Deterministic steps of polymers passing through nanopores

Abstract

To provide characterization of polymers by translocation of polymers through a nanopore.SOLUTION: In a method for controlling translocation of a target polymer molecule through a nanopore, a clamp is reversibly bound to a sequential plurality of polymer subunits along the target polymer molecule length and the molecule and clamp are disposed in an ionic solution that is in fluidic communication with the nanopore. A constant translocation force is applied across the nanopore to induce travel of the target polymer molecule into the nanopore, until the clamp abuts the nanopore aperture and stops further travel of the target polymer molecule into the nanopore. Then a voltage control pulse is applied across the nanopore and / or a thermal control pulse is applied at the nanopore, with a pulse duration that steps the clamp along the target polymer molecule by no more than one polymer subunit in a direction opposite that of travel into the nanopore. No fuel is provided to the clamp.SELECTED DRAWING: Figure 1A-1C

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the interests of U.S. Provisional Patent Application No. 62 / 526,823, filed on 29 June 2017, which is incorporated herein by reference in its entirety.

[0002] Statement regarding federally funded research This invention was made with government support under contract number R01HG003703, awarded by the NIH. The government has certain rights to this invention.

[0003] background This invention relates to the characterization of polymers by the movement of polymers through nanopores, and more specifically, to the control of polymer steps through nanopores. [Background technology]

[0004] Polymers and their subunits can be characterized by measuring the change in the electrical conductivity of a nanopore through which the polymer is stepped. As described in 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 their entirety), the properties of polymers determined using nanopores include chemical and physical properties of the polymer and its monomer units, including concentration, e.g., the number of molecules in a sample solution, the length of the polymer, the number of monomer units along the length of the polymer, and specific sequences of consecutive monomer units. In one example of a nanopore-based characterization method, the polymer under study is supplied to an ionic solution in a cis reservoir that is in fluid communication with the aforementioned nanopores. The polymer is moved from the cis reservoir to a trans-reservoir through the nanopores, and during this time, the movement of the nanopores can be detected, and the properties of the polymer are determined. As described in Kasianowicz et al., Proc. Natl. Acad. Sci., 93:13770-13773, 1996 (which is incorporated herein by reference in its entirety), the electric field generated by applying a constant voltage bias between cis and trans-reservoir electrodes is commonly used to drive polymers through nanopores. However, as described in Lu et al., Nano Lett.. 13:3048-3052, 2013 and Keyser et al., Nature Physics, 2:473-477, 2006 (both of which are incorporated herein by reference in their entirety), other forces can be used instead, such as hydrostatic pressure, optical or magnetic field forces, and combinations of two or more of these forces, to drive polymers through nanopores.

[0005] The concept of binding an active enzyme to a polymer to slow down or delay the polymer, in order to prevent it from being driven freely at an undesirably fast speed through the nanopore, has been proposed and is taught, for example, in Akeson's U.S. Patent No. 7,625,706, Akeson's U.S. Patent No. 7,238,485, Akeson's U.S. Patent No. 7,947,454, Akeson's U.S. Patent No. 8,673,556, Cherf's U.S. 20140051068, Moysey's U.S. 20140335512, and Jayasinghe's U.S. 20150031020 (all of which are incorporated herein by reference in their entirety). Such an active enzyme, e.g., polymerase or helicase, walks along the polymer, depending on the energy derived from a chemical substrate such as adenosine triphosphate (ATP), and accordingly causes the polymer to step through the nanopore. The ATP-dependent movement of the active enzyme drives the polymer through the detection nanopore at a rate corresponding to the enzyme's turnover rate, 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 was found that the turnover rate has little effect on the behavior of the single molecule's activity, even when the time interval between each active step of a single enzyme molecule changes probabilistically, causing the enzyme's turnover rate to fluctuate.

[0006] The probabilistic time intervals between successive steps of a single molecule in and through a nanopore (e.g., the probabilistic time intervals between steps of adjacent monomer units of a polymer passing through a nanopore) pose a significant problem. This is because during these intervals, the electrical 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 so short as to cause errors of omission, whereas others may be so long as to be misinterpreted as a continuous sequence of the same monomer unit when in fact there is no such continuous sequence of the same monomer. Also, it is not possible to distinguish a homopolymer region of length X (a sequence of four identical monomers) from homopolymer regions of length X+1, X+2,... or X+n.

[0007] Summary In a method for controlling the movement of a target polymer molecule through a nanopore provided herein, a clamp reversibly binds to a plurality of successive polymer subunits along the length of the target polymer molecule. The target polymer molecule comprises a plurality of successive polymer subunits along the length of the target polymer molecule. The target polymer molecule and the reversibly bound clamp are disposed in an ionic solution in fluid communication with the nanopore. The nanopore has an aperture diameter smaller than the outer diameter of the clamp.

[0008] To induce the movement of the target polymer molecule in the ion solution into the nanopore, the clamp on the target polymer molecule abuts against the opening of the nanopore, and a constant moving force is applied to the nanopore until the further movement of the target polymer molecule into the nanopore stops. Next, at least one control pulse of the voltage control pulse and the thermal control pulse applied to the nanopore is applied. The control pulse has a control pulse duration that steps the clamp along the target polymer molecule by one polymer subunit in a direction opposite to the direction of movement into the nanopore. No fuel is supplied to the clamp. By the step of the clamp, the target polymer molecule further steps into the nanopore, but by one polymer subunit. The control pulse is repeatedly applied to move a plurality of consecutive polymer subunits of the target polymer molecule through the nanopore.

[0009] This movement control method enables a method for characterizing a target molecule, and when the polymer subunit is in the nanopore, an index representing the characteristics of the polymer subunit is obtained. Here, the control pulse is repeatedly applied to move a plurality of consecutive polymer subunits of the target polymer molecule through the nanopore while obtaining an index representing the characteristics of each polymer subunit moving through the nanopore.

