopores have proven an effective tool in identifying subtle variations at the single molecule limit. This paper reports on the ability of an α-hemolysin (αHL) pore to differentiate between para-, meta-, and ortho- (p-, m-, and o-, respectively) mercaptobenzoic acid ligands attached to gold clusters at the single cluster limit. Detecting differences between p-MBA and m-MBA requires pH-dependent studies that illustrate the role inter-ligand binding plays in stabilizing m-MBA-capped clusters. Additionally, this paper investigates the difference in behavior for these clusters when isolated, and when surrounded by small ligand-Au complexes (AunLm, n = 0, 1, 2… and m = 1, 2,…) that are present following cluster synthesis. It is found that continuous exposure of clusters to freely diffusing ligand complexes stabilizes the clusters, while isolated clusters either disintegrate or exit the nanopore in seconds. This has implications for long term cluster stability.

Single-molecule analysis of i-motif within self-assembled DNA duplexes and nanocircles.

The cytosine (C)-rich sequences that can fold into tetraplex structures known as i-motif are prevalent in genomic DNA. Recent studies of i-motif-forming sequences have shown increasing evidence of their roles in gene regulation. However, most of these studies have been performed in short single-stranded oligonucleotides, far from the intracellular environment. In cells, i-motif-forming sequences are flanked by DNA duplexes and packed in the genome. Therefore, exploring the conformational dynamics and kinetics of i-motif under such topologically constrained environments is highly relevant in predicting their biological roles. Using single-molecule fluorescence analysis of self-assembled DNA duplexes and nanocircles, we show that the topological environments play a key role on i-motif stability and dynamics. While the human telomere sequence (C3TAA)3C3 assumes i-motif structure at pH 5.5 regardless of topological constraint, it undergoes conformational dynamics among unfolded, partially folded and fully folded states at pH 6.5. The lifetimes of i-motif and the partially folded state at pH 6.5 were determined to be 6 ± 2 and 31 ± 11 s, respectively. Consistent with the partially folded state observed in fluorescence analysis, interrogation of current versus time traces obtained from nanopore analysis at pH 6.5 shows long-lived shallow blockades with a mean lifetime of 25 ± 6 s. Such lifetimes are sufficient for the i-motif and partially folded states to interact with proteins to modulate cellular processes.

Ligand-Induced Structural Changes of Thiolate-Capped Gold Nanoclusters Observed with Resistive-Pulse Nanopore Sensing.

Nanopore-based resistive pulse sensing with biological nanopores has traditionally been applied to biopolymer analysis, but more recently, interest has grown in applying the technique to characterizing water-soluble metallic clusters. This paper reports on the use of α-hemolysin (αHL) for detecting a variety of thiolate-capped gold nanoclusters. The ligands studied here are p-mercaptobenzoic acid ( p-MBA), tiopronin (TP), and thiolated PEG7 (S-PEG7). Individual clusters trapped in the cis-side of an αHL pore for extended periods (>10 s) exhibit fluctuations between numerous substates. We compare these current steps between the three different ligands and find that they scale with the mass of the corresponding ligand, which suggests that nanopore sensing could be used to characterize intraparticle surface modifications.

The Utility of Nanopore Technology for Protein and Peptide Sensing.

Resistive pulse nanopore sensing enables label-free single-molecule analysis of a wide range of analytes. An increasing number of studies have demonstrated the feasibility and usefulness of nanopore sensing for protein and peptide characterization. Nanopores offer the potential to study a variety of protein-related phenomena that includes unfolding kinetics, differences in unfolding pathways, protein structure stability, and free-energy profiles of DNA-protein and RNA-protein binding. In addition to providing a tool for fundamental protein characterization, nanopores have also been used as highly selective protein detectors in various solution mixtures and conditions. This review highlights these and other developments in the area of nanopore-based protein and peptide detection.

Infrared Laser Heating Applied to Nanopore Sensing for DNA Duplex Analysis.

