Deterministic stepping of a polymer through a nanopore

Abstract

In a method for controlling translocation of a target polymer molecule (14) through a nanopore (20), a clamp (10) is reversibly bound along a continuous plurality of polymer subunits (12) of the target polymer molecule, and the molecule and clamp are disposed in an ionic solution (30) in fluid communication with the nanopore (20). A constant translocation force (V 驱动 ) is applied across the nanopore to induce the target polymer molecule (14) to travel into the nanopore (20) until the clamp (10) abuts the pore of the nanopore and stops the target polymer molecule from further traveling into the nanopore. A voltage control pulse (V 脉冲 ) and / or a heat control pulse (H 脉冲 ) is then applied across the nanopore, with a pulse duration such that the clamp (10) steps along the target polymer molecule (14) in the opposite direction of travel into the nanopore (20) by no more than one polymer subunit. No fuel is provided to the clamp (10).

Description

[0001] This application is a divisional application of the following application: Application date: June 28, 2018; Application number: 2018800565234 (PCT / US2018 / 040152); Invention title: "Deterministic stepping of polymers through nanopores".

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 526823, filed June 29, 2017, the entire contents of which are incorporated herein by reference.

[0004] Statement on Federally Funded Research

[0005] This invention was made with government support under NIH contract number R01HG003703. The government owns certain rights to this invention. Background Technology

[0006] This invention relates to characterizing polymers by translocating them through nanopores, and more specifically to controlling the stepping of polymers through nanopores.

[0007] Polymers and their subunits can be characterized by measuring changes in the conductivity of nanopores through which the polymer steps. Polymer properties determined using nanopores include concentration (e.g., number of molecules in a sample solution), polymer length, number of monomer units along the polymer length, and chemical and physical properties of the polymer and its monomer units, including specific sequences of continuous monomer units, as described by Deamer et al., Nat. Biotechnol., 34:518-524, 2016; Manrao et al., Nat. Biotechnol., 30:349-353, 2012; Akeson et al., Biophys. J., 77:3227-3233, 1999; and Cherf et al., Nat. Biotechnol., 30:344-348, 2012, all of which are incorporated herein by reference. In one example of a nanopore-based characterization technique, the polymer under investigation is provided in an ionic solution in a cis-reservoir in fluid communication with the nanopore. Polymers translocate from cis-reservoirs to trans-reservoirs through nanopores, during which the translocation of the nanopores can be detected and the properties of the polymer can be determined. While an electric field generated by applying a constant bias voltage between the electrodes of the cis- and trans-reservoirs is commonly used to drive polymers through nanopores, as described, for example, by Kasianowicz et al., Proc. Natl. Acad. Sci., 93: 13770-13773, 1996, the entire contents of which are incorporated herein by reference, other forces, including hydrostatic pressure, optical forces, or magnetic fields, may also be used alternatively to drive polymers through nanopores, as described, for example, by Lu et al., Nano Lett. 13: 3048-3052, 2013; and Keyser et al., Nature Physics, 2: 473-477, 2006; the entire contents of which are incorporated herein by reference.

[0008] Ideas have been proposed to conjugate active enzymes to polymers to slow down or delay the free-moving flow of polymers through nanopores at an undesirable rapid rate, for example, as taught in, for example, by Akeson’s U.S. Patent Nos. 7,625,706, 7,238,485, 7,947,454, 8,673,556; Cherf’s US 2014,005,1068; Moysey’s US 2014,033,5512; and Jayasinghe’s US 2015,003,1020, the entire contents of which are incorporated herein by reference. Such active enzymes, such as polymerases or helicases, depend on energy derived from a chemical substrate, such as adenosine triphosphate (ATP), to travel along the polymer and, accordingly, to step the polymer through the nanopore. The ATP-dependent movement of the active enzyme makes it possible to drive the polymer through a sensing nanopore at a rate that depends on the enzyme’s turnover number, i.e., the maximum number of steps the enzyme takes along its polymer substrate per unit time. However, as with other concepts derived from studies of bulk liquid-phase enzymes, it has been determined that turnover number has little to do with the behavior of individual molecule activity, where the time interval between each activity step of a single enzyme molecule varies randomly around the enzyme's turnover number.

[0009] The random time intervals between each successive step of a single molecule at and through the nanopore (e.g., the time between adjacent monomer units of the polymer stepping through the nanopore) pose significant problems because it is during these intervals that the conductivity of the nanopore is measured and evaluated to characterize the polymer and its polymer subunits within the nanopore. Many of these intervals may be short enough to cause omissions, while others may be long enough to be mistaken for consecutive identical monomer units when such consecutive identical monomer units do not actually exist. They also make it impossible to distinguish a homopolymer region of length X (a sequence of four identical monomers) from those of lengths X+1, X+2, ..., or X+n. Invention Summary

[0010] In the method provided herein for controlling the translocation of target polymer molecules through a nanopore, a clamp is reversibly attached to a plurality of consecutive polymer subunits along the length of the target polymer molecule. The target polymer molecule comprises a plurality of consecutive polymer subunits along its length. The target polymer molecule and the reversibly attached clamp are arranged in an ionic solution in fluid communication with the nanopore. The diameter of the nanopore is smaller than the outer diameter of the clamp.

[0011] A constant dislocation force is applied across the nanopore to induce the target polymer molecule in the ionic solution to travel into the nanopore until a clamp on the target polymer molecule comes into contact with the pore and stops the target polymer molecule from further traveling into the nanopore. A control pulse is then applied, which is at least one of a voltage control pulse across the nanopore and a thermal control pulse at the nanopore. The duration of the control pulse is such that the clamp steps along the target polymer molecule in the opposite direction of travel into the nanopore by no more than one polymer subunit. No fuel is supplied to the clamp. The stepping of the clamp causes the target polymer molecule to further step into the nanopore, but no more than one polymer subunit. The control pulse is applied repeatedly to cause successive polymer subunits of the target polymer molecule to dislocate through the nanopore.

[0012] This translocation control method enables the characterization of target molecules, wherein characteristic indications of polymer subunits are obtained when the polymer subunits are in the nanopore. Here, while obtaining characteristic indications of each polymer subunit translocated through the nanopore, the control pulse is repeatedly applied to induce successive translocations of multiple polymer subunits of the target polymer molecule through the nanopore.

[0013] The nanopore system for characterizing target polymer molecules provided herein includes a first fluid reservoir and a second fluid reservoir, which are in fluid communication with a nanopore that forms a unique fluid path between the two reservoirs. A clamp is provided in the first fluid reservoir. The clamp is abutted against the nanopore and reversibly bound to a plurality of consecutive polymer subunits of the target polymer molecule in an ionic solution within the first fluid reservoir. The target polymer molecule comprises a plurality of consecutive polymer subunits along its length.

[0014] A circuit is provided, comprising electrodes in a first reservoir, electrodes in a second reservoir, and a current amplifier for applying a constant bias voltage across the nanopore between the first and second reservoirs to induce the target polymer molecule to travel into the nanopore. A voltage pulse generator is connected to the circuit to apply a voltage control pulse across the nanopore between the first and second reservoirs to cause the clamp to step along successive polymer subunits of the target polymer molecule in a direction away from the nanopore, further stepping the polymer molecule into the nanopore. The system does not include fuel for the clamp and does not include a fuel source for the clamp. A computer controller is connected to the circuit to collect an electrical indication of the ionic current passing through the nanopore as the successive polymer subunits of the target polymer molecule step through the nanopore.

[0015] Another nanopore system for characterizing target polymer molecules, as described herein, includes a first fluid reservoir and a second fluid reservoir in fluid communication with a nanopore that forms a unique fluid path between the two reservoirs. A clamp is provided in the first fluid reservoir. The clamp is abutted against the nanopore and reversibly bound to a plurality of consecutive polymer subunits of the target polymer molecule in an ionic solution within the first fluid reservoir. The target polymer molecule comprises a plurality of consecutive polymer subunits along its length.