[0010] The nanopore system for characterizing a target polymer molecule provided herein includes a first fluid reservoir and a second fluid reservoir, and the first and second fluid reservoirs are in fluid communication by a nanopore forming the only flow path between the first fluid reservoir and the second fluid reservoir. The clamp is provided in the first fluid reservoir. The clamp abuts against the nanopore and reversibly binds to a plurality of consecutive polymer subunits of the target polymer molecule in the ion solution in the first fluid reservoir. The target polymer molecule includes a plurality of consecutive polymer subunits along the length of the target polymer molecule.

[0011] A circuit is provided, which includes an electrode in the first reservoir, an electrode in the second reservoir, and a current amplifier for applying a constant voltage bias to the nanopore between the first and second reservoirs to induce the migration of the target polymer molecules into the nanopore. A voltage pulse generator is connected to the circuit to apply voltage-controlled pulses to the nanopore between the first and second reservoirs, to step the clamp away from the nanopore with each polymer subunit along a sequence of polymer subunits of the target polymer molecule, and to further step the polymer molecule into the nanopore. The system does not include fuel for the clamp and a fuel source for the clamp. A computer control unit is connected to the circuit to collect an electrical index of the ionic current through the nanopore while a plurality of consecutive polymer subunits of the target polymer molecule step through the nanopore.

[0012] Furthermore, the nanopore system for characterizing the target polymer molecule provided herein comprises a first fluid reservoir and a second fluid reservoir, the first and second fluid reservoirs being fluidly connected by a nanopore forming a single channel between the first and second fluid reservoirs. A clamp is provided within the first fluid reservoir. The clamp abuts against the nanopore and reversibly bonds to a plurality of consecutive polymer subunits of the target polymer molecule. The target polymer molecule comprises a plurality of consecutive polymer subunits along the length of the target polymer molecule.

[0013] A circuit is provided, which includes an electrode in the first reservoir, an electrode in the second reservoir, and a current amplifier for applying a constant voltage bias to the nanopore between the first and second reservoirs, in order to guide the migration of the target polymer molecules into the nanopore. A laser is connected to the computer control unit to generate laser pulses, and an optical system is positioned adjacent to the laser and aligned so that the laser pulses are directed toward the nanopore. A material element that absorbs energy from the laser pulses is placed in the nanopore and heats the ionic solution between each laser pulse to then step the clamp toward the nanopore, polymer subunit by polymer subunit along a sequence of polymer subunits of the target polymer molecule, and further step the polymer molecule toward the nanopore. The system does not include fuel for the clamp and a fuel source for the clamp. The computer control unit is connected to the circuit to collect an electrical indicator of the ionic current passing through the nanopore while a plurality of consecutive polymer subunits of the target polymer molecule step through the nanopore.

[0014] The nanopore systems and methodologies provided herein enable deterministic control of the movement of target polymer molecules through nanopores, and enable a correspondingly superior degree of control in the characterization of target polymer molecules. These and other aspects and embodiments of the disclosure are described below, in the accompanying drawings and in the claims. [Brief explanation of the drawing]

[0015] [Figure 1A-1C] Figures 1A, 1B, and 1C are schematic diagrams of the three deterministic steps of clamp-linked polymer molecules passing through biological nanopores within biological membranes. [Figure 1D-1F] Figures 1D, 1E, and 1F are schematic diagrams of the three deterministic steps of clamp-linked polymer molecules passing through nanopores in a solid film. [Figure 2]Figure 2 is a schematic diagram of a nanopore system containing clamp-linked polymer molecules provided to a cis-reservoir for a deterministic step through the nanopore to a trans-reservoir. [Figure 3] Figure 3 is a flowchart of the methodological steps for performing a deterministic step of clamp-linked polymer molecules through a nanopore. [Figure 4A-4D] Figures 4A, 4B, 4C, and 4D are plots of the nanopore current or applied control pulse voltage measured when performing the steps of the flowchart methodology in Figure 3. [Figure 5] Figure 5 is a schematic diagram of a nanopore system and associated electronic equipment for applying voltage-controlled pulses during the deterministic steps of a clamp-linked polymer passing through a nanopore. [Figure 6] Figure 6 shows an electrical circuit model of a nanopore system including electronic equipment that applies a control pulse voltage. [Figure 7] Figure 7 is a schematic block diagram of the low-noise voltage pulse generator of the system shown in Figure 5. [Figure 8] Figure 8 is a schematic diagram of a nanopore system and related components for implementing the application of thermally controlled pulses in the deterministic steps of clamp-linked polymers through nanopores. [Figure 9A-9B] Figures 9A and 9B are schematic diagrams of gold nanoparticles (NPs) bonded to a clamp and gold nanoparticles (NPs) bonded to a nanopore, respectively, for providing an absorbent material to the nanopore system of Figure 8. [Figure 10] Figure 10 is a schematic diagram of polymerase synthesis. [Figure 11] Figure 11 is a plot of nanopore currents measured as a function of the five surrounding nucleic acid bases adjacent to the detected monomer, for ATP-driven polymer steps and voltage-biased polymer steps without ATP, both using T4 helicase polymer clamps. The DNA sequences shown above the plot are read as a series of overlapping pentamers shown below the horizontal axis. [Figure 12] Figure 12 is a plot of the median measured durations for full-length polymer migration using T4-phase helicase as the polymer clamp, with 2 mM ADPNP and no ATP analog. The vertical capped lines indicate the range of durations for several identical polymers to pass through the nanopore completely. [Figure 13] Figure 13 plots the median monomer step duration as a function of applied bias drive voltage for four different temperatures, using a T4 phase helicase-clamp coupled DNA molecule. [Figure 14A-14C] Figures 14A, 14B, and 14C are schematic diagrams illustrating polymer molecule control by a bias drive voltage applied to overcome the Brownian motion of polymer molecules within a nanopore. [Figures 15A-15B] Figures 15A and 15B are plots of the nanopore current and applied voltage-controlled pulse amplitude, measured as a function of time, for an experimental nanopore system using a bias drive voltage of 160 mV. [Modes for carrying out the invention]

[0016] Detailed explanation This specification provides a structure, system, and corresponding methodology for a deterministic, rather than stochastic, step of a target polymer molecule passing through a nanopore in a nanopore system as shown in Figure 2. In one embodiment, this structure, system, and methodology drives linearly connected consecutive monomer residues of a target polymer molecule through a nanopore by precisely deterministic motion, thereby determining the exact number of identical monomers that have traveled through the nanopore by the time it takes for a repeating sequence of identical monomer units to travel through the nanopore. Specifically, when the time interval between steps of each monomer passing through the nanopore is a known constant k, i.e., when the steps are deterministic as made possible herein, the exact number of identical monomers that have traveled through the nanopore is determined by dividing the total time it takes for a repeating sequence of identical monomer units to traverse the nanopore by the interval constant k.