Temperature studies coupled with resistive-pulse nanopore sensing enable the quantification of a variety of important thermodynamic properties at the single-molecule limit. Previous demonstrations of nanopore sensing with temperature control have utilized bulk chamber heating methodologies. This approach makes it difficult to rapidly change temperatures and enable optical access for other analytical techniques (i.e., single-molecule fluorescence). To address these issues, researchers have explored laser-based methodologies through either direct infrared (IR) absorption or plasmonic assisted heating. In this paper, we demonstrate the use of IR-based direct absorption heating with the DNA sensing capabilities of a biological nanopore. The IR heating enables rapid changes of the temperature in and around an α-hemolysin pore, and we use this to explore melting properties for short (≤50 bp) double-stranded DNA homopolymers. We also demonstrate that the IR heating enables one to measure the percentage of different-sized DNA molecules in a binary mixture.

Microdroplet-Based Potentiometric Redox Measurements on Gold Nanoporous Electrodes.

Potentiometric redox measurements were made in subnanoliter droplets of solutions using an optically transparent nanoporous gold electrode strategically mounted on the stage of an inverted microscope. Nanoporous gold was prepared via dealloying gold leaf with concentrated nitric acid and was chemisorbed to a standard microscope coverslip with (3-mercaptopropyl)trimethoxysilane. The gold surface was further modified with 1-hexanethiol to optimize hydrophobicity of the surface to allow for redox measurements to be made in nanoscopic volumes. Time traces of the open-circuit potential (OCP) were used to construct Nernst plots to evaluate the applicability of the droplet-based potentiometric redox measurement system. Two poised one-electron transfer systems (potassium ferricyanide/ferrocyanide and ferrous/ferric ammonium sulfate) yielded Nernstian slopes of -58.5 and -60.3 mV, respectively, with regression coefficients greater than 0.99. The y-intercepts of the two agreed well to the formal potential of the two standard oxidation-reduction potential (ORP) calibrants, ZoBell's and Light's solution. The benzoquinone and hydroquinone redox couple was examined as a representative two-electron redox system; a Nernst slope of -30.8 mV was obtained. Additionally, two unpoised systems (potassium ferricyanide and ascorbic acid) were studied to evaluate the system under conditions where only one form of the redox couple is present in appreciable concentrations. Again, slopes near the Nernstian values of -59 and -29 mV, respectively, were obtained. All experiments were carried out using solution volumes between 280 and 1400 pL with injection volumes between 8 and 100 pL. The miniscule volumes allowed for extremely rapid mixing (<305 ms) as well. The small volumes and rapid mixing along with the high accuracy and sensitivity of these measurements lend support to the use of this approach in applications where time is a factor and only small volumes are available for testing.

Functional characterization of a melittin analog containing a non-natural tryptophan analog.

Tryptophan (Trp) is a naturally occurring amino acid, which exhibits fluorescence emission properties that are dependent on the polarity of the local environment around the Trp side chain. However, this sensitivity also complicates interpretation of fluorescence emission data. A non-natural analogue of tryptophan, ß-(1-azulenyl)-L-alanine, exhibits fluorescence insensitive to local solvent polarity and does not impact the structure or characteristics of several peptides examined. In this study, we investigated the effect of replacing Trp with ß-(1-azulenyl)-L-alanine in the well-known bee-venom peptide melittin. This peptide provides a model framework for investigating the impact of replacing Trp with ß-(1-azulenyl)-L-alanine in a functional peptide system that undergoes significant shifts in Trp fluorescence emission upon binding to lipid bilayers. Microbiological methods including assessment of the antimicrobial activity by minimal inhibitory concentration (MIC) assays and bacterial membrane permeability assays indicated little difference between the Trp and the ß-(1-azulenyl)-L-alanine-substituted versions of melittin. Circular dichroism spectroscopy showed both that peptides adopted the expected α-helical structures when bound to phospholipid bilayers and electrophysiological analysis indicated that both created membrane disruptions leading to significant conductance increases across model membranes. Both peptides exhibited a marked protection of the respective fluorophores when bound to bilayers indicating a similar membrane-bound topology. As expected, while fluorescence quenching and CD indicate the peptides are stably bound to lipid vesicles, the peptide containing ß-(1-azulenyl)-L-alanine exhibited no fluorescence emission shift upon binding while the natural Trp exhibited >10 nm shift in emission spectrum barycenter. Taken together, the ß-(1-azulenyl)-L-alanine can serve as a solvent insensitive alternative to Trp that does not have significant impacts on structure or function of membrane interacting peptides.

