Capture and analysis of double‐stranded DNA with the α‐hemolysin nanopore: Fundamentals and applications

Abstract

The α‐hemolysin nanopore has attracted much attention as a tool for the single‐molecule analysis of DNA due to its potential as an ultra‐sensitive, specific, and label‐free sensing technique. The vast majority of DNA sensing research with the α‐hemolysin nanopore has focused on interrogating single‐stranded DNA. Nevertheless, the structure of the α‐hemolysin pore, specifically the circa 32.6 cubic nanometer vestibule, is of sufficient size for a short section of double‐stranded DNA (dsDNA) to reside before unzipping into its single‐stranded constituents. In this review, we describe past and current literature relating to the rich information that can be obtained from the interrogation of dsDNA while residing within the α‐hemolysin nanopore vestibule, and the subsequent voltage‐driven unzipping of the residing DNA into its single‐stranded constituents. Applications for dsDNA interrogation and unzipping that have been implemented include DNA sequencing, disease diagnosis, and the identification of epigenetic modifications.

INTRODUCTION

Biological nanopores are self-assembling channel-forming proteins that provide the ability to discriminate, probe, and manipulate single molecules with high specificity and sensitivity through the provision of a nanometer volume for capture, analysis, reactivity, and translocation.^[] DNA capture is achieved through the application of a bias voltage across the pore, which itself is embedded into a lipid bilayer that bridges two separate electrolyte compartments.^[] The potential gradient that results from applying a voltage across the pore drives electrolyte ions and any charged analyte molecules present from one side of the nanopore towards the other, while the ionic current is measured as a function of time.^[] The entry and translocation of molecules of interest through the nanopore are detected by a momentary drop in current relative to that of the open, unblocked pore. The blockage current, blockage duration time interval, and in some instances, the noise of each current blockage event can then be analyzed at the single-molecule level to provide information on the molecule's characteristics.

There are several biological nanopores that can be utilized for nucleic acid analysis, including aerolysin,^[] cytolysin A (ClyA),^[] the phi29 Motor,^[] Mycobacterium smegmatis porin A (MspA),^[] and alpha-hemolysin (α-HL).^[]

Aerolysin is a heptameric β-pore-forming toxin, with a constriction diameter of approximately 1.0 nm. This nanopore contains seven unpaired positively charged residues within its constriction site,^[] which have been shown to enhance the pore interaction with negatively charged molecules.^[] However, its limiting lumen diameter restricts its applications to single-stranded DNA (ssDNA) and RNA.

ClyA is a toxin synthesized by E. coli and Salmonella enterica.^[] It has a diameter of 3 nm at the narrower opening, 6 nm at the wider opening, and is 13 nm in length.^[] As a result of its larger geometry, most of the research using a ClyA nanopore focuses on detecting protein translocation.^[] Furthermore, the pore lumen of wild-type ClyA is extremely negatively charged.^[] ClyA mutants have been engineered to introduce additional positive charges in the lumen and the wide entrance of the pore to allow both ssDNA and DNA translocation at physiological salt concentrations (150 mM NaCl).^[]

The bacteriophage phi29 DNA-packaging motor comprises a dodecamer channel made up of 12 copies of the gp10 protein.^[] It is 3.6 nm wide at the narrow opening, 6 nm at its wider opening, and 7 nm long.^[] Engineering of the channel size, through removing the internal loop segment from the gp10 subunit and forming two subpopulations within the channel,^[] has been shown to facilitate sensitive detection of DNA and RNA.^[] However, its use in DNA sensing has been limited because phi29 also exhibits gating at high potentials, that is, rapidly switching from open to closed states, and this can interfere with the baseline for molecular sensing experiments.^[]

MspA is a funnel-shaped octameric channel pore, with a single constriction region approximately 1.2 nm wide and 0.6 nm long.^[] The constriction size of MspA is particularly attractive in ssDNA analysis, as it is of comparable diameter to circa four DNA bases in length which provides improved sensitivity and spatial resolution.^[,] MspA has also been utilized in unzipping experiments.^[] However, the pore must be engineered to replace the negative charges within the pore cavity with neutral residues.^[]