[0017] As shown in Figures 1A-1C, the Specified Specification provides a deterministic clamp 10 structure which reversibly clamps or reversibly binds to a target polymer molecule 14 such as a DNA strand, or to a plurality of monomers 12, such as a group of 1 to 20 monomers, along the molecular length of another suitable polymer molecule. As shown in Figures 1A-1C, the outer diameter 16 of the clamp 10 is greater than the diameter 18 of the opening or channel extending through the nanopore 20.

[0018] As used herein, the term “polymer molecule” refers to biomolecules, such as biopolymer nucleic acid molecules, polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), synthetic nucleic acids such as peptide nucleic acid (PNA), and proteins, glycopolymers, and other biomolecules. Therefore, the following description is not intended to limit itself to any particular embodiment. Details relating to examples of these molecules, such as polynucleotides, are provided to illustrate the principles provided herein, within the scope of embodiments for characterizing polymer molecules.

[0019] Referring also to the cross-sectional view in Figure 2, this clamp-linked molecular structure can be used in a nanopore system 25 that characterizes polymer molecules. The nanopore system generally includes a structure 26, such as a membrane, on which the nanopores 20 are arranged, and either or both of these can be substantially biological or solid. The membrane or other structure 26 on which the nanopores are arranged can be supported at its ends, for example, by a support structure 28, if necessary. The structure 26 and the support structure 28 are arranged to separate the cis reservoir 30 from the trans reservoir 32, and the nanopores provide a single fluid pathway between the two reservoirs. The cis and trans reservoirs contain an ionic liquid solution, and the cis reservoir contains target molecules 14 that move through the nanopores 20 to the trans reservoir. Each target molecule 14 in the cis reservoir moving through the nanopores has a reversible clamp 10 positioned along the molecular length on multiple polymer subunits, forming a target molecule clamp complex. Such a solution of target molecule clamp complexes can be prepared by conventional methods, for example, as described by Moysey in U.S. Patent Application Publication No. 20140335512, which is incorporated herein by reference in whole. For the analysis of target molecules by migration through nanopores, the target molecule clamp complex is provided in a cis reservoir.

[0020] 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 them. The electric field generated by applying such a voltage bias between the cis and trans reservoir electrodes 34 and 36 can be used in the conventional way to drive charged molecules in the cis reservoir through the nanopore to the trans reservoir. However, it should be recognized that other mobilization forces can be used instead. Therefore, in the description of the step control methodology herein, "voltage bias" or V drive Although the term is used, other forces, including hydrostatic pressure, optical fields, or magnetic fields, and combinations of two or more fields, can also be used to drive the polymer through the nanopore. Hereafter, "constant voltage drive bias" or V driveWhere the term is used herein, it should be understood that one or more of these sites may be substituted. Further details and examples of electrophoretic-driven molecular transfer through nanopores are described in Branton et al., U.S. Patent No. 6,627,067, “Molecular and Atomic Scale Evaluation of Biopolymers,” published September 30, 2003, which is incorporated herein by reference in its entirety.

[0021] Herein, referring to the methodology made possible herein, as shown in Figure 1A, the target polymer molecule 14 under investigation, including a reversibly bonded clamp 10, is shown in the nanopore 20. As shown in Figure 1B, a voltage bias V is applied between the cis and trans reservoirs. drive A voltage is applied, providing conditions that promote the migration of the target polymer 14 into the nanopore 20, as shown in Figure 2. As shown in Figure 1A, the target polymer 14 moves into the nanopore, and as shown in Figure 1B, the migration automatically stops when the clamp 10 contacts a region 40 of the nanopore 20 where the inner diameter 18 is smaller than the outer diameter 16 of the clamp. Under these conditions, one polymer subunit 42 of the polymer 14, for example, one monomer, occupies a portion within the nanopore, and in a preferred embodiment, occupies a portion of a narrow, highly sensitive region 44 of the nanopore 20. As a result, as long as the subunit 42 remains in a suitable location within the nanopore, for example, in a highly sensitive region of the nanopore, the electrical and volumetric properties of the subunit 42 govern the cis-to-trans conductivity of the nanopore in this state. Therefore, by sensing, detecting, measuring, or otherwise determining parameters of the nanopore system related to the conductivity of the nanopore, an index characterizing the subunit 42 under these conditions can be obtained.

[0022] After the indicators characterizing subunit 42 are determined, for example, as shown in Figure 1C, a voltage-controlled pulse V pulse and / or thermal control pulse H pulse The control pulse provided as is the bias drive voltage V applied to the nanopore. driveIn addition to the above, a control pulse is provided, which causes the clamp 10 to step along the polymer 14 away from the nanopore 20 in the direction indicated by arrow 44. The clamp 10 is specifically driven controllly, causing one polymer subunit to step along the polymer and reversibly bond to the next consecutive group 12 polymer subunits. As a result, subunit 42 that was in the nanopore now moves to the solution in the trans reservoir, and the next subunit 46 dominates the cis-to-trans conductivity of the nanopore. This process continues with the deterministic stepping of the clamp away from the nanopore along the polymer, with polymer molecules stepping in and passing through the nanopore one subunit at a time. No energy is supplied to fuel the stepping of the clamp from one polymer subunit to the next. Specifically, neither chemical nor biochemical fuels are supplied to the clamp. Only by the application of a control pulse does the clamp step from one polymer subunit to the next.