[0016] A circuit is provided, comprising electrodes in a first reservoir, electrodes in a second reservoir, and a current amplifier for applying a constant bias voltage across the nanopore between the first and second reservoirs to induce the target polymer molecules to travel into the nanopore. A laser is connected to the computer controller to generate laser pulses, and optics are arranged adjacent to and oriented to guide the laser pulses into the nanopore. Material elements that absorb energy from the laser pulses are arranged at the nanopore to heat the ionic solution during each laser pulse, thereby causing the clamp to step along successive polymer subunits of the target polymer molecules in a direction away from the nanopore, further advancing the target polymer molecules into the nanopore. The system does not include fuel for the clamp and does not include a fuel source for the clamp. The computer controller is connected to the circuit to collect an electrical indication of the ionic current passing through the nanopore as the successive polymer subunits of the target polymer molecules step through the nanopore.

[0017] The nanopore systems and methods provided herein enable deterministic control over the translocation of target polymer molecules through nanopores, and a correspondingly superior degree of control in characterizing the target polymer molecules. These and other aspects and embodiments of this disclosure are described below, in the accompanying drawings, and in the claims. Attached Figure Description

[0018] Figure 1 A, Figure 1 B and Figure 1 C is a schematic diagram of the three stages of the deterministic stepping of clamp-hinged polymer molecules through bio-nanopores in a biomembrane.

[0019] Figure 1 D、 Figure 1 E and Figure 1 F is a schematic diagram of the three stages of the deterministic stepping of polymer molecules through nanopores in a solid film via a clamp-and-hinged mechanism.

[0020] Figure 2 This is a schematic diagram of a nanopore system that includes clamp-hinged polymer molecules provided in a cis reservoir for deterministic stepping through nanopores into an inverse reservoir.

[0021] Figure 3 It is a flowchart of the method steps for deterministic stepping of polymer molecules through nanopores using a clamp-hinged system;

[0022] Figure 4 A, Figure 4 B. Figure 4 C and Figure 4 D is in the process of Figure 3 The flowchart shows the method steps for measuring the nanopore current or applying the control pulse voltage.

[0023] Figure 5 This is a schematic diagram of a nanopore system and associated electronic devices used to implement voltage-controlled pulse application in a deterministic stepping of a clamp-hinged polymer through a nanopore;

[0024] Figure 6 It is a circuit model of a nanopore system that includes an electronic device for controlling the application of pulse voltage;

[0025] Figure 7 yes Figure 5 A schematic block diagram of a low-noise voltage pulse generator for a system;

[0026] Figure 8 This is a schematic diagram of a nanopore system and related component parts used to implement thermal control pulse application in a deterministic stepping of a polymer through a nanopore via a clamp-hinged connection;

[0027] Figure 9A and Figure 9 B are schematic diagrams showing gold nanoparticles (NPs) connected to the clamp and connected to the nanopore, respectively, used in... Figure 8 Provide absorbent materials in nanoporous systems;

[0028] Figure 10 This is a schematic diagram of polymerase synthesis;

[0029] Figure 11 This is a graph showing the variation of the nanopore current with approximately 5 nucleotides adjacent to the sensed monomer under conditions of ATP-driven polymer stepping and bias-driven polymer stepping without ATP (both with T4 helicase polymer clamps), where the DNA sequence shown at the top of the graph is read out as a series of overlapping pentamers shown below the horizontal axis.

[0030] Figure 12 This is a graph showing the median duration of the polymer across the entire length of the polymer, using T4 phage helicase as a polymer clamp for 2 mM ADPNP and for ATP-free analogs, where the vertical capping line represents the duration range of several identical polymers completely traversing the nanopore.

[0031] Figure 13 This is a graph showing the median monomer step duration as a function of the applied bias driving voltage for DNA molecules hinged using a T4 phage helicase clamp at four different temperatures.

[0032] Figure 14 A, Figure 14 B and Figure 14 C is a schematic diagram illustrating polymer molecule control, where the applied bias driving voltage overcomes the Brownian motion of polymer molecules within the nanopores; and

[0033] Figure 15 A and Figure 15 B is a graph showing the changes in the measured nanopore current and the amplitude of the applied voltage control pulse over time for an experimental nanopore system using a 160mV bias driving voltage.

[0034] Detailed description

[0035] This article provides information for, such as Figure 2All structures, systems, and corresponding methods in the illustrated nanopore system represent deterministic rather than random stepping of target polymer molecules through the nanopore. In one embodiment, the structures, systems, and methods are used to drive a linearly linked series of monomer residues of the target polymer molecule through the nanopore with precise deterministic motion, such that the time taken for a repeating sequence of the same monomer unit to traverse the nanopore identifies the precise number of the same monomers that have translocated through the nanopore. Specifically, when the time interval between each monomer step through the nanopore is a known constant k, that is, as described herein, when the stepping is deterministic, the total time taken for a repeating sequence of the same monomer unit to traverse the nanopore divided by the interval constant k identifies the precise number of the same monomers that have traversed the nanopore.

[0036] like Figure 1 A to Figure 1 As shown in Figure C, this paper provides the structure of a deterministic propulsion clamp 10, which reversibly clamps or binds to a plurality of monomers 12 (e.g., a group of 1-20 monomers) along a target polymer molecule 14 (e.g., a DNA strand) or other suitable polymer molecules having polymer subunits along their molecular length. The outer diameter 16 of the clamp 10 is larger than the diameter 18 of the pore or channel extending through the nanopore 20, such as... Figure 1 A to Figure 1 As shown in C.

[0037] As used herein, the term "polymer molecule" is intended to refer to biomolecules such as polynucleotides, biopolymer nucleic acid molecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), synthetic nucleic acids such as peptide nucleic acids (PNA), as well as proteins, glycopolymers, and other biomolecules. Therefore, the following discussion is not intended to be limited to any particular implementation. Details relating to examples of these molecules, such as polynucleotides, are provided in a series of embodiments used for polymer molecule characterization to illustrate the principles presented herein.

[0038] Also refer to Figure 2A cross-sectional schematic diagram shows that the clamp-hinged molecular configuration can be used in a nanoporous system 25 for characterizing polymer molecules. The nanoporous system typically includes a structure 26, such as a membrane, in which nanopores 20 are arranged, which can be biological or solid-state in nature, or both. If desired, the membrane or other structure 26 in which nanopores are arranged can be supported at its edges by, for example, a support structure 28. The arrangement of structure 26 and support structure 28 separates the cis reservoir 30 from the trans reservoir 32, wherein the nanopores provide a single fluid path between the two reservoirs. The cis and trans reservoirs comprise ionic liquid solutions, and the cis reservoir includes a target molecule 14 to be translocated through the nanopore 20 to the trans reservoir. The target molecule 14 in the cis reservoir to be translocated through the nanopore each has a reversible clamp 10 arranged at sites along the molecular length on multiple polymer subunits, forming a target molecule-clamp complex. Solutions of such target-fixture complexes can be generated using conventional methods, such as those described by Moysey in US 20140335512, the entire contents of which are incorporated herein by reference. The target-fixture complexes are provided in cis-reservoirs for the analysis of target molecules that have translocated through nanopores.

[0039] In one embodiment, electrodes 34 and 36 are included in the cis reservoir 30 and the trans reservoir 32, respectively, and a constant bias voltage 38 can be applied between these two electrodes. The electric field generated by applying such a bias voltage between the cis reservoir electrode 34 and the trans reservoir electrode 36 can be used to drive charged molecules in the cis reservoir through the nanopores to the trans reservoir in a conventional manner. However, it should be recognized that other displacement forces can be used alternatively. Therefore, although the term "bias voltage" or V will be used in the step control method description herein. 驱动 However, it should be understood that other forces can also be used to drive the polymer through the nanopores, including hydrostatic pressure, optical or magnetic fields, and combinations of two or more fields. Hereafter, when the term "constant voltage driven bias" or V is used herein... 驱动 In this context, it should be understood that any one or more of these fields may be substituted. Further details and examples of molecular translocations driven by electrophoresis through nanopores are provided in U.S. Patent No. 6,627,067, entitled “Molecular and Atomic Scale Evaluation of Biopolymers,” issued to Branton et al. on September 30, 2003, the entire contents of which are incorporated herein by reference.