The α-HL pore is a 33-kDa leukotoxin secreted by the Staphylococcus aureus bacterium,^[] and from cis to trans side (Figure ) is composed of a broad upper vestibule and a narrow lower β-barrel embedded into a lipid bilayer.^[,] Unlike the ClyA, mspA, and Phi29 pores, α-HL can be used directly for DNA analysis as the wild-type without recourse to complex protein engineering. The absence of a requirement for mutagenesis has enabled a larger number of research groups to utilize the α-HL pore in their research. This, combined with its favorable geometry relative to the diameter of both ssDNA (β-barrel) and double-stranded DNA (dsDNA) (vestibule), has enabled α-HL to remain the best-characterized and most broadly used protein pore for DNA analysis,^[,] since the initial demonstration by Kasianowicz et al. in 1996.^[]

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If the DNA helix is designed with a ‘tail’, that is, a single-stranded section at either or both ends of the helical section, this tail will be capable of passing through the central 1.4 nm constriction, while the dsDNA ‘body’ section cannot. In this scenario, the captured dsDNA is exposed to a locally exerted denaturing force at the narrowest constriction site, resulting in its unzipping/separation into two ssDNA components, as illustrated in Figure . The duration of the blockage event before unzipping can be analyzed to provide information on the duplex's length, composition, and stability.^[] The dynamics of dsDNA unzipping are fundamental to various cellular processes such as DNA replication, RNA transcription, and DNA repair,^[,] and thus, such experiments are interesting from both a fundamental biological perspective and an applied perspective, whereas, in the latter, α-HL is a valuable tool for interrogating DNA structure and stability.^[]

If the DNA helix is designed with a ‘tail’, that is, a single-stranded section at either or both ends of the helical section, this tail will be capable of passing through the central 1.4 nm constriction, while the dsDNA ‘body’ section cannot. In this scenario, the captured dsDNA is exposed to a locally exerted denaturing force at the narrowest constriction site, resulting in its unzipping/separation into two ssDNA components, as illustrated in Figure . The duration of the blockage event before unzipping can be analyzed to provide information on the duplex's length, composition, and stability.^[] The dynamics of dsDNA unzipping are fundamental to various cellular processes such as DNA replication, RNA transcription, and DNA repair,^[,] and thus, such experiments are interesting from both a fundamental biological perspective and an applied perspective, whereas, in the latter, α-HL is a valuable tool for interrogating DNA structure and stability.^[]

With the correct choice of experimental parameters (DNA length, voltage, temperature, etc.), it is possible to manipulate the residence time of a DNA helix within the α-HL pore, controlling the unzipping process. The current measured while the dsDNA resides in the pore prior to unzipping is strongly dependent on base-pair composition, and, for those base-pairs aligned with the “latch” constriction of α-HL (Figure ), local conformational changes in a DNA structure as well. The “latch” constriction of α-HL is the point at which the diameter of the pore is most comparable to the diameter of a DNA helix trapped in the pore vestibule, and comprises a ring of lysine residues that can interact with DNA bases residing in close proximity.^[]

Analysis of dsDNA with the α-HL pore provides a wealth of information about the structure, dynamical processes, and protein-DNA interactions that are distinct from those of ssDNA. This mini-review explores the use of α-HL as an extremely powerful tool for investigating dsDNA, discussing the fundamentals of the unzipping process, and its applications for interrogating DNA structure and monitoring dynamical processes in dsDNA, such as base flipping.