[0023] Figures 1A-1C show a target polymer molecule 14 having a linear sequence of subunits, which are represented as simple circles in the figures. This representation is provided solely for clarity. The target polymer molecule loaded into the cis reservoir may be single-stranded, like ssDNA, or double-stranded, like dsDNA. In either case, the clamp is positioned at a site on the region of the single-stranded polymer molecule. Therefore, a double-stranded target polymer molecule may have a single-stranded region that allows the clamp to be positioned along the single-stranded region of the double-stranded target molecule, and the clamp can be positioned in this single-stranded region in the conventional manner.

[0024] The state in Figure 1A-1C illustrates one embodiment provided as a biological nanopore 20 in membrane 35, the membrane may be biological or organic and can be, for example, a lipid bilayer, a tetraether lipid, or a triblock copolymer. For example, the nanopore can be provided as an octameric protein channel, Mycobacterium smegmatis porin A (MspA), as a nonamermeric protein channel, as bacterial porin CsgG, or as variants thereof, or as other channels, such as those taught by Moysey in U.S. Patent No. 9,617,591 (which is incorporated herein by reference in its entirety). The selected biological nanopore is arranged in a solid structure, such as, for example, a diphytanoylphosphatidylcholine membrane (diPhPC), a tetraether lipid, a triblock copolymer, or a formed solid membrane. Such solid films can be provided, for example, as one or more graphene layers, SiNx, or other solid supports through which ions cannot pass, but which can have any of the aforementioned bio-nanopores arranged in such a way that they obstruct the cis-to-trans flow of ions (except through the channels of the nanopores themselves).

[0025] The polymer capture and stepping state in the nanopore shown in Figures 1A-1C, also shown in Figure 1D-1F, is one embodiment of a solid nanopore system. As shown in Figure 1D-1E, a constant voltage bias V is applied between the cis and trans reservoirs. driveA fixed bias voltage is applied, and as shown in FIG. 2, a state is provided at the nanopore site 20 that promotes the capture of the target polymer 14 including the clamp 10. Here, the nanopore 20 is an opening, hole, channel, or other opening disposed in the solid membrane 37. The membrane can be formed of any suitable solid material as described above, such as graphene or other atomically thin material, or a microelectronics material such as a nitride, oxide, or combination material layer. Next, the solid membrane can be supported by a support structure such as a microelectronic substrate, as in the membrane 26 and support structure 28 of FIG. 2. For clarity, the system 25 of FIG. 2 is shown with the nanopore 20 which is an opening in the membrane 26, but it should be recognized that any suitable combination of biological, organic, and solid nanopores and membranes can be used for the nanopore system 25 of FIG. 2.

[0026] Due to the fixed bias voltage, the target polymer 14 moves through the nanopore. Thus, as shown in FIG. 1E, when the clamp 10 abuts against the nanopore 20, the movement automatically stops. The diameter 18 of the nanopore is smaller than the outer diameter 16 of the clamp, as shown in FIG. 1D. In this state, one subunit 42 of the target polymer 14, for example, one monomer, occupies the most sensitive region of the nanopore. As a result, the electrical and volume characteristics of the subunit 42 dominate the conductivity from the cis to the trans of the nanopore in this state as long as the monomer subunit 42 remains within the nanopore. As a result, by detecting, detecting, measuring, or otherwise determining the parameters of the nanopore system related to the conductivity of the nanopore, an index representing the characteristics related to the subunit 42 in this state is obtained.

[0027] After the index indicating the characteristics of the subunit 42 is determined, as shown in FIG. 1F, a control pulse is applied, for example, as a voltage control pulse V pulse and / or a thermal control pulse H pulse as a bias drive voltage V driveProvided in combination with the clamp 10, the clamp 10 steps away from the nanopore 20 along the target polymer 14 in the direction indicated by the arrow 44. Specifically, the clamp 10 is driven to step along the polymer by one monomer subunit at a time, then to bind to the next group of polymer subunits. As a result, subunit 42 of the nanopore moves to the trans reservoir, and the next subunit 46 governs the cis-to-trans conductivity of the nanopore. This process with respect to the deterministic stepping of the clamp along the target polymer continues, with polymer molecules stepping in and passing through the nanopore one subunit at a time.

[0028] Figure 3 is a flowchart of the steps of the method for carrying out the conditions shown in Figures 1A-C and 1D-F, and Figures 4A-4D are plots of measured electrical signals showing the conditions for each step of the method. Methodology 50 involves assembling a nanopore system and initiating the method 52 with target polymer molecules coupled to clamps placed in a cis reservoir, and in the first step 54, a constant bias drive voltage V drive This is applied between the cis and trans reservoirs. In the next step 56, it is determined whether the target polymer has been trapped in the nanopore. Figure 4A is a plot of the current as a function of time measured for the nanopore, showing that when the target polymer is trapped in the nanopore, the measured current through the nanopore decreases dramatically from the open-pore current level to a lower level indicating polymer molecule trapping in the nanopore. If it is determined that the polymer has not been trapped in the nanopore, the constant voltage bias is maintained and the determination is held until it is confirmed that the polymer has been trapped in the nanopore.

[0029] In the next step 58, in addition to the bias voltage, control pulses such as voltage control pulses and thermal control pulses are applied between the cis and transformer reservoirs. In one embodiment, here with respect to the voltage control pulse, a voltage pulse is applied to the nanopore as shown in the plot in Figure 4B. Next, as shown in the plot in Figure 4C, the total voltage applied to the nanopore includes the sum of the constant bias voltage and the pulse voltage. The timing and duration of each pulse can be controlled manually or by an automated system that can provide continuously repeating pulses. Since the polarity of the bias voltage and the pulse voltage are the same with respect to the nanopore, the sum of the bias voltage and the voltage pulse is additive and directed in the direction that drives the polymer through the nanopore for a given polymer charge.

[0030] In the next step 60, it is determined whether the target polymer is stepping through the nanopore in response to the applied control pulse. The plot in Figure 4D shows an example where current measurement can determine that the target polymer takes a single step of one polymer subunit in response to a single voltage pulse. If it is determined that the polymer is not taking a single step in response to a single voltage pulse or a single thermal pulse, the pulse magnitude and / or duration are increased. If it is shown that the polymer is stepping through the nanopore one polymer subunit at a time in response to a single applied pulse, the next step involves obtaining several representative indices that reflect the characteristics of the polymer subunit at the nanopore detection site. For example, as shown in Figure 3, in this step 62, the measured current through the nanopore can be obtained and, if necessary, recorded to reflect the characteristics of the polymer subunit as the polymer moves through the nanopore in response to the control pulse.

