Controlled Genetic Encoding of Unnatural Amino Acids in a Protein Nanopore

Conventional protein engineering methods for modifying protein nanopores are typically limited to 20 natural amino acids, which restrict the diversity of the nanopores in structure and function. To enrich the chemical environment inside the nanopore, we employed the genetic code expansion (GCE) technique to site-specifically incorporate the unnatural amino acid (UAA) into the sensing region of aerolysin nanopores. This approach leveraged the efficient pyrrolysine-based aminoacyl-tRNA synthetase-tRNA pair for a high yield of pore-forming protein. Both molecular dynamics (MD) simulations and single-molecule sensing experiments demonstrated that the conformation of UAA residues provided a favorable geometric orientation for the interactions of target molecules and the pore. This rationally designed chemical environment enabled the direct discrimination of multiple peptides containing hydrophobic amino acids. Our work provides a new framework for endowing nanopores with unique sensing properties that are difficult to achieve using classical protein engineering approaches.     

Rapid and multiplex preparation of engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing

Acknowledging its unique conical lumen structure, Mycobacterium smegmatis porin A (MspA) was the first type of nanopore that has successfully sequenced DNA. Recent developments of nanopore single molecule chemistry have also suggested MspA to be an optimum single molecule reactor. However, further investigations with this approach require heavy mutagenesis which is labor intensive and requires high end instruments for purifications. We here demonstrate an efficient and economic protocol which performs rapid and multiplex preparation of a variety of MspA mutants. The prepared MspA mutants were demonstrated in operations such as nanopore insertion, sequencing, optical single channel recording (oSCR), nanopore single molecule chemistry and nanopore rectification. The performance is no different from that of pores however prepared by other means. The time of all human operations and the cost for a single batch of preparation have been minimized to 40 min and 0.4$, respectively. This method is extremely useful in the screening of new MspA mutants, which has an urgent requirement in further investigations of new MspA nanoreactors. Its low cost and simplicity also enable efficient preparations of MspA nanopores for both industrial manufacturing and academic research.

A rapid and multiplex approach to prepare engineered Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing and sequencing.     

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized as the main gating loop that closes the pore at low pH values and opens it at high pH values. A method was devised to pin each of the loops to the embedding membrane and measure the single‐pore conductances of the resulting constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighboring and distant loops in a correlated fashion. Pinning loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.     

Single molecule observation of hard–soft-acid–base (HSAB) interaction in engineered Mycobacterium smegmatis porin A (MspA) nanopores

In the formation of coordination interactions between metal ions and amino acids in natural metalloproteins, the bound metal ion is critical either for the stabilization of the protein structure or as an enzyme co-factor. Though extremely small in size, metal ions, when bound to the restricted environment of an engineered biological nanopore, result in detectable perturbations during single channel recordings. All reported work of this kind was performed with engineered α-hemolysin nanopores and the observed events appear to be extremely small in amplitude (∼1–3 pA). We speculate that the cylindrical pore restriction of α-hemolysin may not be optimal for probing extremely small analytes. Mycobacterium smegmatis porin A (MspA), a conical shaped nanopore, was engineered to interact with Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺ and Cd²⁺ and a systematically larger event amplitude (up to 10 pA) was observed. The measured rate constant suggests that the coordination of a single ion with an amino acid follows hard–soft-acid–base theory, which has never been systematically validated in the case of a single molecule. By adjusting the measurement pH from 6.8 to 8.0, the duration of a single ion binding event could be modified with a ∼46-fold time extension. The phenomena reported suggest MspA to be a superior engineering template for probing a variety of extremely small analytes, such as monatomic and polyatomic ions, small molecules or chemical intermediates, and the principle of hard–soft-acid–base interaction may be instructive in the pore design.

The principle of hard–soft-acid–base (HSAB) theory was first validated in single molecule by measurements with engineered Mycobacterium smegmatis porin A (MspA) nanopore reactors.     

Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by a helicase motor for peptide sequencing application

The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone''s size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies.