Mechanism of DNA unzipping

Sauer-Budge et al. conducted the first direct demonstration of dsDNA unzipping with α-HL in 2003. In these inaugural unzipping studies, two synthetic DNA duplexes were designed that comprised of a 50 base pair helical section with a 50-base single-stranded tail. The first of two DNA strands comprised of 100 bases hybridized to 50 complimentary nucleotides (100/50comp), while the second comprised of 100 bases bounded to 50 base pairs featuring six mispaired bases (100/50mis). The mismatched sequence is shown in Figure .^[] In the experiment, a bias upwards of 120 mV bias was applied, and the average blockage time after the DNA was added to the cis chamber was recorded. At the conclusion of the experiment, a polymerase chain reaction was performed on the contents of the trans chamber. Following subsequent gel electrophoresis, only the 100mer was identified, indicating that the 100/50mis dsDNA had denatured into its constituent components, with the 100mer passing through the constriction, as depicted in Figure .^[] A kinetic model was proposed based on the distribution of blockade duration for both duplexes, as is presented in Figure . An exponential distribution was observed for the 100/50comp, while a more complicated shape was reported for the 100/50mis. Accordingly, a two-step model was presented for the 100/50mis unzipping process. The first step (k₁/k_-1) corresponds to the reversible zipping and unzipping of the dsDNA up to the four-base mismatch, followed by the irreversible separation of DNA strands (k₂). Sauer-Budge et al. postulate that a duplex devoid of this mismatch would not exhibit this intermediate and thus function as a first-order reaction. In addition to differences in mechanism, the unzipping times for mismatch containing duplexes were found to be much shorter than those observed for fully complementary duplexes. A similar decrease in unzipping times was also observed for PNA/DNA duplexes in later work by Luchian and co-workers.^[]

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In studies by Mathé et al., the capture and subsequent unzipping of DNA hairpin structures within the α-HL vestibule were investigated.^[] In these experiments, the hairpin molecules consisted of a 3′ single-strand overhang and a 10 base double-stranded section. Analysis of blockade current upon hairpin capture at the micro to millisecond time scale enabled each step to be decoupled from one another. The unzipping of DNA was inferred by the abrupt increase from the blocked current back to the open pore current, as depicted in Figure .^[] Meller and co-workers proposed that the unzipping of DNA hairpin molecules occurs in two consecutive steps: first, the entry and sliding of the ssDNA overhang until the double-stranded portion of the DNA is lodged in the vestibule section of α-HL; followed by the unwinding of the helical part in the confines of the vestibule and the translocation of the fully-unzipped DNA hairpin through the pore.^[]

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The model developed by Mathé et al. is supported through experiments by Akeson and co-workers.^[] In these experiments, DNA hairpin duplexes with varying stem lengths of 3–9 base pairs were studied using α-HL. From the results obtained, a 2-step model was also proposed. Firstly, the hairpin stem is captured in the vestibule, as confirmed by the abrupt reduction in current. Next, unzipping of the duplex takes place, thus allowing translocation of the now unzipped hairpin through the channel, signaled by the end of the blockage and a substantial increase in current.^[]

Meller and colleagues also investigated the reversibility of the DNA unzipping process.^[] Dynamic voltage control programs that apply a different voltage ramp rate to the nanopore were developed, allowing a steady voltage ramp to be applied to individual untethered DNA molecules. They measured the mean critical voltage at which DNA unzipping occurred as a function of the ramp. This technique has since been labeled as nanopore force spectroscopy. The results obtained showed a distinct change between the reversible unzipping at low ramp rates compared to the irreversible unzipping process observed at high voltages.^[]

Burrows and co-workers studied the unzipping process for fishhook (one-tail) and internal (two-tail) hairpin DNA architectures with the α-HL pore.^[] The results presented in Figure  show disparities in current levels and unzipping duration for internal hairpin DNA and fishhook hairpins of analogous sequences, suggesting that they unfold by different mechanisms. At 100 mV bias in 1 M KCl, the duration of internal hairpin unzipping and translocation through the pore was 2.6 ± 0.1 ms with a blockage current (I_D) of 18 ± 1% of the open channel (I_O), while the fishhook hairpin took 64 ± 2 ms, with an I_D of 11 ±1 % of I_O.^[] The fishhook hairpins showed long unzipping times with one deep blockage current level, while the internal hairpins unzipped relatively quickly with a characteristic pulse-like current pattern. The tail length of one of the two ends for the internal hairpin was then varied from 6 to 15 nucleotides in 3 base-pair increments. In these experiments, a voltage ≥ 80 mV was applied, and the duration and current level corresponding to the unzipping time and blockage current, respectively, was recorded. Internal hairpin DNA in which one of the tail lengths was just 6 nucleotides in length unzipped in the same way as fishhook hairpins, whereas those with a tail length of 12 nucleotides or greater behaved more like internal hairpins. The authors attributed these differences to internal and fishhook hairpins unzipping through different mechanisms, where fishhook hairpins were postulated to unzip inside the vestibule and internal hairpins to unzip outside the vestibule (Figure ).^[]