[0040] Now refer to the methods used in this article, such as Figure 1 As shown in Figure A, the target polymer molecule 14 under study is displayed at the nanopore 20, which includes a reversibly bound clamp 10. Figure 1 As shown in Figure B, a bias voltage V is applied between the cis-type reservoir and the inverse-type reservoir. 驱动,like Figure 2 As shown, conditions are provided to facilitate the movement of the target polymer 14 into the nanopore 20. When the target polymer 14 moves into the nanopore, as... Figure 1 As shown in Figure B, when the clamp 10 is pressed against region 40 of the nanopore 20, the displacement automatically stops. The inner diameter 18 of the nanopore 20 is smaller than the outer diameter 16 of the clamp. Figure 1 As shown in Figure A. Under these conditions, a polymer subunit 42 of polymer 14 (e.g., a monomer) occupies a site within the nanopore, for example, in a preferred embodiment, a site in a highly sensitive narrow region 44 of nanopore 20. As a result, for these conditions, as long as subunit 42 remains at a suitable site within the nanopore, such as a highly sensitive region of the nanopore, the electrical and volumetric properties of subunit 42 dominate the cis-to-trans conductivity of the nanopore. Therefore, sensing, detecting, measuring, or otherwise determining parameters of the nanopore system related to the conductivity of the nanopore provides a characteristic indication of subunit 42 under these conditions.

[0041] After determining the characteristic indications of subunit 42, then as follows Figure 1 As shown in C, besides the bias driving voltage V across the nanopores 驱动 In addition, control pulses are also provided, such as voltage control pulse V. 脉冲 and / or thermal control pulse H 脉冲 Provided. A control pulse causes the clamp 10 to step away from the nanopore 20 along the polymer 14 in the direction indicated by arrow 44. Specifically, the clamp 10 is controllably driven to step along the polymer one polymer subunit, and then reversibly bind to the next successive group 12 of the polymer subunit. As a result, subunit 42 in the nanopore now moves into the solution in the trans reservoir, while the next subunit 46 now dominates the cis-to-trans conductivity of the nanopore. This process of deterministic stepping of the clamp away from the nanopore along the polymer continues to cause polymer molecules to enter and step through the nanopore one subunit at a time. No energy is applied to the clamp to fuel the clamp's stepping from one polymer subunit to the next. Specifically, no chemical fuel is supplied to the clamp, and no biochemical fuel is supplied to the clamp. Only the application of a control pulse causes the clamp to step from one polymer subunit to the next.

[0042] Figure 1 A to Figure 1Figure C shows the target polymer molecule 14 with linear subunit sequences, which are represented by simple circles in the figure. This representation is provided for clarity only. The target polymer molecule loaded into the cis reservoir can be single-stranded (e.g., ssDNA) or double-stranded (e.g., dsDNA). In either case, the clamp is positioned at a site on the single-stranded polymer molecule region. Thus, the double-stranded target polymer molecule provides a single-stranded region to which the clamp can be positioned in a conventional manner, such that the clamp can be positioned at a point along the single-stranded region of the double-stranded target molecule.

[0043] Figure 1 A to Figure 1 Condition C shows an embodiment in which nanopores 20 are provided as bio-nanopores in membrane 35, wherein membrane 35 may be biological or organic, such as a lipid bilayer, tetraether lipid, or triblock copolymer. For example, the nanopores may be provided as mycobacterium smegmatis porin A (MspA), an octamer protein channel; as bacterial porin CsgG, a nonamer protein channel; or as mutants of these and other channels, such as those taught by Moysey in U.S. Patent No. 9,617,591, the entire contents of which are incorporated herein by reference. The selected bio-nanopores are arranged in, for example, diphyllylphosphatidylcholine membranes (diPhPC), tetraether lipids, triblock copolymers, or solid structures (e.g., composed of solid membranes). Such solid membranes may be provided as, for example, one or more graphene layers, SiN… x Alternatively, other solid supports may be used to prevent ions from flowing trans-propagating through the solid membrane, but any of the aforementioned bio-nanopores may be arranged within the solid membrane in a manner that prevents ions from flowing from cis to trans unless through the nanopore's own channels.

[0044] exist Figure 1 A to Figure 1 The conditions for polymer trapping and stepping at the nanopores shown in C are also... Figure 1 D to Figure 1 As shown in F, but for implementation methods of solid-state nanoporous systems. (See also...) Figure 1 D to Figure 1 As shown in E, a constant bias voltage V is applied between the cis-type reservoir and the inverse-type reservoir. 驱动 ,like Figure 2 As shown, conditions are provided to facilitate the capture of the target polymer 14, including the clamp 10, at sites in the nanopore 20. The nanopore 20 is here a pore, hole, channel, or other opening arranged in the solid film 37. This film can be formed from any suitable solid material as described above, such as graphene or other atomically thin materials, or as a microelectronic material, such as a combination of nitrides, oxides, or material layers. The solid film can further employ… Figure 2The membrane 26 and the support structure 28 are supported by a support structure such as a microelectronic substrate. It should be understood that, for clarity, Figure 2 System 25 exhibits nanopores 20, which are pores in membrane 26, but can employ any suitable combination of biological, organic, and solid nanopores and membranes, and is intended for use in Figure 2 25. Nanoporous systems.

[0045] When the target polymer 14 displaces through the nanopore due to a constant bias voltage, such as Figure 1 As shown in Figure E, the displacement automatically stops when the clamp 10 is pressed against the nanopore 20. The diameter 18 of the nanopore is smaller than the outer diameter 16 of the clamp, as shown in Figure E. Figure 1 As shown in Figure D, under these conditions, one subunit 42 of the target polymer 14 (e.g., a monomer) occupies the most sensitive region of the nanopore. Consequently, under these conditions, as long as the monomeric subunit 42 remains within the nanopore, the electrical and volumetric properties of subunit 42 dominate the cis-to-trans conductivity of the nanopore. Consequently, sensing, detecting, measuring, or otherwise determining parameters of the nanopore system related to its conductivity provides an indication of the properties related to subunit 42 under these conditions.

[0046] After determining the characteristic indications of subunit 42, then as follows Figure 1 As shown in F, the bias drive voltage V 驱动 The combination provides control pulses, such as voltage control pulses V. 脉冲 and / or thermal control pulse H 脉冲 The clamp 10 is driven to step along the target polymer 14 in the direction indicated by arrow 44, away from the nanopore 20. Specifically, the clamp 10 is driven to step along the polymer one monomer subunit, and then bind to the next set of polymer subunits. As a result, subunit 42 in the nanopore moves into the trans reservoir, while the next subunit 46 now dominates the cis-to-trans conductivity of the nanopore. This process of deterministic stepping of the clamp along the target polymer continues to allow polymer molecules to enter and step through the nanopore one subunit at a time.

[0047] Figure 3 It is used to implement Figure 1 AC and Figure 1 The flowchart shows the method steps for the conditions shown in DF. Figure 4 A-4D is a graph of measured electrical signals indicating the conditions of each method step. In method 50, after assembling the nanopore system to begin method 52 (where the target polymer molecules are hinged together with clamps arranged in the cis reservoir), then in step 54, a constant bias driving voltage V is applied between the cis and trans reservoirs. 驱动 In the next step, 56, it is determined whether the target polymer has been captured at the nanopores. Figure 4A is a graph showing the change in current measured through the nanopore over time. It illustrates that when the target polymer is captured at the nanopore, the current measured through the nanopore drops sharply from the opening current level to a lower level indicating the capture of polymer molecules in the nanopore. If it is determined that the polymer has not yet been captured at the nanopore, a constant bias voltage is maintained and measurements continue until it is confirmed that the polymer has been captured at the nanopore.

[0048] In the next step 58, in addition to the bias voltage, a control pulse, such as a voltage control pulse and / or a thermal control pulse, is applied between the cis- and anti-cis-cell reservoirs. In one embodiment, the voltage control pulse here is as follows: Figure 4 As shown in Figure B, a voltage pulse is applied across the nanopore. Then, as... Figure 4 As shown in Figure C, the total voltage across the nanopore comprises a constant bias voltage and a pulsed voltage. The timing and duration of each pulse can be controlled manually or by an automated system that can provide continuously repeating pulses. The polarity of the bias voltage and the pulsed voltage is the same relative to the nanopore, so for a given polymer charge, the sum of the bias voltage and voltage pulses is additive and oriented in the direction that drives the polymer through the nanopore.

