Detection of 5-methylcytosine and 5-hydroxymethylcytosine in DNA via host–guest interactions inside α-hemolysin nanopores

Cytosine methylation and hydroxymethylation are both important epigenetic modifications of DNA in mammalian cells. Therefore, profiling DNA (hydroxy)methylation across the genome is vital for understanding their roles in gene regulation. Here, we report a nanopore-based approach for quick and reliable detection of 5-methylcytosine and 5-hydroxymethylcytosine in DNA at the single-molecule level. The single-stranded DNA containing 5-methylcytosine or 5-hydroxymethylcytosine was first selectively modified on the epigenetic base to attach a host–guest complex. Threading of the modified DNA molecules through α-hemolysin nanopores causes unbinding of the host–guest complex and generates highly characteristic current signatures. Statistical analysis of the signature events affords quantitative information about 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Our results suggest that other DNA modifications could also be detected with the developed method. Furthermore, we anticipate our nanopore sensing strategy to be generally useful in biochemical analysis and to find applications in the early diagnosis of diseases.     

Processive replication of single DNA molecules in a nanopore catalyzed by phi29 DNA polymerase

Coupling nucleic acid processing enzymes to nanoscale pores allows controlled movement of individual DNA or RNA strands that is reported as an ionic current/time series. Hundreds of individual enzyme complexes can be examined in single-file order at high bandwidth and spatial resolution. The bacteriophage phi29 DNA polymerase (phi29 DNAP) is an attractive candidate for this technology, due to its remarkable processivity and high affinity for DNA substrates. Here we show that phi29 DNAP-DNA complexes are stable when captured in an electric field across the α-hemolysin nanopore. DNA substrates were activated for replication at the nanopore orifice by exploiting the 3'-5' exonuclease activity of wild-type phi29 DNAP to excise a 3'-H terminal residue, yielding a primer strand 3'-OH. In the presence of deoxynucleoside triphosphates, DNA synthesis was initiated, allowing real-time detection of numerous sequential nucleotide additions that was limited only by DNA template length. Translocation of phi29 DNAP along DNA substrates was observed in real time at ?ngstrom-scale precision as the template strand was drawn through the nanopore lumen during replication.     

Spatial blockage of ionic current for electrophoretic translocation of DNA through a graphene nanopore

Graphene nanopore has been promising the ultra‐high resolution for DNA sequencing due to the atomic thickness and excellent electronic properties of the graphene monolayer. The dynamical translocation phenomena and/or behaviors underneath the blocked ionic current, however, have not been well unveiled to date for the translocation of DNA electrophoretically through a graphene nanopore. In this report, the assessment on the sensitivity of ionic current to instantaneous statuses of DNA in a 2.4 nm graphene nanopore was carried out based on the all‐atom molecular dynamics simulations. By filtering out the thermal noise of ionic current, the instantaneous conformational variations of DNA in a graphene nanopore have been unveiled from the fluctuations of ionic current, because of the spatial blockage effect of DNA against ionic current. Interestingly, the neighborhood effect of DNA against ionic current was also observed within a distance of 1.5 nm nearby the graphene nanopore, suggesting the further precise control for DNA translocation through a graphene nanopore in gene sequencing. Moreover, the sensitivity of the blocked ionic current toward the instantaneous conformations of DNA in a graphene nanopore demonstrates the great potential of graphene nanopores in the dynamics analysis of single molecules.     

生物大分子纳米孔分析技术研究进展

脱氧核糖核酸穿越纳米孔动力学研究以及利用纳米孔开展新型DNA测序技术研究是后人类基因组计划的热点之一。本文对生物纳米孔、固态纳米孔以及纳米孔生物大分子识别技术的研究现状进行了归纳和总结,并对该领域的发展趋势进行展望。     

Editorial Board: Electrophoresis 8'14

Electrophoresis 2014, 35, 1144–1151. DOI: 10.1002/elps.201300501 The center stage of nanopore sequencing is to extract gene information from the translocation of DNA through a nanopore. Graphene nanopore technology has been promising ultra‐high resolution for gene sequencing owing to the atomic thickness and excellent electronic properties of the graphene monolayer. By filtering out the thermal noise of ionic current, the instantaneous conformational variations of DNA in a graphene nanopore could be unveiled from undulates of the blocked ionic current, because of the spatial blockage effect of DNA against ionic migration. It supplies a theoretical basis for the monitor of dynamical information of DNA in a graphene nanopore during sequencing from the ionic current fluctuation.