Voltage and blockade state optimization of cluster-enhanced nanopore spectrometry.

Recent work described the use of thiolate-capped gold clusters (Au25(SG)18) with nanopore sensing to increase the residence time of polyethylene glycol (PEG) in an alpha hemolysin pore [Anal. Chem., 2014, 86, 11077]. It was shown that the residence time enhancement narrows the peaks in the PEG-induced current blockade distribution, thus increasing the resolving power of the single molecule nanopore spectrometry (SMNS) technique. Here, we further study the interaction between the cluster and PEG with the goal of optimizing the residence time enhancement for SMNS detection. Specifically, we report the voltage dependence of the enhancement effect and show that, under the conditions studied, the cluster-enhanced residence time is maximized at an applied transmembrane potential near 60 mV. Additionally, we show that the PEG residence time depends on the degree to which the cluster blocks current through the pore and that the PEG on-rate to the pore can be more accurately measured with a cluster in the pore. Finally, we develop a model that describes the cluster-induced shift of the PEG current blockade distribution. We use this model to characterize the interaction between the cluster and PEG and show that it scales linearly with the applied voltage as expected from the proposed enhancement mechanism.

Improving the prospects of cleavage-based nanopore sequencing engines.

Recently proposed methods for DNA sequencing involve the use of cleavage-based enzymes attached to the opening of a nanopore. The idea is that DNA interacting with either an exonuclease or polymerase protein will lead to a small molecule being cleaved near the mouth of the nanopore, and subsequent entry into the pore will yield information about the DNA sequence. The prospects for this approach seem promising, but it has been shown that diffusion related effects impose a limit on the capture probability of molecules by the pore, which limits the efficacy of the technique. Here, we revisit the problem with the goal of optimizing the capture probability via a step decrease in the nucleotide diffusion coefficient between the pore and bulk solutions. It is shown through random walk simulations and a simplified analytical model that decreasing the molecule's diffusion coefficient in the bulk relative to its value in the pore increases the nucleotide capture probability. Specifically, we show that at sufficiently high applied transmembrane potentials (≥100 mV), increasing the potential by a factor f is equivalent to decreasing the diffusion coefficient ratio D(bulk)/D(pore) by the same factor f. This suggests a promising route toward implementation of cleavage-based sequencing protocols. We also discuss the feasibility of forming a step function in the diffusion coefficient across the pore-bulk interface.

Enhanced single molecule mass spectrometry via charged metallic clusters.

Nanopore sensing is a label-free method for characterizing water-soluble molecules. The ability to accurately identify and characterize an analyte depends on the residence time of the molecule within the pore. It is shown here that when a Au25(SG)18 metallic cluster is bound to an α-hemolysin (αHL) nanopore, the mean residence time of polyethylene glycol (PEG) within the pore is increased by over 1 order of magnitude. This leads to an increase in the range of detectable PEG sizes and improves the peak resolution within the PEG-induced current blockade distribution. A model describing the relationship between the analyte residence time and the width of the peaks in the current blockade distribution is included. Finally, evidence is presented that shows the Coulombic interaction between the charged analyte and cluster plays an important role in the residence time enhancement, which suggests the cluster-based approach could be used to increase the residence time of a wide variety of charged analyte molecules.