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Another example where unzipping location was found to be dependent on the helical structure is that of A-form DNA-RNA or DNA-PNA duplex and B-form DNA-DNA duplex.^[] The external diameter of an A-form duplex (d = 2.4 nm) is larger than the B-form duplex (d = 2.0 nm) due to differences in the conformation of the sugar ring. In B-form, the sugar ring adopts a C2’ endo conformation, whereas the A-form has a C3’ endo conformation due to the presence of a 2′-OH in RNA.^[] At 120 mV, the larger A-form duplex had a 15-fold faster unzipping time and a larger residual current than the B-form duplex. This was attributed to the B-form duplex moving through the latch zone and into the vestibule, where they slowly unzip in the sterically constrained cavity. In contrast, the broader A-form duplexes were postulated to unzip outside the pore, with far fewer steric constraints. Differences in the hydration structure, which is well-ordered and less mobile around the A-form duplexes relative to the B-form duplexes, were also proposed to serve as a barrier for entry into the nanopore for the A-form helical structure.^[]

More recently, Dragomir et al. synthesized two synthetic duplexes: a blunt DNA-PNA duplex and a polyarginine-conjugated DNA-PNA duplex.^[] In their experiment, a force of 200 mV was applied to drive the duplexes through α-HL, and the ionic current blockage signature of each duplex unzipping and translocation event was collected. The polyarginine conjugated DNA-PNA duplex was found to undergo unzipping and translocation faster than its DNA-PNA counterpart. This was attributed to the fact that 15 base pairs of the duplex fully occupy the α-HL vestibule, leaving the polyarginine overhang outside the pore and increasing the overall pulling force on the duplex. From this information, Dragomir et al. proposed a kinetic model in which, upon applying a trans-positive potential to the negatively charged duplexes, they are captured within the nanopore. The presence of a polyarginine overhang caused the poly-arginine conjugated DNA-PNA to enter the nanopore vestibule in the 5′ direction. In contrast, in its absence, the DNA-PNA duplex could enter with either the 5′ and 3′ end and be found to unzip slower through the pore, which further highlights the importance of interactions with the α-HL vestibule prior to β-barrel entry. This was then proceeded by force-induced unzipping in the constriction site and finally the translocation of the unhybridized ssDNA fragment.^[]

It should be noted that in the works of Dragomir and later Luchian,^[] the DNA/PNA helix is proposed to unzip within the nanopore vestibule, while White and co-workers postulate the DNA/PNA helix is postulated to unzip outside of the vestibule.^[] The different proposed mechanisms in these studies highlight that there is not currently a clear consensus in the research field regarding the location of unzipping for non-DNA/DNA helicases.

Kinetics of DNA unzipping

The duration of the current blockage can be exploited to extract kinetic information related to DNA unzipping. As previously mentioned, in 2003, Sauer-budge et al. utilized a single-molecule approach to elucidate the kinetics of DNA strand separation. This was achieved by comparing two synthetic DNA constructs, 100/50com and 100/50mis. A distribution of blockage duration for the two constructures was recorded to propose a kinetic model for DNA unzipping. As shown above in Figure , for 100/50mis, an intricate shape with a mean time of 185 ms was observed, corresponding to a two-step mechanism of reversible unzipping up to 4 bp mismatch before the complete and irreversible unzipping. Whereas for 100/50comp, an exponential distribution with a longer mean time of 435 ms was recorded, inferring that force-inducing strand separation follows a first-order reaction in which the single-stranded overhang must initially enter the pore for unzipping to occur. Furthermore, the enthalpy barrier for each step in the separation of the 100/50mis was calculated, which proved that k₂ < k₁.^[]

Following this, Mathé et al. introduced the study of unzipping kinetics of individual DNA hairpins as a function of voltage ranging from 30 mV to 150 mV. Again, an exponential distribution of times was observed. This voltage-dependent study demonstrated that unzipping duration has an inverse correlation with the applied voltage.^[]