[0049] In the next step 60, it is determined whether the target polymer responds to the applied control pulse and steps through the nanopore. Figure 4 Figure D provides an example showing how a single step of a polymer subunit in response to a single voltage pulse can be determined by current measurement. If it is determined that the polymer does not take a single step in response to a single voltage pulse or a single thermal pulse, the amplitude and / or duration of the pulse are increased. When it is indicated that the polymer is positively stepping a polymer subunit through the nanopore in response to an applied pulse, some representative indication of the polymer subunit characteristics at the sensing site of that nanopore is obtained in the next step. For example, as... Figure 3 As shown, in step 62, the measured current through the nanopore can be obtained and recorded (if needed) to reflect the properties of the polymer subunits as the polymer responds to a control pulse and steps through the nanopore.

[0050] In the final step 64, it is determined whether the nanopore has opened, for example, by determining whether the nanopore current has returned to the opening current. If not, step 62, recording the polymer sequence, continues. If the nanopore is determined to be open, step 54 is performed again, applying a constant bias voltage only across the nanopore while waiting for the capture of target polymer molecules at the nanopore.

[0051] In method 50, a step of measuring the nanopore current can be performed to obtain a series of different characterizations of the polymer subunits in the nanopore. Typically, characterizations of the polymer subunits can be obtained as follows: for example, detecting the presence of polymer subunits in the nanopore; counting polymer subunits in a successive plurality of subunits; identifying the number of identical subunits in a successive plurality of subunits; identifying the polymer subunits; determining the chemical aspects of the subunits or other characterizations of the subunits. Subunits can be characterized in this way along a continuous sequence throughout the polymer molecule, providing information on the quantity and identity of the entire polymer molecule sequence.

[0052] This controlled stepping method can be implemented using any suitable electronic control system. Figure 5 This is a schematic diagram of one implementation, in which the arrangement system 75 is used to implement [the system] using control pulses. Figure 3 The method involves using a voltage control pulse. In system 75, electrodes 34 and 36 are provided in the cis- and trans-cell receptacles and connected to a low-noise voltage pulse generator 70 and a current amplifier 72. The current amplifier 72 is controlled to apply a constant bias voltage V between the cis- and trans-cell receptacles. 驱动 When triggered by a control signal from the control computer 74, the pulse generator 70 generates a voltage control pulse V. 脉冲 The voltage control pulse V 脉冲 The bias voltage V applied between electrodes 34 and 36 is 驱动 Above. Current amplifier 72 provides an analog output measurement of the nanopore current, which is digitized by analog converter 76 and provided to computer 74 for recording. The circuit formed by cis and trans reservoirs, nanopore, current amplifier, A / D converter, pulse generator and computer is capable of measuring the current in the circuit, which indicates the ion current passing through the nanopore.

[0053] The system can be temperature-controlled as needed and in any suitable manner, such as by using a thermoelectric heater / cooler. No component of the system supplies energy to the clamp to fuel its stepping along the polymer molecules, and the system does not contain any energy source. As described above, only voltage control pulses cause the clamp to step along the polymer molecules.

[0054] The system 75 can be used as follows Figure 6 The circuit model 80 shown is electrically modeled. The nanopores in the membrane cause pore resistance and membrane capacitance. Figure 7 yes Figure 5 A schematic diagram of one embodiment of a low-noise voltage pulse generator 70. The low-noise pulse generator includes a control element that determines the duration between control pulses and the duration and amplitude of voltage control pulses. This embodiment is particularly preferred to enable precise generation of voltage control pulses. Specifically, in a preferred embodiment, such as... Figure 7 As shown, there exists a capacitor compensation +V generated by the voltage follower-inverter. 脉冲 and -V 脉冲 To reduce the "overshoot" caused by capacitance, when the time interval between voltage pulses is set to the minimum interval (e.g., to maximize nanopore data output), the duration of this "overshoot" can make it impossible to measure the actual current passing through the nanopore.

[0055] Figure 8 This is a schematic diagram of one embodiment, in which the nanopore system 85 is arranged to be implemented using control pulses. Figure 3 The method involves a thermal control pulse. In system 85, electrodes 34 and 36 are provided in the cis- and anti-cis-cell reservoirs and connected to a current amplifier 72. The current amplifier 72 provides a constant bias voltage V between the cis- and anti-cis-cell reservoirs. 驱动 A thermal control pulse is generated under control from computer 74. A trigger pulse control signal is provided to laser 82 and optics 84, which are arranged to guide the pulse of laser energy 86 to the site of the nanopore in the film, where material absorbing the laser energy 86 is arranged. The absorption of laser energy by the absorbing material causes a temperature rise near the nanopore, as explained below. Current amplifier 72 provides an analog output measurement of the nanopore current, which is digitized by analog converter 76 and provided to computer 74 for use in... Figure 5 The voltage pulse control system is recorded. Similar to this system, it can be used as needed and in any suitable manner, such as by utilizing a thermoelectric heater / cooler; here, system 85 can be temperature-controlled.

[0056] Figure 9 A to Figure 9 B indicates that it is used in Figure 8 The implementation of the system 85 provides absorbent material at the sites of nanopores. Figure 9 In the first embodiment shown in A, absorbent particles 90, such as gold, silver, or other suitable nanoparticles (NP), are provided and attached to the clamp 10 via molecular connectors 92. Figure 9 In another embodiment shown in B, absorbent particles 90 (e.g., gold NP) are attached to nanopores 20 via molecular connectors 92.

[0057] In either of these embodiments, irradiation of the particles, preferably gold nanoparticles (NPs), with a laser is particularly effective for rapid localized heating of the solution near the nanopores, the wavelength of which utilizes the plasmon resonance effect of the gold particles. Because the highly confined surface plasmon resonance effect in the gold nanoparticles enhances the absorption of light by the particles, visible laser light incident on the nanoparticles causes a rapid and significant increase in the temperature of the particles and the adjacent solution. This temperature rise can be estimated by the change in the ionic conductivity of the nanopores. For example, a 532 nm wavelength laser operating at a beam energy of 300 mW can easily raise the temperature of gold nanoparticles and the region immediately surrounding the NPs from about 20 °C to about 50 °C in just nanoseconds. Because the heated volume can be as small as a few liters, this small volume can quickly return to the temperature of the surrounding solution volume as its thermal energy rapidly dissipates into the surrounding solution, which is typically at least 16 orders of magnitude larger.

[0058] In another embodiment, Figure 5 The control system 75 and Figure 8 The control system 85 is configured to combine voltage control pulses with thermal control pulses. An absorbent, such as gold NP, is used at an appropriate location at the nanopore, employing both voltage and thermal pulses for deterministic monomer movement in the manner described above. The timing and duration of each voltage and thermal pulse can be controlled individually, either manually or through an automated system, coordinating thermal and voltage pulse control.

[0059] Now turn to the details of the clamp 10 used with the nanopore system. Figure 1 A to Figure 1 The clamp shown in C can be constructed and arranged in any suitable manner, such that it is generally capable of reversibly clamping or binding to multiple polymer subunits or other subunits of the target polymer to be translocated through the nanopore. While clamps can be molecules used as engines for enzyme-dependent or biofuel-dependent processes in other systems, the term "clamp" as used herein refers to any biomolecule or non-biomolecule component, i.e., a solid component, that reversibly binds to a polymer and can be controlled by applying a voltage pulse V. 脉冲 and / or thermal control pulse H 脉冲 Instead of using biofuel-dependent action-dependent deterministic drive, it steps or moves in single, continuous monomer steps along the length of the polymer. No chemical or other fuel is supplied to the fixture to step along the sequence of polymer subunits of the target polymer molecule.

[0060] In one embodiment, the clamp is a helicase, such as T4 phage helicase. In other embodiments, other enzymes may be used as clamps, including mutants of T4 phage helicase, as well as other proteins and biochemical or inorganic chemical complexes that bind to nucleic acid polymers.