Droplet-based optical trapping for cell separation in mock forensic samples.

Optical tweezers have a wide range of uses for mechanical manipulation of objects in the microscopic range. This includes both living and static cells in a variety of biomedical and research applications. Single-focus optical tweezers, formed by focusing a laser beam through a high numerical aperture immersion objective, create a significant force, which enables controlled transport of a variety of different cell types and morphologies in three dimensions. Optical tweezers have been previously reported to capture and separate spermatozoa from a reconstituted simulated postcoital sample. We report herein the development of a simplified, more efficient cell transfer protocol that can separate and isolate both spermatozoa as well as leukocytes, with similar efficiencies as those previously reported. The new cell transfer method was used to separate sperm cells from a reconstituted mixture of spermatozoa and vaginal epithelial cells, with complete STR profiles developed from 50 cells with little evidence of contribution from the female contributor to the mixture. This modified protocol was then used to separate 21 samples of enriched leukocytes, with trapped cells ranging from 5 to 22 cells. Complete STR profiles were developed from as few as 10 leukocytes. Thus, with minimal sample preparation and a short trapping time, this method has the potential to provide an alternative to traditional differential extraction methods for separation of sperm:nonsperm mixtures while also providing versatility for separation of cells with differing morphologies.

Cluster-Enhanced Nanopore Sensing of Ovarian Cancer Marker Peptides in Urine.

The development of novel methodologies that can detect biomarkers from cancer or other diseases is both a challenge and a need for clinical applications. This partly motivates efforts related to nanopore-based peptide sensing. Recent work has focused on the use of gold nanoparticles for selective detection of cysteine-containing peptides. Specifically, tiopronin-capped gold nanoparticles, trapped in the cis-side of a wild-type α-hemolysin nanopore, provide a suitable anchor for the attachment of cysteine-containing peptides. It was recently shown that the attachment of these peptides onto a nanoparticle yields unique current signatures that can be used to identify the peptide. In this article, we apply this technique to the detection of ovarian cancer marker peptides ranging in length from 8 to 23 amino acid residues. It is found that sequence variability complicates the detection of low-molecular-weight peptides (<10 amino acid residues), but higher-molecular-weight peptides yield complex, high-frequency current fluctuations. These fluctuations are characterized with chi-squared and autocorrelation analyses that yield significantly improved selectivity when compared to traditional open-pore analysis. We demonstrate that the technique is capable of detecting the only two cysteine-containing peptides from LRG-1, an emerging protein biomarker, that are uniquely present in the urine of ovarian cancer patients. We further demonstrate the detection of one of these LRG-1 peptides spiked into a sample of human female urine.

Theory of polymer-nanopore interactions refined using molecular dynamics simulations.

Molecular dynamics simulations were used to refine a theoretical model that describes the interaction of single polyethylene glycol (PEG) molecules with α-hemolysin (αHL) nanopores. The simulations support the underlying assumptions of the model, that PEG decreases the pore conductance by binding cations (which reduces the number of mobile ions in the pore) and by volume exclusion, and provide bounds for fits to new experimental data. Estimation of cation binding indicates that four monomers coordinate a single K(+) in a crown-ether-like structure, with, on average, 1.5 cations bound to a PEG 29-mer at a bulk electrolyte concentration of 4 M KCl. Additionally, PEG is more cylindrical and has a larger cross-section area in the pore than in solution, although its volume is similar. Two key experimental quantities of PEG are described by the model: the ratio of single channel current in the presence of PEG to that in the polymer's absence (blockade depth) and the mean residence time of PEG in the pore. The refined theoretical model is simultaneously fit to the experimentally determined current blockade depth and the mean residence times for PEGs with 15 to 45 monomers, at applied transmembrane potentials of -40 to -80 mV and for three electrolyte concentrations. The model estimates the free energy of the PEG-cation complexes to be -5.3 kBT. Finally the entropic penalty of confining PEG to the pore is found to be inversely proportional to the electrolyte concentration.