Similarly, Ohara et al. studied the unzipping kinetics of hairpin DNA using a lipid bilayer array.^[] Analysis of this data supports that the voltage-driven unzipping of DNA could be modeled as a first-order dissociation reaction described by the following scheme: 1 $\begin{equation}\left[ {{\rm{h}}pDNA + \alpha HL{\rm{ }}} \right] \xrightarrow{k} \left[ {ssDNA} \right] + \left[ {\alpha HL{\rm{ }}} \right]\end{equation}$Here the rate constant k (s^–1) is the inverse of t and 1/t the reaction frequency per unit of time of the single-molecule reaction.^[]

In an experiment to study the unzipping kinetics of damaged DNA duplexes in the α-HL nanopore, Jin et al. compared the unzipping of analogous duplexes which contained oxidatively damaged guanine product 8-oxo-7,8-dihydroguanine (OG), or its oxidized products guanidinohydantion (Gh) and spiroiminodihydantion (SP) in their target sequence, relative to a control containing an undamaged guanine nucleotide.^[] These studies are of great interest due to the implication of oxidative DNA damage in various diseases, including cancer, Alzheimer's, and cardiovascular diseases. Figure  shows the observed single-exponential decay duration histogram obtained for the undamaged guanine, which is consistent with previous studies, indicating that this process follows first-order kinetics. These histograms can be mathematically represented using the kinetic equation for a first-order reaction as shown in Equation (), where C/T is event count in a time increment, k is the rate constant of the unzipping process, t is unzipping duration, and $$\Delta t$$ is time, t divided by the total counts.^[]

The histograms of unzipping duration for Gh:C and Sp:C exhibited non-trivial exponential distributions, implying that these oxidatively weakened duplexes follow a different kinetic model. The researchers propose that this is due to the duplexes’ inability to form stable hydrogen bonds with cytosine, resulting in decreased stability. Their histograms were consistent with a multistep model occurring from the presence of an intermediate and the unzipping of two consecutive first-order reactions, according to Equation ().^[] 2 $\begin{equation}\frac{C}{T} = \;\frac{{{k_1}{k_2}}}{{{k_2} - {k_1}}}\left( {{e^{ - {k_1}t}} - {e^{ - {k_2}t}}} \right)\Delta t\end{equation}$In this equation, k₁ and k₂ represent the rate constants for the two sequential steps of the unzipping process respectively.

Meng et al. have utilized unzipping in α-HL to determine the effects that an isomer of a DNA modification has on structural stability.^[] Azobenzene, as a photosensitive molecule, presents an opportunity to be utilized as a photo-regulator in DNA functions and nanomachines. However, it is difficult to quantify the individual contribution of the different cis and trans isomers to the overall stability of the duplex. A melting curve cannot be used because, as an ensemble measurement, it does not differentiate the individual contributions of the two isomers.^[] Using the α-HL nanopore, the kinetics of the unzipping process of a DNA duplex modified with azobenzene was monitored, and trans isomer modified duplexes were found to have a longer residence time. After comparison with the native duplex, it was found that the cis isomer, which is present after exposure to UV light, causes instability in the duplex and has a shorter unzipping time than both the native and trans modified duplexes.

The effect of temperature and electrolyte composition on DNA unzipping

The mechanism and kinetics of DNA unzipping are governed by the environment that the DNA experiences. Johnson et al. explored the influence of temperature on blockage current and DNA unzipping by studying the unzipping processes of two duplexes: one with a furan group at the latch region of α-HL during unzipping and one with the corresponding base.^[] At an applied potential of 120 mV, the current-time traces for both duplexes were monitored as a function of temperature (12–35°C). As shown in Figure , the current of the open pore and the unzipping events increased when the temperature was raised from 12 to 35°C. However, the disparity initially observed due to the missing base at the latch zone decreased with increasing temperature. The increase in current was coupled with a high signal/noise ratio, thus reducing the accuracy of the determined blockage current. Lower temperatures also displayed the most significant difference in current for the furan-present duplex and its reference, making it easier to distinguish between the two duplexes. This is an important finding that should not be understated; however, this advantage is counterbalanced by the fact that as temperature decreases, the time taken for DNA unzipping increases exponentially.^[]