A new technique for single molecular peptide sequencing is demonstrated by translocation of ssDNA-conjugated-peptide through MspA nanopore which is regulated by a DNA helicase motor.     

基于仿生膜的功能化单纳米通道在分析化学中的应用

灵感来源于蛋白质离子通道的仿生功能化单纳米通道,已逐渐成为一种成熟的单分子检测技术和离子整流器。功能化纳米通道包括两种:基因改造的蛋白质纳米通道和固体加工的纳米通道。常用的固体纳米通道有三种:在纳米氮化硅或石墨烯上用聚焦离子束(FIB)或电子束(FEB)轰击得到的纳米通道,化学腐蚀聚合物薄膜中的重金属离子轨迹得到的锥形纳米通道和拉制毛细管或激光刻蚀得到的玻璃纳米孔。相对于蛋白质纳米通道,固态的人工纳米通道具有更优越的机械稳定性,并可用于各种功能基团的修饰。经过近十年的发展,包括蛋白质纳米通道在内的各种仿生的纳米通道已广泛用于对小分子、蛋白质和聚合物等其他一些对象的定性和定量检测。本综述详细介绍了近年来国内外该领域的发展,并对未来的发展方向作了简要的展望。     

Electrochemical Quantitative Evaluation of the Surface Charge of a Poly(1-Vinylimidazole) Multilayer Film and Application to Nanopore pH Sensor

Nanopore pH sensing is based on the interaction between the surface charge of the nanopore and ions passing through the nanopore. The nanopore surface charge can be derived from the acid-base dissociation equilibrium of the modified polyelectrolyte. Various polyelectrolytes have been selected based on the acid dissociation constant of the monomer units, and various techniques have been applied to modify nanopores. However, they have been developed without clear guidelines for characterizing the surface modification status or surface charge. One reason has been the difficulty in accurately estimating the surface charge of nanopores in solution. Thus, in this study, the dissociation constant (pKa_app) of the surface charge of a modified polyelectrolyte nanopore was quantitatively estimated via electrochemical measurements. Previously, the modification status of nanopores has been evaluated using the ion current response. In addition, we monitored in real-time the polyelectrolyte modification status using a quartz crystal microbalance (QCM). Some polyelectrolytes were difficult to immobilize directly on the nanopore surface, and those polymers could be effectively modified by the layer-by-layer (LbL) technique. Therefore, we produced a guideline for the fabrication of a nanopore sensor for pH measurements under physiological conditions by quantitative evaluation of the pKa_app via electrochemical methods, the monitoring of the modification status by QCM, and the development of an effective modification method via the LbL technique.     

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.     

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.     

Lipid bilayer membrane technologies: A review on single-molecule studies of DNA sequencing by using membrane nanopores

Nanopores based on α-hemolysin and MspA represent attractive sensing platforms due to easy production and operation with relatively low background noise. Such characteristics make them highly favorable for sequencing nucleic acids. Artificial lipid bilayer membranes, also referred to as black lipid membranes, in conjunction with membrane nanopores, can be applied to both the detection and highly efficient sequencing of DNA on a single-molecule level. However, the inherently weak physical properties of the membrane have impeded progress in these areas. Current issues impeding the ultimate recognition of the artificial lipid bilayer as a viable platform for detection and sequencing of DNA include membrane stability, lifespan, and automation. This review (with 105 references) highlights attempts to improve the attributes of the artificial lipid bilayer membrane starting with an overview on the present state and limitations. The first main section covers lipid bilayer membranes (BLM) in general. The following section reviews the various kinds of lipid bilayer membrane platforms with subsections on polymer membranes, solid-supported membranes, hydrogel-encapsulated membranes, shippable and storable membrane platforms, and droplet interface bilayers. A further section covers engineered biological nanopore sensor applications using BLMs with subsections offering a comparative view of different DNA sequencing methods, a detailed look at DNA Sequencing by synthesis using alpha-hemolysin nanopores, sequencing by synthesis using the MspA nanopore and quadromer map, and on limitations of sequencing based on synthesis technology. We present an outlook at the end that discusses current research trends on single-molecule sequencing to highlight the significance of this technology and its potential in the medical and environmental fields.

