αHL Nanopore Research Articles

Microscopic Mechanism of Macromolecular Crowder-Assisted DNA Capture and Translocation through Biological Nanopores.

Biological nanopore sensors are widely used for genetic sequencing as nucleic acids and other molecules translocate through them across membranes. Recent studies have shown that the transport of these polymers through nanopores is strongly influenced by macromolecular bulk crowders. By using poly(ethylene glycol) (PEG) molecules as crowders, experiments have shown an increase in the capture rates and translocation times of polymers through an α-hemolysin (αHL) nanopore, which provides high-throughput signals and accurate sensing. A clear molecular-level understanding of how the presence of PEGs offers such desirable outcomes in nanopore sensing is still missing. In this work, we present a new theoretical approach to probe the effect of PEG crowders on DNA capture and translocation through the αHL nanopore. We develop an exactly solvable discrete-state stochastic model based on the cooperative partitioning of individual polycationic PEGs within the cavity of the αHL nanopore. It is argued that the apparent electrostatic interactions between the DNA and PEGs control all of the dynamic processes. Our analytical predictions find excellent agreements with existing experiments, thereby strongly supporting our theory.

Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

Ultra-low concentration detection of DNA without its amplification using a biological nanopore.

Nanopore sensing is a powerful tool for single-molecule detection. A single molecule is recognized and detected from the information obtained when the target molecule passes through the nanoscale pore by electrophoresis. However, practically it is challenging to measure the target molecules at ultra-low concentrations because it takes a long time for the target molecule to enter the pore. Several elaborate techniques with a biological nanopore, α-hemolysin (αHL), have been proposed to detect the subpicomolar or femtomolar nucleic acids. Wang et al. used a salt gradient between solutions that sandwiched the αHL pore to enhance the electric field and increased the capture frequency of the targets, resulting in the detection of nucleic acid molecules at 0.1 pM. Zhang et al. showed the detection of target nucleic acids at 1 fM using the αHL pore with isothermal amplification of the targets. In contrast, we have recently reported the detection of 0.5 fM nucleic acids without their amplification reaction in the presence of a 500 nM probe complementary binding to the target. The excess amount of the probe relative to the target molecules was assumed to increase the capture frequency of the target/probe duplexes. Here, we attempt to reveal the mechanism of detecting DNA molecules at ultra-low concentrations with the αHL nanopore using probes with complementary sequences to the target molecules. We found that the complementary strand of the target in the probe sequence enhances the capture frequency of the target/probe duplexes by interacting with the target/probe molecules. In addition, higher concentrations of the probe relative to the target molecule (>107-fold) resulted in more frequent capture. This approach potentially advances nanopore technology toward its application in the direct detection of nucleic acid biomarkers at ultra-low levels in body fluids.

MicroRNA detection at femtomolar concentration using a probe-based nanopore technique

Circulating nucleic acids (CNA) in body fluids have proven to be potential non-invasive biomarkers. One approach for detecting CNA at a single-molecule level is nanopore sensing. In the nanopore measurements, we can observe characteristic changes in the ion current passing through the pore depending on the analyte. In order to detect nucleic acids that are similar in length or sequence, DNA probes selectively binding to the target are widely used. After the target/probe duplex is captured into the pore, the probe passes through the αHL nanopore by electrophoresis with the target sequences unzipping, generating their specific electrical signatures.

Highly sensitive detection of paraquat with pillar[5]arenes as an aptamer in an α-hemolysin nanopore

We report a highly sensitive biological nanopore sensor by introducing carboxylatopillar[5]arene (CP[5]A) molecule as an aptamer into the lumen of αHL nanopore for the single-molecule detection of paraquat (PQ) via host–guest interactions.

Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin.

Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the α-hemolysin (αHL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180°. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside αHL are used to derive thermodynamic (ΔH, ΔS) and kinetic (EA) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of αHL out of the helix (18 ± 1 kcal mol-1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the αHL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level.