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The effect of electrolyte concentration on DNA unzipping through the α-HL nanopore was also investigated. A series of unzipping experiments were carried out at 120 mV using duplexes containing a furan in the latch position and one further away from the latch area, in KCl solutions ranging from 0.15 to 1 M, to determine the relationship between electrolyte concentration and dsDNA unzipping. Up to ∼0.3 M, there was a linear relationship between measured current during unzipping and electrolyte concentration, which was followed by a plateau region, suggesting that ions inside the pore which carry current during an unzipping event are weakly dependent on the external electrolyte concentration between 0.3 and 1 M (Figure ). From this study, Johnson et al. hypothesized that an electrolyte concentration of less than 0.15 M will result in decreased current towards a limiting value based on counterions associated with fixed charges on the DNA duplex wall.^[] Furthermore, this study highlights that it is unnecessary to utilize high supporting electrolyte concentrations to achieve maximum resolution of current blockades events for differing DNA structures. Indeed, utilization of high electrolyte concentrations in many cases may be disadvantageous due to the stabilization of secondary structures that are readily captured by the pore and can complicate and/or interfere with data analysis and interpretation.

In addition to electrolyte concentration, the electrolyte composition (specifically, the type of cation used) has also been found to play an important role in both the ability to discriminate between blocking events of different current magnitudes and the timescale of the unzipping process.^[] In a follow-up study to that above, Johnson and co-workers showed that a DNA duplex with a single missing base, relative to the wild-type control, showed the maximum difference in the blocking current magnitude when rubidium chloride was utilized for the supporting electrolyte, and minimum difference when lithium chloride was used as the supporting electrolyte. Broadly, the capacity to discriminate a duplex containing the missing base improves with electrolyte conductivity. In a similar trend, unzipping times were found to be shorter for higher conductivity electrolytes (e.g., RbCl), and substantially longer for lower conductivity electrolytes (e.g., LiCl)

The noise characteristics of DNA unzipping

Current-carrying ions produce a signal and noise for every single blockage current event. Noise is impacted by factors such as temperature, electrolyte concentration, and applied voltage.^[] Therefore, the signal-to-noise ratio is optimized by controlling these factors, and the presence of excessive noise can limit a nanopore's sensitivity and reliability.^[] However, noise analysis can also be useful. Noise analysis of DNA capture events in α-HL has been successfully used to gain insights into the structure of captured DNA and found to reflect the stability of a DNA duplex to localized and short time scale conformational changes such as DNA breathing.^[]

Johnson et al. demonstrated that the noise observed during an ion-channel recording differs in the presence of a missing base. The melting temperature (T_m) of the DNA duplexes with the base and without the base was calculated and related to the stability of the polynucleotide. The results indicated that the current measured depends on the duplex's stability; as the T_m of a DNA duplex decreases, greater noise in the current blockade is observed.^[] They also showed that the noise associated with the current measured during unzipping increased with increasing temperature and electrolyte concentration; thus, the optimum conditions for obtaining a high signal-noise ratio are expected at electrolyte concentrations ⩽0.5 M and low-temperature conditions of ∼12°C.^[]

INTERROGATING dsDNA STRUCTURE

Commercial, benchtop sequencing devices using nanopores are now available that claim to be able to sequence entire genomes, detect chromatin conformation, and even determine epigenetic information present within genes.^[,] However, these types of devices are limited to the interrogation of ssDNA structure at the point of measurement. Using the α-HL protein nanopore to capture and hold single molecules of dsDNA for milliseconds up to seconds in duration permits the opportunity to interrogate and identify structural and conformational changes that are unique to DNA in its helical form, such as mispairing base-flipping and crosslinking, that are not otherwise accessible with currently available commercial technologies.

Gu and co-workers have demonstrated multiple novel uses for α-HL nanopores in detecting and characterizing cross-linked DNA.^[] Inter-strand cross-links are covalent bonds formed between two strands of DNA by an exogenous molecule. This can prevent DNA unzipping and cell replication.^[] Previously, detecting cross-links required multiple time-consuming techniques such as nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry, and X-ray crystallography. Over the course of numerous studies, a characteristic “current signature” was determined for various cross-linking events as they pass through the nanopore, allowing the determination of modifications across the DNA duplex. Each cross-link has a specific residence time in the pore as it unzips to allow a single strand to pass through, and this residence time combined with the degree of current that is blocked allows for the specific determination of the type of cross-linking present in the pore. The group expanded upon this method in 2018 in a study that used mechlorethamine to selectively cross-link a probe strand of DNA to a mutant strand of interest.^[] It was found that this cross-link could be identified as it passed through the α-HL nanopore, presenting a potential method for detecting small strands of cancer-driving genes.