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Graphical abstract Sequencing by Synthesis, a novel method of sequencing DNA, uses the αHL biological nanopore and the artificial lipid bilayer membrane platform. Polymer tags attached to nucleotides bind to the polymerase-primer–template complex and are characterized by the nanopore upon release.

     

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.     

Conductance‐based profiling of nanopores: Accommodating fabrication irregularities

Solid‐state nanopores are nanoscale channels through otherwise impermeable membranes. Single molecules or particles can be passed through electrolyte‐filled nanopores by, e.g. electrophoresis, and then detected through the resulting physical displacement of ions within the nanopore. Nanopore size, shape, and surface chemistry must be carefully controlled, and on extremely challenging <10 nm‐length scales. We previously developed a framework to characterize nanopores from the time‐dependent changes in their conductance as they are being formed through solution‐phase nanofabrication processes with the appeal of ease and accessibility. We revisited this simulation work, confirmed the suitability of the basic conductance equation using the results of time‐dependent experimental conductance measurements during nanopore fabrication by Yanagi et al., and then deliberately relaxed the model constraints to allow for (i) the presence of defects; and (ii) the formation of two small pores instead of one larger one. Our simulations demonstrated that the time‐dependent conductance formalism supports the detection and characterization of defects, as well as the determination of pore number, but with implementation performance depending on the measurement context and results. In some cases, the ability to discriminate numerically between the correct and incorrect nanopore profiles was slight, but with accompanying differences in candidate nanopore dimensions that could yield to post‐fabrication conductance profiling, or be used as convenient uncertainty bounds. Time‐dependent nanopore conductance thus offers insight into nanopore structure and function, even in the presence of fabrication defects.     

Surface-charge induced ion depletion and sample stacking near single nanopores in microfluidic devices

We report integrated nanopore/microchannel devices in which single nanopores are isolated between two microfluidic channels. The devices were formed by sandwiching track-etched conical nanopores in a poly(ethylene terephthalate) membrane between two poly(dimethylsiloxane) microchannels. Integration of the nanopores into microfluidic devices improves mass transport to the nanopore and allows easy coupling of applied potentials. Electrical and optical characterization of these individual nanopores suggests double layer overlap is not required to form an ion depletion region adjacent to the nanopore in the microchannel; rather, excess surface charge in the nanopore contributes to the formation of this ion depletion region. We used fluorescent probes to optically map the ion depletion region and the stacking of fluorescein near the nanopore/microchannel junction, and current measurements confirmed formation of the ion depletion region.     

Investigation of entrance effects on particle electrophoretic behavior near a nanopore for resistive pulse sensing

Resistive pulse sensing using solid-state nanopores provides a unique platform for detecting the structure and concentration of molecules of different types of analytes in an electrolyte solution. The capture of an entity into a nanopore is subject not only to the electrostatic force but also the effect of electroosmotic flow originating from the charged nanopore surface. In this study, we theoretically analyze spherical particle electrophoretic behavior near the entrance of a charged nanopore. By investigating the effects of pore size, particle–pore distance, and salt concentration on particle velocity, we summarize dominant mechanisms governing particle behavior for a range of conditions. In the literature, the Helmholtz–Smoluchowski equation is often adopted to evaluate particle translocation by considering the zeta potential difference between the particle and nanopore surfaces. We point out that, due to the difference of the electric field inside and outside the nanopore and the influence from the existence of the particle itself, the zeta potential of the particle, however, needs to be at least 30% higher than that of the nanopore to allow the particle to enter into the nanopore when its velocity is close to zero. Accordingly, we summarize the effective salt concentrations that enable successful particle capture and detection for different pore sizes, offering direct guidance for nanopore applications.     