[0031] In the final step 64, it is determined whether the nanopore has opened, for example, by determining whether the nanopore current has returned to an open-hole current. If not, step 62, which records the polymer sequence, continues. If it is determined that the nanopore is open, step 54 is performed again, and only a constant bias voltage is applied to the nanopore while waiting for the target polymer molecules to be captured in the nanopore.

[0032] Methodology 50 involves a step of measuring nanopore current to obtain a range of indices indicating various properties of polymer subunits within the nanopore. Generally, obtaining indices indicating the properties of polymer subunits can be carried out, for example, by detecting the presence of polymer subunits within the nanopore, by counting polymer subunits within a series of consecutive subunits, by identifying the number of identical subunits within a series of consecutive subunits, by identifying polymer subunits, by determining the chemical aspects of the subunits, or by determining other characteristic indices of the subunits. A series of subunits along the entire polymer molecule can be characterized in this way, providing the number and identification information of the entire polymer molecule sequence.

[0033] This controlled stepping method can be implemented with any suitable electronic control system. Figure 5 is a schematic diagram of one embodiment in which a system 75 is arranged for implementing the methodology of Figure 3 using a control pulse, which is a voltage-controlled pulse. In system 75, electrodes 34 and 36 are provided in the cis and transformer reservoirs and connected to a low-noise voltage pulse generator 70 and a current amplifier 72. The current amplifier 72 applies a constant bias voltage V between the cis and transformer reservoirs. drive The pulse generator 70 is controlled to apply a bias voltage V when triggered by a control signal from the control computer 74. drive A voltage-controlled pulse V is added. pulseThe current amplifier 72 provides an analog output measurement of the nanopore current, which is digitized by the analog-to-digital converter 76 and provided to the computer 74 for recording. The circuit formed by the cis and transformer reservoir, nanopore, current amplifier, A / D converter, pulse generator, and computer allows for the measurement of the current in the circuit that represents the ionic current passing through the nanopore.

[0034] The system can be made temperature-controllable, if necessary, using an appropriate method such as a thermoelectric heater / cooler. No element of the system supplies energy to the clamp and fuels the clamp's steps along the polymer molecule; no energy source is included in the system. As described above, only voltage-controlled pulses cause the clamp to step along the polymer molecule.

[0035] This system 75 can be electrically modeled in an electrical circuit model 80, as shown in Figure 6. Nanopores in the film give rise to pore resistance and film capacitance. Figure 7 is a schematic diagram of one embodiment of the low-noise voltage pulse generator 70 of Figure 5. The low-noise pulse generator includes control elements that determine the duration between control pulses, as well as the duration and amplitude of the voltage-controlled pulses. This embodiment may be particularly preferred to enable the precise generation of voltage-controlled pulses. In particular, a preferred embodiment, as shown in Figure 7, includes capacitance compensation, +V pulse and -V pulse A voltage is generated, which is provided by a voltage follower-inverter, reducing the "overshoot" caused by capacitance. If the time interval between voltage pulses is set to the minimum interval, for example, to maximize the data output of the nanopore, the duration of the "overshoot" may make it impossible to measure the actual current flowing through the nanopore.

[0036] Figure 8 is a schematic diagram of one embodiment in which a nanopore system 85 is arranged to implement the methodology of Figure 3 with a control pulse, which is a thermally controlled pulse. In system 85, electrodes 34 and 36 are provided in the cis and transformer reservoirs and connected to a current amplifier 72. The current amplifier 72 provides a constant bias voltage V between the cis and transformer reservoirs. drive The system provides the following. A thermal control pulse is generated by control from the computer 74 and provides a trigger pulse control signal to the laser 82 and the optical system 84. Here, the laser 82 and the optical system 84 are positioned such that pulses of laser energy 86 are directed towards the nanopore areas of the film where the material that absorbs the laser energy 86 is located. As described below, the absorption of laser energy by the absorbent material generates pulses of temperature rise near the nanopores. The current amplifier 72 provides an analog output measurement of the nanopore current, which is digitized by the analog-to-digital converter 76 and provided to the computer 74 for recording in the manner of the voltage pulse control system in Figure 5. Similar to that system, the system 85 hereby can control the temperature in any suitable way as needed, for example, using a thermoelectric heater / cooler.

[0037] Figures 9A-9B show embodiments for providing an absorbent material to the nanopore portion of system 85 in Figure 8. In the first embodiment shown in Figure 9A, absorbent particles 90 such as gold, silver, or other suitable nanoparticles (NPs) are provided and attached to the clamp 10 by a molecular linker 92. In a further embodiment shown in Figure 9B, absorbent particles 90 such as gold NPs are attached to the nanopore 20 by a molecular linker 92.

[0038] In both of these embodiments, irradiation of particles, preferably gold nanoparticles (NPs), with laser light is particularly effective for rapid local heating of the solution near the nanopore, as the wavelength of the laser light utilizes the plasmon resonance effect on the gold particles. Due to the highly confined surface plasmon resonance effect of the gold nanoparticles, the absorption of light by the particles is enhanced, so visible laser light incident on the nanoparticles causes a rapid and significant increase in the particle temperature and the adjacent solution temperature. This temperature increase can be estimated from the change in the ionic conductivity of the nanopore. For example, a 532 nm wavelength laser operating at a beam energy of 300 mW can easily raise the temperature of the surrounding region near nanoscale gold particles and NPs from room temperature by about 20°C to about 50°C in nanoseconds. Because the volume heated is small, only a few yoctoliters, this small volume can rapidly return to the temperature of the surrounding solution volume. This is because the thermal energy is rapidly dissipated into the surrounding solution, whose volume is typically at least 16 orders of magnitude larger.

[0039] In a further embodiment, the control system 75 in Figure 5 is combined with the control system 85 in Figure 8 to enable a combination of voltage and thermal control pulses. When an absorber such as gold NP is placed in the nanopore, both voltage and thermal pulses are used for the deterministic movement of monomers in the above method. The timing and duration of each voltage and thermal pulse can be individually controlled by adjusting the thermal pulse control and voltage pulse control, either manually or automatically.