     

Gating of single synthetic nanopores by proton-driven DNA molecular motors

Switchable ion channels that are made of membrane proteins play different roles in cellular circuits. Since gating nanopore channels made of proteins can only work in the environment of lipid membrane, they are not fully compatible to the application requirement as a component of those nanodevice systems in which lipid membranes are hard to establish. Here we report a synthetic nanopore-DNA system where single solid-state conical nanopores can be reversibly gated by switching DNA motors immobilized inside the nanopores. High- (on-state) and low- (off-state) conductance states were found within this nanopore-DNA system corresponding to the single-stranded and i-motif structures of the attached DNA motors. The highest gating efficiency indicated as current ratio of on-state versus off-state was found when the length of the attached DNA molecule matched the tip diameter of the nanopore well. This novel nanopore-DNA system, which was gated by collective folding of structured DNA molecules responding to the external stimulus, provided an artificial counterpart of switchable protein-made nanopore channels. The concept of this DNA motor-driven nanopore switch can be used to build novel, biologically inspired nanopore machines with more precisely controlled functions in the near future by replacing the DNA molecules with other functional biomolecules, such as polypeptides or protein enzymes.     

Resistive-pulse DNA detection with a conical nanopore sensor

In this paper, we describe resistive-pulse sensing of two large DNAs, a single-stranded phage DNA (7250 bases) and a double-stranded plasmid DNA (6600 base pairs), using a conically shaped nanopore in a track-etched polycarbonate membrane as the sensing element. The conically shaped nanopore had a small-diameter (tip) opening of 40 nm and a large-diameter (base) opening of 1.5 microm. The DNAs were detected using the resistive-pulse, sometimes called stochastic sensing, method. This entails applying a transmembrane potential difference and monitoring the resulting ion current flowing through the nanopore. The phage DNA was driven electrophoretically through the nanopore (from tip to base), and these translocation events were observed as transient blocks in the ion current. We found that the frequency of these current-block events scales linearly with the concentration of the DNA and with the magnitude of the applied transmembrane potential. Increasing the applied transmembrane potential also led to a decrease in the duration of the current-block events. We also analyzed current-block events for the double-stranded plasmid DNA. However, because this DNA is too large to enter the tip opening of the nanopore, it could not translocate the pore. As a result, much shorter duration current-block events were observed, which we postulate are associated with bumping of the double-stranded DNA against the tip opening.     

Investigating the translocation of λ-DNA molecules through PDMS nanopores

We investigate the translocation of λ-DNA molecules through resistive-pulse polydimethylsiloxane (PDMS) nanopore sensors. Single molecules of λ-DNA were detected as a transient current increase due to the effect of DNA charge on ionic current through the pore. DNA translocation was found to deviate from a Poisson process when the interval between translocations was comparable to the duration of translocation events, suggesting that translocation was impeded during the presence of another translocating molecule in the nanopore. Characterization of translocation at different voltage biases revealed that a critical voltage was necessary to drive DNA molecules through the nanopore. Above this critical voltage, frequency of translocation events was directly proportional to DNA concentration and voltage bias, suggesting that transport of DNA from the solution to the nanopore was the rate limiting step. These observations are consistent with experimental results on transport of DNA through nanopores and nanoslits and the theory of hydrodynamically driven polymer flow in pores.     

Advances in DNA Origami Nanopores: Fabrication,Characterization and Applications

In recent years, bio‐nanopore and solid‐state nanopore have been greatly improved for molecule bio‐sensing. Whereas, the development of this scientific field seems to have encountered a bottleneck due to their respective limitations. The small pore size of the former impedes the detection of large single molecule, and the latter is difficult to achieve similar accuracy and functional control. DNA origami plays a novel role to bring more opportunities for the development of nanopore technology since it is relatively easy to synthesize and modify. This review mainly focuses on introducing the DNA origami nanopore fabrication methods, characterization and application. Meanwhile, the challenges in the present DNA origami nanopore research are also discussed.     

Graphene nanopores toward DNA sequencing: a review of experimental aspects

Nanopores for DNA sequencing have drawn much attention due to their potentials to achieve amplification-free, low-cost, and high-throughput analysis of nuclei acids. The material configuration and fabrication of the nanopore has become one important consideration in the nanopore based DNA sequencing research. Among various materials, the newly emerged graphene has brought more opportunities to the development of sequencing technology because of its unique structures and properties. This review mainly focuses on the experimental aspects of graphene nanopore research including the nanopore fabrication methods and processes. Meanwhile, the challenges in the present graphene nanopore research including hydrophobicity, translocation velocity and noise are also addressed and discussed.     