Electrostatic-gated transport in chemically modified glass nanopore electrodes

Electrostatic-gated transport in chemically modified glass nanopore electrodes with orifice radii as small as 15 nm is reported. A single conical-shaped nanopore in glass, with a approximately 1 microm radius Pt disk located at the pore base, is prepared by etching the exposed surface of a glass-sealed Pt nanodisk. The electrochemical response of the nanopore electrode corresponds to diffusion of redox-active species through the nanopore orifice to the Pt microdisk. Silanization of the exterior glass surface with Cl(Me)(2)Si(CH(2))(3)CN and the interior pore surface with EtO(Me)(2)Si(CH(2))(3)NH(2) introduces pH-dependent ion selectivity at the pore orifice, a consequence of the electrostatic interactions between the redox ions and protonated surface amines. Nanopore electrodes with very small pore orifice radii (< approximately 50 nm) display anion permselectively at pH < 4, as demonstrated by electrochemical measurement of transport through the pore orifice. Ion selective transport vanishes at pH > 6 or when the pore radius is significantly larger than the Debye screening length, consistent with the observed ion selectivity resulting from electrostatic interactions. The ability to introduce different surface functionalities to the interior and exterior surfaces of glass nanopores is demonstrated using fluorescence microscopy to monitor the localized covalent attachment of 5- (and 6)-carboxytetramethylrhodamine succinimidyl ester to interior pore surfaces previously silanized with EtO(Me)(2)Si(CH(2))(3)NH(2).     

Chemically-modified nanopores for sensing

Sensing with chemically-modified nanopores is an emerging field that is expected to have major impact on bioanalysis and fundamental understanding of nanoscale chemical interactions down to the single-molecule level. The main strength of nanopore sensing is that it implies the prospect of label-free single-molecule detection by taking advantage of the built-in transport-modulation-based amplification mechanism. At present, fabrication and application of solid-state nanopores are becoming the focus of attention because, compared with their biological counterparts, they offer greater flexibility in terms of shape, size, and surface properties, as well as superior robustness. A breakthrough in label-free nanopore sensing for real-world applications is therefore expected from implementing solid-state nanopores, an area that is still developing. Without claiming comprehensiveness, the focus of this review comprises recent results and trends in nanopore-based sensing (i.e. emerging technologies for fabricating solid-state nanopores, their chemical functionalization, and detection methods for quantitative analysis).     

Sensing with ion current rectifying solid-state nanopores

This review gives a current opinion on the state of the art of ion rectifying solid-state nanopore sensors, as well as on the recent directions and challenges of the field, focusing in particular on the progress made in the last two to three years. The review explains the phenomenon of ion current rectification in geometrically asymmetric nanopores and the principle of sensing with these systems. Aside from the conventional approach of analyte immobilization onto the pore surface, some intriguing sensing schemes that decouple the pore selectivity from the surface and promise a more flexible sensing approach are also presented. Lastly, an overview of the recent effort towards amplifying the ion currents and the rectification of these sensors is given, followed by a brief discussion of future perspectives for the field.     

A glass nanopore electrode for single molecule detection

<正>We have developed a simple method for fabricating robust and low noise glass nanopore electrodes with pore size 10±5 nm to detect single molecules.β-Cyclodextrin was used as model compound for characterization.In 1.0 mol/L NaCl solution,the molecules generated current pulses of 2-5 pA with noise level less than 0.8 pA.A slide mode and a plug mode were suggested for the way ofβ-cyclodextrin single molecule moving into the glass nanopores.     

Protein detection by nanopores equipped with aptamers

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.     

A storable encapsulated bilayer chip containing a single protein nanopore

A robust, portable chip containing a single protein nanopore would be a significant development in the practical application of stochastic sensing technology. Here, we describe a chip in which a single alpha-hemolysin (alphaHL) pore in a planar phospholipid bilayer is sandwiched between two layers of agarose gel. These encapsulated nanopore chips remain functional after storage for weeks. The detection of the second messenger inositol 1,4,5-trisphosphate (IP3) was demonstrated with a chip containing a genetically engineered alphaHL pore as the sensor element.