[0061] Using the aforementioned clamp and control system, the polymer stepping method presented herein avoids the random movement of enzyme-active molecules through the use of the clamp. The movement of the clamp does not require an input of ATP or chemical or biochemical fuels; instead, it is driven by a constant bias voltage V. 驱动 Superimposed voltage control pulse V 脉冲 and / or thermal control pulse H 脉冲 Deterministic driving. As a result, this paper provides a method in which a linear sequence of target polymer monomers translocates through nanopores in a precisely timed stepwise motion. Two features in particular make this method possible.

[0062] First, unlike the biochemical fuel alignment used for enzymes (e.g., helicases and polymerases) to control the random movement of polymers through nanopores, the method presented here requires a deterministically driven gripper that does not use chemical energy to induce movement of the gripper along the monomer subunits of the target polymer. Instead, a constant driving bias V is used here. 驱动 Controllable application of voltage control pulse V 脉冲 and / or thermal control pulse H 脉冲 When a polymer is driven to step through a nanopore into a monomer, such as Figure 1 As shown in C, the clamp bound to the polymer is released or released very briefly, causing the clamp to slide along the polymer in one and only one monomer step. Therefore, except as... Figure 1 As shown in Figure C, when the clamp moves relative to the target polymer, the clamp prevents or stops the target polymer from being driven through the nanopore by a driving force that would otherwise rapidly drive the polymer along its full length through the nanopore without the clamp. In effect, the clamp acts as a pawl on a clock ratchet, while the bias voltage V... 驱动 A spring or electric motor used to drive a ratchet.

[0063] Secondly, voltage-controlled pulses and / or thermal-controlled pulses, preferably with a duration of nanoseconds to microseconds (e.g., one millisecond or less), cause the target polymer chain to step through the nanopores by causing the clamping component to slide one monomer unit along the polymer with each pulse. This is achieved due to a constant bias voltage V. 驱动 Continuous action is applied to cause polymer electrophoresis or drive the polymer through a nanopore. The attached clamp, taking into account its large diameter, cannot move through the nanopore. As the clamping component reversibly but deterministically slides and then reversibly re-clamps the polymer at another plurality of monomer units, the polymer determinately steps through the nanopore one monomer unit at a time.

[0064] In a preferred embodiment, a single voltage control pulse V 脉冲 and / or thermal control pulse H 脉冲 The duration is adjusted to be no greater than the duration required for instantaneous release of the clamp from the polymer, therefore in Figure 1In C, the upward direction is indicated by the arrow. As the clamp slides along the polymer one monomer step, a constant bias voltage V... 驱动 The polymer is driven through a nanopore to bind with the next successive monomer unit or group of monomer units. This causes the next successive monomer unit of the polymer to occupy the nanopore, preferably the narrowest region of the most sensitive nanopore, such that the conductivity of the nanopore is now dominated by the electrical and / or volumetric properties of that next successive monomer or group of monomers. Therefore, a series of these voltage-controlled pulses and / or thermal-controlled pulses operate in a ratchet-release mechanism of a clock system. The continuous voltage-controlled pulses and / or thermal-controlled pulses thus enable the full-length stepping of the target polymer to be driven through the nanopore.

[0065] Note that even when the clamp is implemented as a molecular clamp enzyme, which could potentially utilize ATP or another biofuel for turnover and stepping along the polymer, no ATP or chemical energy is provided in the method described herein, and it is only controlled by voltage-controlled pulses and thermal-controlled pulses (V). 脉冲 and / or H 脉冲 This allows the clamp to slide along its polymer substrate. The clamp is controlled by precisely timed voltage control pulses V. 脉冲 and / or thermal control pulse H 脉冲 Driven along the polymer, the stepping motion of the polymer through the nanopores is thus deterministic.

[0066] Deoxynucleotide triphosphate (dNTP)-driven polymerases and ATP-dependent helicases have both been used to randomly step DNA through nanopores for sequencing. The stepping rate of these and other chemically driven enzymes is known to be accelerated, decelerated, or even stopped by mechanical forces. It is also known that, in the presence of ATP or dNTPs, nucleic acid stepping enzymes (such as helicases and polymerases) step along the polymer substrate monomer by monomer, without skipping or jumping several monomers, even when mechanically accelerated or decelerated. For example, if... Figure 1 Clamp 10 shown in AC is a helicase, which, in the presence of ATP, ... Figure 1 The direction indicated by the upward arrow in C is typically along the polymer substrate. This stepping becomes faster, but because the bias driving the polymer through the nanopores from the cis reservoir to the trans reservoir increases, it still steps monomer by monomer without skipping, as taught by Moysey in U.S. Patent No. 9,617,591, the entire contents of which are incorporated herein by reference.

[0067] Conversely, reference Figure 10If the clamp is a progressive polymerase 85, then in the presence of dNTPs, in the direction of arrow 86, the polymerase tends to cause the polymer to step out of the nanopore more slowly along the polymer's stepping path, but because the electrophoretic force driving the polymer from cis to trans becomes larger, the polymer still steps monomer by monomer without skipping, as described by Lieberman et al., J. Am. Chem. Soc., 132: 17961-17972, 2010, the entire contents of which are incorporated herein by reference. Finally, the fact that F1-ATPases, which typically consume ATP and step counterclockwise, synthesize ATP when driven clockwise by purely mechanical force clearly demonstrates that mechanical forces acting at a specific point on a protein machine can drive chemical reactions at catalytic sites. These examples show that properly applied mechanical forces do not break or disrupt the normal conformational changes of enzymes. Furthermore, these examples provide clear evidence that purely mechanical forces can delay, promote, or even drive conformational changes that are normally driven by chemical energy.

[0068] The fact that mechanical force can promote, delay, or substitute for conformational modifications of enzymes (causing steps) in the presence of ATP raises the following problem addressed by the structure provided herein. In the absence of any ATP, i.e., in the absence of any fuel, purely mechanical force alone can induce conformational modifications in polymer stepping enzymes to characterize the same monomer-by-monomer precision of ATP-dependent steps, causing them to slide along the polymer. When chemical energy derived from ATP hydrolysis is used, enzymes (such as helicases) undergo conformational changes that allow the helicase to precisely advance one and only one nucleobase per step along the polymer. If the mechanical force acting alone simply pushes the enzyme clamp in each step to slide randomly over an indeterminate number of nucleobases, the nucleobase sequence sensed by the nanopore will not report the actual sequence of nucleobases in the polymer, because the recognition of each consecutive nucleobase through the nanopore occurs in the time interval between each consecutive step.

[0069] Now for reference Figure 11 The inventors discovered in this paper that the applied force can push the enzyme clamp in a manner achieved by ATP, but entirely in the absence of ATP, i.e. without providing any fuel to the clamp, each step precisely advances one and only one nucleobase. Figure 11 The figure shows nanopore current blockage due to approximately five nucleobases directly adjacent to the monomers that occupy the narrowest region of the nanopore in each step of the DNA strand. The DNA sequence is read as a series of overlapping pentamers, as shown on the horizontal axis.

[0070] like Figure 11 As shown in the figure, for the applied bias voltage V 驱动=160mV, the current through the nanopore at each step of the bias-driven DNA strand (circle) is the same as the current reading at each step of the ATP-driven strand (square) (within the error limit of such current readings). This shows that mechanically forced motion can propel a polymer (e.g., a DNA strand) forward one nucleotide through the nanopore, just like ATP-fuel force, and can do so without the use of ATP. Therefore, voltage-forced motion can faithfully move the target polymer molecule through the nanopore, and thus when the movement of the polymer through the nanopore is controlled by, for example, an ATP-fuel helicase, the current level sequence is identical. This clearly shows that a sufficiently large pure mechanical force causes what is typically used as a clamp by an ATP-dependent enzyme, which, in the absence of ATP, can be driven by voltage and / or thermal control to move along the length of the DNA strand with the same monomer-by-monomer step precision as an ATP-dependent DNA motor enzyme.