[0040] Focusing on the details of the clamp 10 used in the nanopore system, the clamp shown in Figures 1A-1C can take any suitable configuration and arrangement, thereby allowing the clamp to reversibly clamp or reversibly bind to multiple polymer subunits of a target polymer moving through the nanopore, or to subunits of other common molecules. While the clamp may be a molecule that functions as an enzyme or biochemical fuel-dependent motor in other systems, in this specification, the term “clamp” refers to any biomolecule or non-biomolecular assembly. That is, reversibly binding to a polymer and acting instead of a biochemical fuel-dependent action, using a voltage-controlled pulse, V pulse and / or thermal control pulse H pulse It is a solid assembly that, upon application, can be deterministically driven to step or move in a single, continuous monomer step along the polymer length. No chemicals or other fuels are supplied to the clamp to cause it to step along the sequence of polymer subunits of the target polymer molecule.

[0041] In one embodiment, the clamp is a helicase such as the T4 phage helicase enzyme. In other embodiments, other enzymes, including variants of the T4 phage helicase enzyme, other proteins that bind to nucleic acid polymers, and biochemical or inorganic complexes can also be used as clamps.

[0042] With such clamps and the control system described above, the polymer step methodology provided herein avoids the stochastic movement of enzyme-active molecules by using clamps. Here, the clamps do not require the input of ATP or chemical or biochemical fuels, but instead use voltage-controlled pulses V pulse , and / or thermal control pulse H pulse Also, constant voltage bias V drive The superposition of these elements is deterministically driven. As a result, a method is provided in which a linear sequence of monomers of the target polymer moves through the nanopore in precisely timed step motions. Two features, in particular, make this methodology possible.

[0043] Firstly, unlike the biochemical fuel arrangements for enzymes such as helicases and polymerases that have been used to control the stochastic motion of polymers through nanopores, the methodology here requires that the deterministically driven clamp does not use a chemical energy source to move the clamp along the monomer subunits of the target polymer. Instead, this specification uses a voltage-controlled pulse V pulse and / or thermal control pulse H pulse However, constant drive voltage bias V drive Along with being controllably applied, the polymer bond clamp is very easily released or loosened, thereby allowing the clamp to slide along the polymer by one monomer step, and the polymer is driven, as shown in Figure 1C, so that only one monomer steps through the nanopore. Thus, unless the clamp moves relative to the target polymer as shown in Figure 1C, the bonded clamp prevents or stalls the target polymer from being driven through the nanopore by the driving force that, without the clamp, would rapidly drive the entire length of the polymer through the nanopore. In practice, the clamp acts as a pawl in a clock ratchet wheel, and the bias voltage V drive It functions as a spring or motor that propels the ratchet wheel.

[0044] Secondly, the voltage-controlled pulse and / or thermal-controlled pulse preferably have a duration of nanoseconds to microseconds, e.g., less than 1 millisecond, and the clamping component causes the target polymer chain to pass through the nanopore and step by "sliding" one monomer unit along the polymer with each pulse. Constant voltage bias V drive The components act continuously to electrophoretically or drive the polymer through the nanopore. Here, the bonded clamps cannot move if their diameter is larger than the nanopore. Therefore, the polymer deterministically steps through the nanopore, one monomer unit at a time, as the clamp components slide reversibly, but deterministically, and then reversibly reclamp to another set of monomer units of the polymer.

[0045] In a preferred embodiment, a single voltage-controlled pulse V pulse and / or thermal control pulse H pulse The duration is adjusted so as not to be longer than the time required to instantaneously release the clamp from the polymer, with a constant bias voltage V drive The clamp drives the polymer through the nanopore so that it slides along the polymer by one monomer step in the direction indicated by the upward arrow in Figure 1C, coupling to the next consecutive monomer unit or group of monomer units. This causes the next consecutive monomer unit of the polymer to occupy the nanopore, preferably the narrowest region of the nanopore, which is the most sensitive, and therefore the conductivity of the nanopore is governed by the electrical and volumetric properties of this next consecutive monomer or group of monomers. Thus, these series of voltage and / or thermal control pulses function in the manner of a pawl-release mechanism of the clock system. This causes the series of voltage and / or thermal control pulses to step the entire length of the target polymer being driven through the nanopore.

[0046] Even if clamping is performed as a molecular clamping enzyme and can turn over and step along the polymer using ATP or other biochemical fuels, ATP or a chemical energy source is not supplied in this methodology, and the clamping is V pulse Ya H pulse Note that it can only be slid along its polymer substrate by voltage and thermal control pulses using the following precisely timed voltage control pulses V pulse and / or thermal control pulse H pulse Because the clamp is driven along the polymer, the stepwise motion of the polymer through the nanopore is deterministic.

[0047] Deoxynucleotide triphosphate (dNTP)-driven polymerases and ATP-dependent helicases are both used to probabilistically step DNA through nanopores for sequencing. The step rates of both these and other chemically driven enzymes are known to be accelerated, decelerated, or even stopped by mechanical forces. Furthermore, in the presence of ATP or dNTPs, nucleic acid step enzymes such as helicases and polymerases are known to step monomer by monomer along the polymer substrate monomer without skipping or jumping some monomers, even when mechanically accelerated or decelerated. For example, if clamp 10, shown in Figures 1A-C, is a helicase and steps along the polymer substrate in the direction indicated by the upward arrow in Figure 1C in the presence of ATP, this step becomes faster (but still steps monomer by monomer without skipping) as the voltage bias driving the polymer from the cis reservoir through the nanopore to the trans reservoir increases. This is taught by Moysey in U.S. Patent No. 9,617,591 (which is incorporated herein by reference in its entirety).