Detection of structured single‐strand DNA via solid‐state nanopore

Nanopore is a single‐molecule analysis method which also employed electrophoresis has achieved promising single‐molecule detections. In this study, we designed two kinds of confined spaces by fabricating solid‐state nanopores with desirable diameters to study the structured single‐strand DNA of C‐rich quadruplex. For the nanopore whose diameter is larger than the quadruplex size, the DNA molecule could directly translocate through the nanopore with extremely high speed. For the nanopore whose diameter is smaller than the quadruplex size, DNA molecule which is captured by nanopore could return to the solution without translocation or unzip the quadruplex structure into single‐strand and then pass the nanopore. This study certifies that choosing a suitable sensing interface is the vital importance of observing detailed single‐molecule information. The solid‐state nanopores hold the great potential to study the structural dynamics of quadruplex DNA molecule.     

Reversing current rectification to improve DNA‐sensing sensitivity in conical nanopores

Herein, we report the ultrasensitive DNA detection through designing an elegant nanopore biosensor as the first case to realize the reversal of current rectification direction for sensing. Attributed to the unique asymmetric structure, the glass conical nanopore exhibits the sensitive response to the surface charge, which can be facilely monitored by ion current rectification curves. In our design, an enzymatic cleavage reaction was employed to alter the surface charge of the nanopore for DNA sensing. The measured ion current rectification was strongly responsive to DNA concentrations, even reaching to the reversed status from the negative ratio (?6.5) to the positive ratio (+16.1). The detectable concentration for DNA was as low as 0.1 fM. This is an ultrasensitive and label‐free DNA sensing approach, based on the rectification direction‐reversed amplification in a single glass conical nanopore.     

DNA‐Based Nanopore Sensing

Nanopore sensing is an attractive, label‐free approach that can measure single molecules. Although initially proposed for rapid and low‐cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing. In this Minireview, we highlight the recent research efforts of nanopore sensing based on DNA‐mediated characteristic current events. As nanopore sensing is becoming increasingly important in biochemical and biophysical studies, DNA‐based sensing may find wider applications in investigating DNA‐involving biological processes.     

DNA conformation and base number simultaneously determined in a nanopore

When dsDNA polymers containing identical number of base pairs were electrophoresed through a nanopore in a voltage biased silicon nitride membrane, the measured time integral of blocked ionic current (the event-charge-deficit, ecd, Fologea, D., Gershow, M., Ledden, B., McNabb, D. S. et al.., Nano Lett. 2005, 5, 1905-1909) for each translocation event was the same regardless of whether the molecules were in a linear, circular relaxed, or supercoiled form. Conversely, when DNA polymers containing different numbers of base pairs were electrophoresed through a nanopore, the ecd depended strongly on, and predicted the value of, the molecule's number of base pairs. Measurements showed that the magnitude of the current blockages was strongly affected by a molecule's form. The current blockages exhibited characteristic differences that distinguished among single-stranded linear, double-stranded linear, circular relaxed, and supercoiled forms. Because the data that establish ecd are usually determined concomitantly with current blockade measurements, our results show that a single nanopore assay can simultaneously determine both DNA conformation and base number.     

纳米孔道单分子电化学测量的低噪音控温系统研究

纳米孔道检测技术是一种利用单个分子测量界面实现在单分子水平上测量DNA、RNA、蛋白、多肽等生物分子的高灵敏的单分子检测技术. 由于单个分子与纳米孔道的相互作用受热力学控制,亟需精准控制纳米孔道单分子分析的实验温度. 因此,本文研制了一种低噪音控温系统用于具有皮安级电流分辨的纳米孔道单分子实验,以实现精确调控测量时的环境温度. 该系统利用半导体制冷片的热电效应对检测池环境加热/制冷,通过对高精度热敏电阻进行电磁屏蔽以实现在温度反馈的同时避免噪音的引入. 利用比例-积分-微分算法进行控制,达到高精度快速控温的要求. 该系统控温精度为±1 °C,无额外噪音引入至超灵敏纳米孔道单分子测量,获得了25 °C到5 °C下Poly(dA)5与单个气单胞菌溶素（Aerolysin）分子界面间作用产生信号的差异,应用于研究单分子与纳米孔道相互作用的热力学行为.     