[0071] Although Figure 11 The figure illustrates the normal 5' to 3' step of ATP-driven T4 helicase and the mechanically driven 5' to 3' T4-helicase as a clamp in the absence of ATP. However, T4-helicase can also be mechanically driven in the reverse direction of the DNA strand (i.e., from 3' to 5'). Indeed, considering the observations of Mulkidjanian et al., Nat. Rev. Microbiol., 5: 892-899, 2007, which are incorporated herein by reference in full, when driven in the reverse direction by mechanical force, ATP can be synthesized using F1-ATPase instead of hydrolyzing ATP, as described by Ito et al., Nature, 427: 465-468, which are incorporated herein by reference in full. It should be understood that ATP can be produced rather than consumed by driving an ATP-dependent nucleic acid stepper motor enzyme in the reverse direction of its normal 5' to 3' or 3' to 5' movement along the DNA strand. As a result, in another embodiment provided herein, a bias drive voltage and voltage control pulses and / or thermal control pulses can be used to move the clamp along the polymer molecular length in either of two possible directions.

[0072] Because the method for controlling pulses provided in this paper includes a bias drive voltage V 驱动 Add an additional voltage control pulse V 脉冲 Very brief intervals and / or in thermal control pulse H 脉冲 The combination of short intervals at higher temperatures provides an understanding of how higher bias and higher temperature affect the step rate. Again using T4-helicase as an example of a DNA strand clamp, experimental results are produced for pulseless conditions, for voltage and temperature parameters. (See reference...) Figure 12The figure shows that, in the absence of ATP, when a DNA polymer with a binding clamp is driven through a nanopore by a constant cis-to-trans bias, the bias voltage V across the nanopore increases with the change in the bias voltage V. 驱动 The increase in [something] shortens the median time spent by the polymer's full length stepping through the nanopores. See also [other sources]. Figure 13 The result is that, for the representatives Figure 12 The data in the figure are based on the conditions of using a T4 helicase clamp, with the bias voltage V. 驱动 The increase in shortens the median duration of the time interval between each unit step (referred to here as the median step duration). By Figure 12 The duration of the complete molecular translocation shown is divided by the number of monomers known to constitute the full length of the molecule undergoing the translocation, for... Figure 12 Data to determine Figure 13 The median step duration data in the graph.

[0073] Several other factors can affect the median step duration or the time interval between each step. For example, the addition of low concentrations of the non-hydrolyzable ATP analog 5'-(β,γ-imino)triphosphate (ADPNP) significantly reduces the median step duration when... Figure 11 The figure shows the range of times that many of the same polymers can completely traverse the nanopores during stepping under the conditions illustrated. Other factors that can affect the median stepping duration include the direction of polymer movement through the jig, such as the 3' to 5' or 5' to 3' direction, and variations in the amino acid composition of the jig, or the formation of disulfide bonds between jig components or other structures.

[0074] Refer again Figure 13 It was found that the temperature of the fixture and its closest surrounding environment had a particularly significant impact on the median step duration, such as Figure 13 The plotted data are shown below. For example, using T4 phage helicase as a clamp, while lowering the temperature below room temperature significantly prolonged the median step time, raising the temperature from 25°C to 37°C shortened the median step time by more than three times. It was found that neither the increased voltage nor the increased temperature altered the accuracy of the clamp's monomer-by-monomer stepping along the length of the observed DNA polymer.

[0075] The implementation of voltage and thermal control pulses recognizes that the characteristics of the nanopore system, the clamping components, and the duration and amplitude of the voltage and / or thermal pulses of the method should be selected in coordination with each other and with other commonly used characteristics of nanopore sensing known to those skilled in nanopore sensing and nanopore sequencing methods. In one implementation, a strategy for achieving control pulses that generate deterministic steps is as follows:

[0076] 1) First step, set a constant bias drive voltage V 驱动And all other conditions of the nanopore system, such that the median step duration is as long as possible, and the frequency of short-duration steps is as low as possible. For example, by using the bias drive voltage V 驱动 This can be achieved by setting a voltage of approximately 20mV to approximately 140mV and maintaining the solution medium in the reservoir at a temperature of approximately 10°C to approximately 15°C. It is understood that, although the bias drive voltage V... 驱动 Longer median step durations can be achieved at or below approximately 120 mV, but bias voltages below approximately 120 mV may not optimally prevent the passage of the duration, during which successive monomer units may diffuse outside the sensing pore region of the nanopore, as explained in Lu et al., Biophys. J., 109: 1439-1445, 2015, which is incorporated herein by reference in its entirety. As explained below, this reduces the ability of the nanopore to distinguish between different subunits of the polymer. As a result, in the preferred embodiment, the bias drive voltage V 驱动 It is at least about 120mV, and in a preferred embodiment, the bias drive voltage V 驱动 It ranges from approximately 120mV to approximately 140mV.

[0077] 2) In the next step, set the voltage control pulse V. 脉冲 Or thermal control pulse H 脉冲 or V 脉冲 and H 脉冲 Simultaneously control the pulse frequency so that the duration between pulses is significantly less than when only a bias drive voltage V is applied. 驱动 The duration of the shortest duration step measured in the absence of a pulse. It is recognized that, although the duration between pulses is preferably significantly shorter than the duration of the shortest duration step observed in the absence of a pulse, the duration between pulses is preferably long enough so that the nanopore system electronics can accurately evaluate the ion current flowing through the nanopore between each pulse. If the duration between pulses is short enough compared to the duration of the shortest duration step observed in the absence of a pulse, it will be guaranteed that the probability of driving the jig to random step by bias alone will become imperceptibly small. Therefore, essentially each jig step is due to the intentional application of a voltage control pulse V. 脉冲 and / or thermal control pulse H 脉冲 Furthermore, it ensures that the polymer's stepping motion through the nanopores is deterministic, controlled, and repeatable.

[0078] In other words, to ensure that the target polymer advances through the nanopore in a deterministic stepping motion, the duration of each voltage-controlled pulse and / or thermal-controlled pulse is selected to overcome the normal force binding the jig to the monomer unit of the polymer, achieving a value less than the bias drive voltage V. 驱动The length of time it takes for the polymer to move forward through the nanopores to the next monomer unit.

[0079] Depending on the chosen study using the nanopore system, the details of the target polymer under investigation, and the availability of clamping components and suitable nanopores, several preferred embodiments are provided herein. Two embodiments are given below illustrating how those skilled in the art of nanopore sensing and nanopore sequencing can select suitable features of the systems provided herein, and how to set their parameters, as well as the parameters of other commonly used features for nanopore sensing, so that they work in harmony. DNA will be used in the embodiments, but other linearly linked (i.e., continuous) charged polymers, such as RNA, proteins, and the molecules described above, can be similarly probed using suitable clamping components that are reversibly bound to the polymer.

[0080] In the various embodiments provided herein, single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) is the target polymer molecule. In any embodiment, the nanopore is configured with a restrictive pore through which only one strand of the target polymer will pass, but the strand with the bound clamping member cannot pass. Among readily available nanopores, for many embodiments, organic nanopores (e.g., proteins) or inorganic solid nanopores (e.g., pores in membranes) are preferred, in either case having a channel pore diameter smaller than the diameter of the clamp, but larger than about 1 nm.

[0081] The production, arrangement, and configuration of nanopores, membranes, and support structures in nanoporous systems can be achieved in any suitable manner, for example, as described in Moysey’s U.S. Patent No. 9,617,591; as described in Golovchenko’s U.S. Patent No. 7,468,271; as described in Lieber’s U.S. Patent No. 8,698,481; as described in Garaj’s US Patent No. 20,120,234,679; as described in Lieber’s U.S. Patent No. 9,702,849; as described in Russo’s US Patent No. 9,611,140; as described in Golovchenko’s U.S. Patent No. 9,815,082; and as described in Xie’s US Patent No. 20,160,231,307, the entire contents of which are incorporated herein by reference.

[0082] Implementation with example parameters for counting monomers along the length of the target polymer

[0083] In this exemplary embodiment, a nanoporous membrane formed of a lipid bilayer is specified. For example, a diPhPC lipid bilayer extending across pores in a support structure can be used, wherein the diameter of the pores in the support structure is from about 10 micrometers to about 20 micrometers. Suitable nanopores, such as CsgG nanopores, can be used here. Because diPhPC lipid bilayer membranes are known to rupture under constant or long-duration bias voltages greater than about 300 mV, a relatively low bias driving voltage (e.g., V) is required here. 驱动 =120mV) is sufficient as a constant bias voltage to be maintained during and between voltage control pulses, even though this relatively low bias voltage does not optimally reduce the length of time each consecutive monomer unit spends outside the most sensitive sensing aperture of the nanopore, as explained above.