[0048] Conversely, referring to Figure 10, when the clamp is processive polymerase 85, as the electrophoretic force of the voltage bias driving the polymer from cis to trans becomes greater, the polymerase stepping along the polymer in the direction of arrow 86 in the presence of dNTPs tends to cause the polymer to step out of the nanopore more slowly (but stepping monomer by monomer without skipping). This is explained in Lieberman et al., J. Am. Chem. Soc., 132:17961-17972, 2010 (the whole of which is incorporated herein by reference). Finally, the fact that F1-ATPase, which normally steps counterclockwise consuming ATP, synthesizes ATP when driven clockwise by purely mechanical force, clearly demonstrates that mechanical forces acting at certain points in a protein machine can drive chemical reactions at catalytic sites. These examples show that appropriately applied mechanical forces do not disrupt or destroy the normal conformational changes of enzymes. Furthermore, these examples provide clear evidence that purely mechanical forces can “delay,” “accelerate,” or even “drive” standard chemical energy-driven steric changes.

[0049] The fact that, in the presence of ATP, mechanical forces can "promote," "delay," or "substitute" changes in the conformation of an enzyme raises the following question addressed by the structures provided herein: In the absence of ATP, i.e., in the absence of fuel, can purely mechanical forces alone cause a conformational change in a polymer-step enzyme, thereby allowing it to slide along the polymer with monomer-by-monomer precision, possessing the same characteristics as ATP-dependent steps? When using the chemical energy derived from the hydrolysis of ATP, enzymes such as helicases undergo conformational changes, thereby advancing the helicase precisely one nucleic acid base at a time along the polymer. If a mechanical force acting alone merely pushes the enzyme clamp at each step, causing an indeterminate number of nucleic acid bases to slide randomly, then the sequence of nucleic acid bases detected by nanopores cannot report the actual sequence of nucleic acid bases within the polymer. This is because the identification of each consecutive nucleic acid base by nanopores occurs in the time interval between each consecutive step.

[0050] Referring here to the plot in Figure 11, the inventors herein have discovered that, in a manner made possible by ATP, but entirely in the absence of ATP, i.e., without fuel supplied to the clamp, the applied force can push the enzyme clamp and advance precisely by one nucleic acid base portion at each step. The plot in Figure 11 shows the current interruption of the nanopore by the vicinity of five nucleic acid bases adjacent to the monomer occupying the narrowest region of the nanopore at each step of the DNA strand. The DNA sequence is read as a series of overlapping pentamers, as shown on the horizontal axis.

[0051] As shown in the plot in Figure 11, V driveFor an applied voltage bias of 160mV, the current flowing through the nanopore at each step of the voltage-biased driven DNA strand (circle) is the same as the current measured at each step of the ATP-driven strand (square) (within the error limits of such current measurements). This indicates that mechanically forced motion, like force with ATP fuel, can advance polymers such as DNA strands by one nucleotide through nanopores, and can do so without using ATP. Therefore, voltage-forced motion can faithfully move target polymer molecules through nanopores, thus enabling a range of current levels similar to when the movement of polymers through nanopores is controlled, for example, by a helicase enzyme with ATP fuel. This clearly demonstrates that, by a sufficiently large, purely mechanical force, what is normally an ATP-dependent enzyme can function as a clamp and move along the length of the DNA strand with monomer-by-monomer step precision, in the absence of ATP, driven by voltage and / or thermal control, just as ATP-dependent DNA motor enzymes do.

[0052] The plot in Figure 11 shows the 5'-to-3' step of a normal, ATP-driven T4 helicase and the 5'-to-3' step of a mechanically driven T4 helicase that functions as a clamp in the absence of ATP, and that the T4 helicase is also mechanically driven in the reverse direction along the DNA strand, i.e., from 3' to 5'. Indeed, considering what was observed by Mulkidjanian et al. (Mulkidjanian et al., Nat. Rev. Microbiol., 5:892-899, 2007, which is incorporated herein by reference in its entirety), it is possible to synthesize ATP by mechanically driving it in the opposite direction using F1-ATPase instead of hydrolyzing it. It is understood that ATP can be produced rather than consumed by driving an ATP-dependent nucleic acid step motor enzyme in the opposite direction to the standard 5'-to-3' or 3'-to-5' movement along DNA. This is described in Ito et al., Nature, 427:465-468, 2004, (which is incorporated herein by reference in its entirety). As a result, in further embodiments provided herein, a bias drive voltage and voltage-controlled pulses and / or thermal-controlled pulses can be used to move the clamp along the polymer molecular length in either of two possible directions.

[0053] The methodology provided herein for control pulses includes a bias drive voltage, V drive , and additional voltage control pulse V pulse Very short intervals, and / or thermal control pulse H pulse Since a combination of shorter intervals at higher temperatures is relevant, an understanding of how higher voltage bias and higher temperature affect the step rate is provided here. Experimental results for these conditions were prepared for voltage and temperature parameters under conditions without pulsing, and T4-helicase was again used as an example of clamping on the DNA strand. Referring to the plot in Figure 12, in the absence of ATP, when a DNA polymer with a fixed clamp is driven through a nanopore by a constant cis-to-trans bias, the median time taken to step the entire length of the polymer through the nanopore is given by the voltage bias V applied to the nanopore.drive It becomes shorter as increases. See also Figure 13. As a result, the median time interval between each monomer step (referred to here as the median step duration) is for the conditions with a T4 helicase clamp and representing the data in the plot in Figure 12, with a voltage bias V drive The duration shortens as the value increases. The median step duration data in the plot in Figure 13 are for the data in Figure 12, and were determined by dividing the measured duration of total molecule movement shown in Figure 12 by the number of known monomers that make up the total length of the moved molecule.

[0054] Several other factors may affect the time interval between each step or the median step duration. For example, the addition of low concentrations of 5'-(β,γ-imide) triphosphate (ADPNP), a non-hydrolyzable analog of ATP, significantly shortens the range of durations for complete passage through the nanopore when many identical polymers step through the nanopore, as shown in the plot in Figure 11. Other factors that may affect the median step duration include, for example, the direction of polymer movement through the clamp, such as 3' to 5' or 5' to 3', and changes in the amino acid composition of the clamp, or the formation of disulfide links between clamp components, or other structures.