Gu et al. have also outlined an approach to detect the presence of mutations in a dsDNA sample by using a nanolock-nanopore system.^[] A “nanolock” refers to metal ions and ligands designed to bind to specific base pairs. These ligand-bound structures hybridize the DNA duplex, leading to longer unzipping times. Normal nanopore analysis of DNA strands tagged with such nanolocks increases the residence time of DNA strands but does not offer a clear enough separation from the wild-type DNA, so the authors examined another unique quality of the tagged nucleotides: a distinctive, two-step unzipping process observable in the blocking current data (Figure ). The nanolock molecule, in this case, Hg²⁺, will exclusively bind to the thymine-thymine mismatch present in the mutant DNA during hybridization with a probe strand. Using this approach, the frequency of cancer mutations in a patient's sample can be quantified, and with false positives significantly reduced, the presence of the mutation of interest can be definitively detected and diagnosed with a binary yes/no result. In a later study, the same group demonstrated that a locked nucleic acid, in which the ribose sugar is prevented from rotating, could be used to achieve a similar level of improved discrimination of a duplex containing a mismatch relative to the wild-type based on the unzipping times.^[]

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By analyzing the unzipping time and blocking current, Liu et al. were able to detect single base mutations in oligonucleotides passing through an α-HL nanopore.^[] Using a complementary probe, it was possible to slow the DNA's passage through the nanopore to the extent that an accurate base by base read could be achieved. The probe was separated from the strand of interest while passing through the nanopore, allowing the segments of interest to be examined. Using this, small mismatches in the code could be detected. This offers a distinct advantage over the nanolock methodology presented previously, as not only is the presence of the mutation detectable, but the specific location on the strand can be determined. Figure  depicts how mutations at varying positions on a DNA strand can be distinguished by their characteristic unzipping times. Figure  gives rationale to the P3T probes design, explaining that a poly-A tail on the 3′ end of the strand increases both the unzipping time and the blocking ratio. Figure  shows the results of this extended residence time as the multiple point mutations shown in Figure  are differentiated from each other based on their residence time and blocking ratio. This technique is proposed as an alternative to liquid biopsy for detecting circulating tumor DNA in the bloodstream, often indicative of conditions such as lung cancer.^[]

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White and co-workers have demonstrated that it is possible to detect single base mismatches from helical DNA structures held inside the hemolysin vestibule.^[] By examining modulation in the blocking current at the millisecond timescale, it was possible to distinguish different types of single-base mismatch (e.g. C:C, C:A) from wildtype (C:G). Mismatched nucleotides have been found to rotate to an exposed, extrahelical position more often than the typical Watson-Crick base pairs.^[] The measurement of these base-flipping events has previously been quite difficult, requiring either NMR or single-molecule fluorescence.^[] Previous work by this group has shown that it is possible to measure changes in the residual current when there are structural changes to DNA bases situated at the latch constriction of the hemolysin pore during residence in the vestibule immediately prior to unzipping.^[] They report that C:C and C:A mismatches leave distinct, two-state modulating current signals that are distinguishable both from a control duplex (i.e., a wild type without a mismatch) and from each other. It is hypothesized that the two-state signature arises due to interactions between the extrahelical nucleotide and amino acid residues present in the latch site of the α-HL vestibule.

Using a similar approach to that outlined above, Johnson et al. have demonstrated the detection of epigenetic modifications held at the α-HL latch constriction.^[] An epigenetic modification was introduced to one of the cytosines in the mismatched pair, leaving cytosine paired with either methylcytosine (mC), hydroxymethylcytosine (hmC), formylcytosine (fmC), or carboxycytosine (caC). Each epigenetic modification was found to have a characteristic signal while confined in the pore, which could be used to easily differentiate between the bases. Figure  depicts how these signals are sufficiently distinct and that it is possible in most cases to differentiate the type of epigenetic base from just a single capture event.