[0084] At the selected driving voltage, to ensure the target polymer advances through the CsgG nanopores in the diPhPC with a defined stepping motion, the amplitude and duration of each voltage control pulse and / or thermal control pulse are selected to overcome the normal force binding the jig to the monomer unit of the target polymer, with a bias driving voltage V less than 120 mV. 驱动 The length of time it takes to drive a polymer forward through a nanopore to a single monomer unit. The amplitude and duration of each voltage control pulse and / or thermal control pulse are also selected to avoid rupture or otherwise damage to the membrane. Typically, for most embodiments, because the voltage control pulse is not constant, the amplitude of the voltage control pulse is greater than the amplitude of the constant driving voltage.

[0085] The relatively brittle diPhPC film can be damaged by a constant bias voltage greater than about 180 mV, but can withstand short-duration pulses of about 1 microsecond with applied voltage amplitudes up to about 500 mV. Therefore, in this example, the voltage control pulses and / or thermal control pulses specified for briefly overcoming the forces binding the clamping component to the monomer units of the polymer in the absence of ATP are less than about 1 microsecond in duration and less than about 500 mV in voltage.

[0086] For this embodiment, the clamp can be implemented as any of a variety of highly progressive DNAases that bind to single strands of DNA. For example, translocation enzymes (e.g., helicases or polymerases) can be used as the clamping component. Among helicases, many helicases that bind to unpaired single-stranded regions of additional dsDNA are available, as explained in von Hippel et al., Cell, 104:177-190, 2001, which are incorporated herein by reference in their entirety.

[0087] In this embodiment and various embodiments described herein, it is preferable to use only one clamp rather than several clamps to attach to the DNA polymer. As a result, helicases that bind to unpaired single-stranded DNA regions are very suitable clamps, because those skilled in the art of molecular biology understand how to prevent multiple helicases from binding to ssDNA samples by, for example, temporarily converting most of the ssDNA to dsDNA.

[0088] SF1 family helicases, such as T4 Dda helicase, are preferred clamp choices for this embodiment and many other embodiments because the translocation patterns of T4 Dda helicase along the 5' to 3' of ssDNA have been studied at high resolution, and it is clear that most of the approximately 12-14 amino acid contacts of these helicases with the bound DNA are with the sugar-phosphate backbone of the DNA, as explained in Saikrishnan et al., Cell, 137: 849-859, 2009, which is incorporated herein by reference in its entirety. Therefore, it should be understood herein that SF1 family helicases bind equally well to all nucleotide sequences encountered in the DNA strand they bind to.

[0089] Furthermore, members of this transloase family are found in many organisms, and the force required to make the bound helicase slide along DNA in the absence of ATP can vary significantly depending on the organism from which the SF1 family helicase is purified. For example, a clamp that cannot be overcome by a constant bias voltage of less than about 180 mV, but can be overcome by a voltage pulse of more than about 200 mV (optionally coordinated with a thermal pulse, both lasting for a duration of less than about 500 μs) may be preferred.

[0090] In many embodiments, a 532 nm wavelength laser operating at a beam energy of at least about 300 mW can be used to apply a thermal control pulse, either in conjunction with or in place of a voltage control pulse. As explained above, this laser raises the temperature of an ionic solution with a volume of less than about 10 kiloliters directly surrounding a fixture incorporating one or more gold, silver, or other light-absorbing nanoparticles. In this embodiment, the constant background temperature of the medium is preferably maintained at a deviation temperature of about 15°C, and then raised to about 50°C by laser emission synchronized with the voltage control pulse duration.

[0091] The duration between the voltage control pulse and / or thermal control pulse is greater than the minimum time required for single-cell identification at the desired accuracy level, but less than the time required for bias voltage V. 驱动 The shortest time for a clamp to slide a monomer unit under certain conditions. This shortest measured time varies depending on the specific helicase selected. For example, in the absence of ATP, and using the preferred T4 Dda helicase as the clamp, it was found that when V... 驱动At 120mV, durations of less than 1 second between steps are extremely rare. Therefore, the interval between voltage control pulses can be set to be equal to or less than 100 milliseconds, a condition that can be easily achieved. This avoids undetectable slippage due to the driving voltage, thus ensuring that the pulse count applied to drive the full length of the target DNA polymer through the nanopore will be equal to the number of monomer subunits (i.e., nucleobases) in the polymer length.

[0092] In another embodiment, an implementation with example parameters for determining the chemical and monomer sequence along the length of the target polymer involves selecting a nanopore system, fixture, and control pulse to determine the chemical properties of the target polymer and the sequence of monomer subunits along the target polymer. As described above, this embodiment considers the target polymer molecule DNA, whose nucleobases are monomer subunits of the polymer. Because more detailed information about the monomer subunits is required here, a larger bias voltage than in existing embodiments is needed to drive the polymer through the nanopore to minimize the effects of Brownian motion, which would otherwise degrade the detailed information. As a result, some parameters that are the subject of this embodiment and those of other commonly used features of nanopore sensing differ from those in previous embodiments.

[0093] Because this implementation requires a bias drive voltage V greater than that of the previous implementation. 驱动 And therefore due to V 驱动 +V 脉冲 The amplitude of the combined voltage is correspondingly greater than that of the previous embodiments, therefore a less fragile and more robust membrane is preferred here than that used in the previous embodiments. Suitable membranes include solid membranes, such as graphene membranes, silicon nitride membranes or other suitable solid membranes, or amphiphilic triblock copolymer membranes, as described by Zhao et al., Science, 279:548-552, 1998, which are incorporated herein by reference in their entirety, such as poly(dimethylsiloxane)-block-poly(2-methyloxazoline)-poly(dimethylsiloxane) or mycoic acid membranes, as described by Langford et al., J. Lipid Res., 52:272-277, 2011, which are incorporated herein by reference in their entirety. All of these can form a more robust ion-impermeable membrane compared to diPhPC lipid bilayers.

[0094] Membranes extending across relatively small pores in the support structure (e.g., support structure pores less than 20 micrometers in diameter) are also preferred because they are more robust than membranes formed across more conventional support structure pores ranging from 10 to 50 micrometers in diameter. Nanopores of the selected diameter (e.g., greater than 1 nm in diameter, or no more than about 2 nm in diameter) can directly pierce the membrane, such as graphene membranes, or bioporous nanopores, such as mutants of the CsgG porin, can be preferred protein nanopores provided in the membrane. Any of these is more easily distinguishable between DNA nucleobases than α-hemolysin, as described by Howorka in US20180148481, the entire text of which is incorporated herein by reference.

[0095] Here, a bias drive voltage of approximately 140mV to approximately 250mV can be preferred. 驱动 This minimizes the number of monomers that contribute to each ion current measurement by minimizing Brownian motion and by minimizing the time each continuous monomer unit spends outside the most sensitive, narrow, high-resistivity region of the nanopore. For an exemplary drive voltage of approximately 120 mV, refer to... Figure 14 A to Figure 14 C can understand this condition. By applying a 120mV driving voltage 38 across the nanopore 20, a monomer unit 42 is positioned at the highly sensitive site 44 of the nanopore. Brownian motion can rapidly drive the monomer unit away from the highly sensitive site, such as... Figure 14 As shown in B. By applying a large bias drive voltage, for example greater than 125mV, such as... Figure 14 As shown in Figure C, subunit 42 returns to the highly sensitive site more quickly, thereby improving monomer recognition. Therefore, a relatively high bias drive voltage greater than about 120 mV is preferably used, or a variable voltage to counteract Brownian motion can be used.

[0096] In this embodiment, to apply the thermal pulse, for example, a 532 nm wavelength laser operating with a beam energy of at least about 300 mW can be used to raise the temperature of an ionic solution with a volume of less than about 10 kiloliters directly surrounding the fixture, wherein the gold nanoparticles are bonded to the fixture or nanopores in the manner described above. The constant background temperature of the solution, preferably maintained at about 10°C, is raised to about 50°C by the laser, and the pulse is controlled in the manner described above to be consistent with the voltage control pulse.