Electrokinetic particle translocation through a nanopore containing a floating electrode

Electrokinetic particle translocation through a nanopore containing a floating electrode is investigated by solving a continuum model, composed of the coupled Poisson-Nernst-Planck (PNP) equations for the ionic mass transport and the modified Stokes equations for the flow field. Two effects due to the presence of the floating electrode, the induced-charge electroosmosis (ICEO) and the particle-floating electrode electrostatic interaction, could significantly affect the electrokinetic mobility of DNA nanoparticles. When the electrical double layers (EDLs) of the DNA nanoparticle and the floating electrode are not overlapped, the particle-floating electrode electrostatic interaction becomes negligible. As a result, the DNA nanoparticle could be trapped near the floating electrode arising from the induced-charge electroosmosis when the applied electric field is relatively high. The presence of the floating electrode attracts more ions inside the nanopore resulting in an increase in the ionic current flowing through the nanopore; however, it has a limited effect on the deviation of the current from its base current when the particle is far from the pore.     

Discrimination of single‐stranded DNA homopolymers by sieving out G‐quadruplex using tiny solid‐state nanopores

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single‐base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G‐quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20‐nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105‐nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore‐based DNA sequencing at single‐base level.     

Increased dwell time and occurrence of dsDNA translocation events through solid state nanopores by LiCl concentration gradients

Solid‐state nanopore based biosensors are cost effective, high‐throughput engines for single molecule detection of biomolecules with the added benefit of size modification. Progress in the translation of the science into a viable diagnostic tool is impeded by inadequate sensitivity of data acquisition systems in detection of fast DNA translocations through the pore. To combat this, slowing the transport of DNA through the nanopore by use of various media or by altering experimental parameters is common. Applying a concentration gradient of KCl in the experimental ionic solution has been shown to effectively prolong dwell times as well as increase the capture rate of DNA by the nanopore. Our previous work has corroborated the ability of LiCl ionic solution to slow down the transport of dsDNA through the nanopore by up to 10‐fold through cation‐DNA interactions. However, this drastically reduced the event occurrence frequency, thus hindering the efficacy of this system as a reliable biosensor downstream. Here, we present the use of a concentration gradient of lithium chloride ionic solution to increase the event frequency of single molecule dsDNA translocation through a solid state nanopore. By using 0.5 M/3 M LiCl on the cis/trans chambers respectively, average dwell times experienced up to a 3‐fold increase when compared to experiments run in symmetric 1 M LiCl. Additionally, experiments using the 0.5 M/3 M displayed a greater than 10‐fold increase in event frequency, confirming the capture propensity of the asymmetric conditions.     

生物纳米孔分析技术研究进展

生物纳米孔传感技术因其快速、低成本、无需荧光标记等优点,在化学和生物等诸多研究领域得到广泛应用,已发展成为一种新颖的、独具特色的单分子分析手段。该技术目前主要应用于DNA测序研究,同时在单分子分析领域也取得了令人瞩目的成就。该文简要介绍了生物纳米孔分析技术的原理和生物孔的种类,主要总结了近20年来生物纳米孔在DNA测序和单分子分析中的研究进展并予以了展望。     

单分子电泳:纳米孔道电化学的新认识

该文旨在从电泳分离技术的角度认识纳米孔道电化学单分子分析技术,这种技术可以作为"单分子电泳"来理解和研究。纳米孔道电化学单分子分析技术与电泳的本质都是采用外加电场使待测分子产生电迁移。待测分子性质不同,且与介质材料孔道外露基团相互作用不同,使得分子移动速度具有差异,据此实现分离识别。气单胞菌溶素（Aerolysin）纳米孔道,由于其孔径与待测分子尺寸相匹配,其孔道内壁可以看作是由氨基酸组成的具有调控单个分子电迁移能力的特异性孔道界面。每一个氨基酸残基都相当于一个探测单元,在电场力的作用下,待测分子逐一进入孔道时与每一个探测单元相互作用方式、程度与时长不同,从而形成了单个待测分子特征的迁移速度和迁移运动轨迹。在纳米孔道实验中,每秒可以有上千个待测分子穿过孔道,产生特征阻断电流信号。通过对这些信号的阻断电流、阻断时间、阻断频率、信号特征等进行统计分析,可以从"单分子电泳"水平对单个待测物实现高通量的分辨和识别。该文以Aerolysin纳米孔道分辨仅有一个核苷酸差异的寡聚核苷酸（5'-CAA-3'、5'-CAAA-3'、5'-CAAAA-3'）为例,详细阐述了纳米孔道"单分子电泳"的单核苷酸分辨能力,展现了电化学限域空间在电泳单分子水平分离技术上的应用。