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Jin et al. have demonstrated that DNA damage can be detected through differences in current blockade signatures when a DNA damage site is situated at the latch constriction of α-HL.^[] Single-nucleotide recognition was achieved between uracil-containing duplexes and abasic sites containing duplexes. The presence of an abasic site in a duplex residing within the α-HL vestibule, when situated such that it is aligned to the latch constriction of α-HL, was found to produce blockade events of the lower current magnitude. The result is identical to that outlined earlier in this review for furan, which is commonly used as a synthetic mimic for an abasic site. This enabled the authors to record the conversion of uracil duplexes into abasic duplexes in a solution containing UDG as DNA passed through the nanopore. This same principle was applied in 2016 when the kinetics of T3-DNA ligase-catalyzed phosphodiester bond formation was observed in the latch region.^[] Nicks in the phosphodiester backbone of DNA were detected and differentiated from DNA strands that had been repaired using the change in blockade currents observed between the two states.

DNA damage prior to repair by enzymatic mechanisms can also be elucidated from dsDNA confined in the α-HL vestibule. Spiroiminodihydantoin (SP) is a chiral form of guanine that results from a reaction with a reactive oxygen species. When monitored in the latch zone, a blockade current difference of ∼0.8 pA can be achieved between the (–R)-SP:G and (+-S)-SP:G forms.^[] Duplexes containing (S)-SP consistently gave larger blockage amplitudes and produced more blockade noise, relative to DNA structures without damage.

CONCLUSIONS AND FUTURE OUTLOOK

The studies described above demonstrate the efficacy of α-HL as a protein nanopore, in which DNA unzipping and translocation can be recorded by measuring ion-current as a function of time in the presence of a voltage bias. The process of unzipping in such a constricted space produces unique and rich information that can be used to interrogate DNA structure and has the potential to provide insights into important biophysical processes as well as a platform for DNA-based diagnosis of disease. Analysis of information such as blockage current and duration can then be used to extract information about the DNA duplex such as the length, composition, and stability. Researchers across many groups have studied the mechanism of dsDNA unzipping. From the works reviewed here, the mechanism of unzipping appears to vary depending on the structure of the DNA complex, as structural variations between complexes result in differences in the capturing, unzipping, and translocation processes. The timescales of unzipping, and possibly the mechanisms, are also dependent on experimental conditions such as temperature and electrolyte composition.

Optimal sensitivity and resolution for DNA duplexes of differing compositions held within the α-HL pore are influenced by factors such as electrolyte concentration, composition, and temperature. Optimum conditions for obtaining a high signal-noise ratio are anticipated to be at electrolyte concentrations ⩽0.5 M and low-temperature conditions, while the capacity to discriminate between current blockade events that relate to DNA duplexes of differing structures is greatest when rubidium chloride is used as a supporting electrolyte. This highlights how the experimental conditions that have traditionally been used for the analysis of ssDNA with α-HL, that automatically have been applied to studies with dsDNA, may not be optimal. Moving forward, further investigations are therefore required into how experimental parameters affect the interactions of dsDNA with α-HL, including unzipping mechanisms. In addition, many reports for detecting structural changes in DNA rely on utilizing the 2.6 nm diameter latch constriction, situated near the center of the α-HL vestibule. The discovery of other sensing zones that are also specific to dsDNA, and/or the generation of such through mutagenesis is a potential area of future exploration in the field that could improve the capacity for dsDNA analysis further.

From an application viewpoint, while there is now a good volume of work in the field concerning dsDNA analysis, the breadth, and depth of applications when compared to those studies that describe the analysis of ssDNA (typically translocation studies) is lagging. Much of the work to date has focused on the structural changes in short synthetic DNA duplexes that comprise sequences from human genomes such as KRAS. No doubt the expansion of this work to investigate structural and/or conformational changes in oligonucleotide sequences distinct to other organisms, such as bacteria and double-stranded RNA viruses, where structural changes such as mismatches, and repair pathways and dominant epigenetic markers are often different, will no doubt yield interesting results.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

Not applicable (Mini Review).

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