Temperature sculpting in yoctoliter volumes.

The ability to perturb large ensembles of molecules from equilibrium led to major advances in understanding reaction mechanisms in chemistry and biology. Here, we demonstrate the ability to control, measure, and make use of rapid temperature changes in fluid volumes that are commensurate with the size of single molecules. The method is based on attaching gold nanoparticles to a single nanometer-scale pore formed by a protein ion channel. Visible laser light incident on the nanoparticles causes a rapid and large increase of the adjacent solution temperature, which is estimated from the change in the nanopore ionic conductance. The temperature shift also affects the ability of individual molecules to enter into and interact with the nanopore. This technique could significantly improve sensor systems and force measurements based on single nanopores, thereby enabling a method for single molecule thermodynamics and kinetics.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Theory for polymer analysis using nanopore-based single-molecule mass spectrometry.

Nanometer-scale pores have demonstrated potential for the electrical detection, quantification, and characterization of molecules for biomedical applications and the chemical analysis of polymers. Despite extensive research in the nanopore sensing field, there is a paucity of theoretical models that incorporate the interactions between chemicals (i.e., solute, solvent, analyte, and nanopore). Here, we develop a model that simultaneously describes both the current blockade depth and residence times caused by individual poly(ethylene glycol) (PEG) molecules in a single alpha-hemolysin ion channel. Modeling polymer-cation binding leads to a description of two significant effects: a reduction in the mobile cation concentration inside the pore and an increase in the affinity between the polymer and the pore. The model was used to estimate the free energy of formation for K(+)-PEG inside the nanopore (approximately -49.7 meV) and the free energy of PEG partitioning into the nanopore ( approximately 0.76 meV per ethylene glycol monomer). The results suggest that rational, physical models for the analysis of analyte-nanopore interactions will develop the full potential of nanopore-based sensing for chemical and biological applications.

Engineering Biological Nanopore Approaches toward Protein Sequencing.

Biotechnological innovations have vastly improved the capacity to perform large-scale protein studies, while the methods we have for identifying and quantifying individual proteins are still inadequate to perform protein sequencing at the single-molecule level. Nanopore-inspired systems devoted to understanding how single molecules behave have been extensively developed for applications in genome sequencing. These nanopore systems are emerging as prominent tools for protein identification, detection, and analysis, suggesting realistic prospects for novel protein sequencing. This review summarizes recent advances in biological nanopore sensors toward protein sequencing, from the identification of individual amino acids to the controlled translocation of peptides and proteins, with attention focused on device and algorithm development and the delineation of molecular mechanisms with the aid of simulations. Specifically, the review aims to offer recommendations for the advancement of nanopore-based protein sequencing from an engineering perspective, highlighting the need for collaborative efforts across multiple disciplines. These efforts should include chemical conjugation, protein engineering, molecular simulation, machine-learning-assisted identification, and electronic device fabrication to enable practical implementation in real-world scenarios.

The effects of diffusion on an exonuclease/nanopore-based DNA sequencing engine.

Over 15 years ago, the ability to electrically detect and characterize individual polynucleotides as they are driven through a single protein ion channel was suggested as a potential method for rapidly sequencing DNA, base-by-base, in a ticker tape-like fashion. More recently, a variation of this method was proposed in which a nanopore would instead detect single nucleotides cleaved sequentially by an exonuclease enzyme in close proximity to one pore entrance. We analyze the exonuclease/nanopore-based DNA sequencing engine using analytical theory and computer simulations that describe nucleotide transport. The available data and analytical results suggest that the proposed method will be limited to reading <80 bases, imposed, in part, by the short lifetime each nucleotide spends in the vicinity of the detection element within the pore and the ability to accurately discriminate between the four mononucleotides.