[0097] When selecting a relatively robust membrane with a support structure aperture diameter of less than approximately 10 micrometers, a constant bias voltage greater than approximately 500 mV can cause the membrane to rupture, but it can withstand voltage control pulses with a pulse duration of at most approximately 1 to approximately 3 microseconds and an amplitude of less than approximately 900 mV. Therefore, in the absence of ATP, the voltage control pulse used to briefly overcome the force binding the clamping component to the polymer monomer unit preferably has a duration of less than approximately 3 microseconds and a voltage amplitude of less than approximately 900 mV.

[0098] In the embodiments described herein, SF1 family helicases are preferred because members of this transloase family are found in many organisms, and the force required for helicases to slide along DNA in the absence of ATP varies significantly depending on the organism from which the SF1 family helicase is purified. For this example, binding to the DNA strand can be overcome with a control pulse duration of less than about 500 ns and an amplitude of less than about 900 mV, but a clamp that cannot overcome DNA binding with a bias drive voltage of less than about 500 mV may be preferred.

[0099] As in the previous embodiments, the duration between control pulses is selected here to be greater than the shortest time required for monomer recognition at the desired level of accuracy, but less than the shortest time known to be characteristic of the sliding clamp only under bias drive voltage conditions. This shortest measured time of sliding will vary depending on the specific helicase selected. In the absence of ATP and using a preferred SF1 family helicase as the clamp, the average duration between slides is greater than about 100 ms and decreases exponentially, making it possible to achieve the desired accuracy only under bias drive voltage conditions. 驱动 Sliding from one nucleus base to the next in a span of approximately 15 ms under a bias drive voltage of 300 mV is extremely rare. Therefore, setting the interval between voltage control pulses to occur within an easily implementable duration, such as from approximately 5 to approximately 10 ms, minimizes random sliding and provides deterministic stepping to ensure that each consecutive nucleus base in the DNA sequence is detected and identified by its current signal at the nanopore.

[0100] Experimental Examples

[0101] like Figure 2 As shown, MspA nanopores were provided in a diphyllylphosphatidylcholine membrane and arranged in the nanopore system. DNA with Dda helicase clamps was loaded into the system's cis reservoir in an ionic solution of 1M KCl, 25mM phosphate buffer, 2mM MADPNP, 2mM MgCl2, pH 8.0. The solution was maintained at 37°C. The bias driving voltage across the nanopores was 160mV. Figure 5 Voltage pulse control system and Figure 3A voltage control pulse was applied using a control method. In the first experiment, the duration of the voltage control pulse was 500 microseconds, and in the second experiment, the duration was 1 millisecond. The amplitude of the voltage control pulse was 300 mV in both experiments. The nanopore current was measured and filtered at 2 kHz.

[0102] Figure 15 A and Figure 15 B is a graph showing the measured nanopore current over time when a voltage control pulse is applied, and it displays the change of the applied voltage across the nanopore over time for voltage control pulses of 500 microseconds and 1 millisecond durations, respectively. The measured nanopore ion current is shown as the upper line, with its axis on the left side of the graph, while the applied voltage pulse is shown as the lower line, with its axis marked on the right side of the graph. As shown in the two data graphs, the change in the measured ion current through the nanopore versus the applied voltage control pulse V 脉冲 Consistent in time. This indicates a response to a single applied voltage control pulse V. 脉冲 The helicase clamp advances one monomer along the length of the DNA strand. Figure 15 The capacitive current spike overshoot shown in the applied pulse in Figure B is a result of insufficient compensation by the electronic equipment during a voltage control pulse of relatively long duration of 1 millisecond.

[0103] This description, the embodiments provided herein, and experimental examples demonstrate that the nanopore system, fixture control, and methods presented herein enable deterministic rather than random translocation of target polymer molecules through nanopores, and allow for the characterization of linearly connected, continuous polymer subunits of the target polymer using nanopores. This makes accurate target polymer evaluation, which has been difficult to achieve until now, possible.

[0104] It should be understood that although preferred embodiments have been described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, etc., may be made without departing from the spirit of the invention, and therefore such modifications, additions, substitutions, etc., are considered to be within the scope of the invention as defined in the appended claims.

Claims

1. A nanopore system for characterizing target polymer molecules, said target polymer molecules being selected from nucleic acid polymer molecules and protein polymer molecules, and comprising a plurality of continuous polymer subunits along the length of the target polymer molecule, said system comprising: A first fluid reservoir and a second fluid reservoir are in fluid communication with a nanopore, wherein the nanopore forms a unique fluid path between the first fluid reservoir and the second fluid reservoir. A clamp comprising a biomolecular clamp that can bind to a nucleic acid polymer and is selected from enzyme clamps, biochemical complex clamps, and protein clamps, the clamp being provided in a first fluid reservoir, the clamp being adjacent to the nanopore and reversibly bound to a plurality of successive polymer subunits of a target polymer molecule in an ionic solution in the first fluid reservoir, the clamp having an outer diameter greater than the diameter of the nanopore; A circuit comprising electrodes in a first fluid reservoir, electrodes in a second fluid reservoir, and a current amplifier for applying a constant bias voltage across the nanopore between the first and second fluid reservoirs to induce the target polymer molecules to travel into the nanopore. A voltage pulse generator, connected to the circuit, to apply voltage pulses to the first fluid reservoir and the... A control pulse is applied across the nanopore between the second fluid reservoirs to cause the clamp to step along the continuous polymer subunits of the target polymer molecule in a direction away from the nanopore, further stepping the target polymer molecule into the nanopore. The system does not include fuel for the clamp and does not include a fuel source for the clamp. A computer controller, connected to the circuit, is configured to collect an electrical indication of the ionic current passing through the nanopore as a series of polymer subunits of the target polymer molecule step through the nanopore.

2. The nanopore system of claim 1, wherein the clamp comprises a helicase.

3. The nanopore system of claim 2, wherein the clamp comprises an SF1 family helicase.

4. The nanopore system of claim 3, wherein the clamp comprises T4 Dda helicase.

5. The nanopore system of claim 1, wherein the clamp comprises a polymerase.

6. The nanopore system of claim 1, wherein the nanopore comprises a bio-nanopore disposed in a membrane, the membrane being selected from triblock copolymer membranes, mycoic acid membranes, lipid bilayer membranes, and tetraether lipid membranes.

7. The nanoporous system of claim 6, wherein the membrane comprises a diphyllylphosphatidylcholine (diPhPC) membrane.

8. The nanopore system according to claim 6, wherein the nanopore comprises a bio-nanopore selected from CsgG bacterial porin nanopores and Mycobacterium smegmatis porin A (MspA) nanopores.

9. The nanopore system according to claim 1, wherein the diameter of the nanopore does not exceed 2 nm.

10. The nanopore system of claim 1, wherein the current amplifier provides an analog output measurement of the nanopore current.

11. The nanopore system of claim 10, further comprising an analog-to-digital converter connected in the circuit to the current amplifier to digitize the analog output measurement of the nanopore current.

12. The nanopore system of claim 11, wherein the controller is connected in circuitry to the analog-to-digital converter to control the recording of digital indications of polymer subunits of the target polymer molecule.

13. The nanopore system of claim 1, wherein the target polymer molecule is a double-stranded target polymer molecule, the double-stranded target polymer molecule having single-stranded regions, and the clamp reversibly attaching to the single-stranded regions along the double-stranded target polymer molecule.

14. The nanopore system according to claim 1, wherein the target polymer molecule is a single-chain target polymer molecule.

15. The nanopore system of claim 1, wherein the target polymer molecule is a polymer molecule selected from DNA, RNA and PNA.

16. The nanopore system of claim 1, wherein the nanopore comprises channels in an atomically thin solid material.

17. The nanopore system of claim 1, wherein the sequential plurality of polymer subunits reversibly coupled by the clamp comprises 1 to 20 polymer subunits.

18. The nanoporous system of claim 1, wherein the ionic solution comprises ADPNP.

19. The nanoporous system of claim 1, wherein the ionic solution comprises a buffer solution.

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