Quantifying Short-Lived Events in Multi-State Ionic Channel Measurements

We present a new technique that considerably improves the resolution and accuracy of single molecule measurements with nanopores. Molecular interactions with nanopores are characterized by electrical measurements of discrete changes in the channel conductance. By representing physical components of the system with electrical equivalents, we used circuit theory to model response of the system to a stimulus (e.g., a molecule entering the channel). This allowed us to characterize short-lived events where the ionic current does not reach a steady state value, and were previously not analyzed. Applying this technique to measurements of poly(ethylene glycol) (PEG) molecules with the α-hemolysin (αHL) nanopore resulted in remarkable improvements in accuracy and the number of detected events. When measuring polydisperse PEG (mean molecular weights of 400 g/mol and 600 g/mol), the new method recovered ≈ 18-fold more events per unit time, compared with existing techniques, and discriminated molecules with as few as 8 monomers (PEG8). We validated the measurement of PEG with an αHL nanopore using results from a recently published study (Balijepalli et. al, J Am Chem Soc, 135: 7064, 2013) that refined a previous analytical theory with molecular dynamics simulations. Fitting this model to the newly obtained experimental data resulted in excellent agreement of both the blockade depth (the ratio of the ionic current when a molecule occupies the pore to the open channel current) and the residence times of the molecule in the channel, over the entire measurement range (PEG8 to PEG19). Finally, we applied the new analysis technique to recover the sequence of a known DNA strand with 26 bases, from a published ionic current trace (Manrao et. al, Nat. Biotechnol. 30: 349, 2012). The technique detected systematic fluctuations in the ionic current that were as small as 0.9 ± 0.04 pA.

Detection of 3′-End RNA Uridylation with a Protein Nanopore

Post-transcriptional modifications of the 3'-ends of RNA molecules have a profound impact on their stability and processing in the cell. Uridylation, the addition of uridines to 3'-ends, has recently been found to be an important regulatory signal to stabilize the tagged molecules or to direct them toward degradation. Simple and cost-effective methods for the detection of this post-transcriptional modification are not yet available. Here, we demonstrate the selective and transient binding of 3'-uridylated ssRNAs inside the β barrel of the staphylococcal α-hemolysin (αHL) nanopore and investigate the molecular basis of uridine recognition by the pore. We show the discrimination of 3'-oligouridine tails on the basis of their lengths and propose the αHL nanopore as a useful sensor for this biologically relevant RNA modification.

Single Molecule RNA Base Identification with a Biological Nanopore

The sequencing of individual DNA and RNA strands with nanopores is under investigation as a rapid, low-cost platform. In this approach, bases are identified in order as the strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as α-hemolysin (αHL), are advantageous because they can be precisely manipulated by genetic modification. Previously, it has been shown that the αHL nanopore contains three recognition sites, capable of discriminating between single DNA bases when oligonucleotides are immobilized within the nanopore. Here we extend these investigations into discrimination of nucleobases in RNA. By immobilizing RNA homopolymers within the αHL pore using a terminal biotin-streptavidin complex, we achieve sharply defined current distributions that enable clear discrimination of the nucleobases. This is further extended by investigating individual RNA bases in a DNA polyC background, which gives comparable results. Additionally, with a view to “exo sequencing”, an engineered protein nanopore is successfully used to detect and discriminate all four ribonucleoside monophosphates (rNMPs) by using, am7-β-cyclodextrin as both a transient and covalent molecular adapter.

Kinetic Dynamcs in an αHl-Based Nanopore DNA Sequencer

The prospect of DNA sequencing by a nanopore device has received significant attention in the past decade. One suggested approach involves tethering an exonuclease enzyme to an α-hemolysin (αHL) monomer, while the assembled and embedded pore maintains nominal function. In this proposal, processive cleavage of 2’-deoxynucleoside 5’-monophosphates (dNMPs) by the exonuclease enables identification of the cleaved units. This strategy aims to accomplish rapid and robust DNA sequencing by employing time vs. electrical-current recording of individual dNMPs that are sequentially captured at a constriction site located within the nanopore. If achieved, this accomplishment would mark a significant milestone towards the $1000-genome target set by the NIH.Here we present a Brownian dynamics simulation of the αHL nanopore that mimics the proposed conditions for an exonuclease sequencing device. Analyte motion is described by combining diffusion, electrophoretic drift, and electroosmotic flow over relevant distances and kinetic time scales, ranging from nanoseconds to microseconds. By implementing the algorithm on a distributed computer network, we achieve sufficient iterations to explore the impact of variable parameters such as dNMP size, pore geometry, applied electric field strength, the pore's ion selectivity, the analyte's electrophoretic mobility, and the analyte's diffusive behavior inside and outside the pore. We also decouple the driving factors that give rise to analyte translation and detection. The data show that thermal motion typically overwhelms unidirectional forces at nanoscale dimensions. The analysis also suggests ways to potentially improve DNA sequencing fidelity.