[0055] Referring again to Figure 13, as shown in the plotted data in Figure 13, the temperature of the clamp and its immediate surroundings has a particularly significant effect on the median step duration. For example, when using T4 phage helicase as the clamp, it was observed that lowering the temperature below room temperature significantly increased the median step duration, and raising the temperature from 25°C to 37°C shortened the median step duration by more than three times. Neither increasing the voltage nor increasing the temperature altered the precision with which the clamp stepped per monomer along the observed DNA polymer length.

[0056] Focusing on the embodiments of voltage-controlled and thermal-controlled pulses, it is understood that the nanopore system features, clamping components, and the duration and amplitude of the voltage and / or thermal pulses of this methodology are selected together with other commonly used nanopore detection features known to those familiar with nanopore detection and nanopore sequencing methodologies. In one embodiment, the procedure for implementing a control pulse that generates a deterministic step is as follows:

[0057] 1) In the first step, constant bias drive voltage, V drive And all other conditions of the nanopore system are set so that the median step duration is as long as possible and the frequency of short steps is as low as possible. This is, for example, the bias drive voltage V drive This is achieved by setting the voltage between approximately 20mV and 140mV and maintaining the reservoir solvent temperature between approximately 10°C and 15°C. The bias drive voltage V is approximately 120mV or less. drive While it may be possible to achieve a longer median step duration by using [a specific bias], it is understood that biases below approximately 120 mV may not optimally prevent the passage of duration during which consecutive monomer units may diffuse outside the detection aperture region of the nanopore. This is described in Lu et al., Biophys. J., 109:1439-1445, 2015 (which is incorporated herein by reference in its entirety). As described below, such conditions can reduce the nanopore's ability to distinguish different subunits of the polymer. Consequently, in preferred embodiments, the bias voltage V drive The voltage is at least about 120mV, and in a preferred embodiment, it is about 120mV to about 140mV.

[0058] 2) In the next step, the voltage-controlled pulse V pulse or thermal control pulse H pulse or V pulse and H pulseSet the frequency of the simultaneous control pulses. These frequencies depend on the time length between pulses and the bias drive voltage V. drive By simply applying the bias voltage, the step length is set to be significantly shorter than the shortest duration step length measured when no pulse is present. The time between pulses is preferably sufficiently short compared to the shortest duration step length observed when no pulse is present, but preferably sufficiently long so that the electronics of the nanopore system can accurately assess the ion current flowing through the nanopore between each pulse. If the time between pulses is sufficiently short compared to the shortest duration observed when no pulse is present, it is guaranteed that the probability of the clamp being driven to step probabilistically by the bias voltage alone is very small. Therefore, as a result, substantially all clamp steps are driven by the voltage-controlled pulse V pulse and / or thermal control pulse H pulse This is due to the intentional application of a specific force, and the stepwise movement of the polymer through the nanopore is guaranteed to be deterministic, controlled, and repeatable.

[0059] In other words, the duration of each voltage-controlled pulse and / or thermal-controlled pulse is such that the target polymer is reliably advanced through the nanopore in a deterministic step-like motion, depending on the bias drive voltage V drive However, to make it less time-consuming than the time it takes to drive the polymer through the nanopore and advance it by just one monomer unit, the clamp is selected to overcome the standard forces binding the polymer's monomer units.

[0060] Depending on the selected investigation performed in the nanopore system, the details of the target polymer being investigated, and the availability of clamp components and suitable nanopores, several embodiments are provided herein. The following two embodiments illustrate how those skilled in the art of nanopore detection and nanopore sequencing can select suitable features of the systems provided herein and how to set their parameters and other commonly used features of nanopore detection so that they work together to function with one another. DNA is used in the embodiments, and other linearly linked charged polymers, i.e., continuous monomer residues, such as RNA, proteins, and the above molecules, are similarly investigated with suitable clamp components reversibly bound to the polymer.

[0061] In the various embodiments provided herein, single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) is the target polymer molecule. In each embodiment, the nanopore is configured to have a restricting opening through which only one strand of the target polymer can pass, but which has a bound clamp component that prevents this single strand from passing. Among readily available nanopores, organic nanopores such as protein nanopores, or inorganic solid nanopores such as membrane openings, in either case, having a channel opening diameter smaller than the clamp diameter but larger than about 1 nm, is preferred for many embodiments.

[0062] The fabrication, arrangement, and configuration of the nanopores, membranes, and support structures of the nanopore system can be achieved in any suitable way, for example, in the manner described in U.S. Patent No. 9,617,591 of Moysey, U.S. Patent No. 7,468,271 of Golovchenko, U.S. Patent No. 8,698,481 of Lieber, U.S. Patent No. 20120234679 of Garaj, U.S. Patent No. 9,702,849 of Lieber, U.S. Patent No. 9,611,140 of Russo, U.S. Patent No. 9,815,082 of Golovchenko, and U.S. Patent No. 20160231307 of Xie, all of which are incorporated herein by reference.

[0063] Exemplary Parameters for Counting Monomers Along the Length of a Target Polymer In this exemplary embodiment, a nanopore film formed from a lipid bilayer is identified. For example, a diPhPC lipid bilayer extending across an opening in a support structure can be used, the opening of which has a diameter between approximately 10 microns and approximately 20 microns. A suitable nanopore, e.g., a CsgG nanopore, can be used here. Since diPhPC lipid bilayer films are known to be susceptible to breakdown under constant or prolonged voltage biases exceeding approximately 300 mV, a relatively low bias drive voltage is used here as the constant bias voltage, e.g., V drive =120mV is appropriate, and as described above, this relatively low bias voltage is maintained both during and between voltage-controlled pulses, even if it does not optimally reduce the length of time each successive monomer unit spends outside the most sensitive sensing aperture of the nanopore.

[0064] With this drive voltage selected, the amplitude and duration of each voltage-controlled pulse and / or thermal-controlled pulse are then set to ensure that the target polymer advances through the CsgG nanopores of diPhPC in a deterministic step-like motion, under a bias drive voltage of 120mV V drive However, to ensure that the clamp overcomes the standard forces binding to the monomer units of the target polymer, the clamp is selected to overcome the standard forces binding to the monomer units of the target polymer, so that the time required to drive the polymer through the nanopore is less than the length of time it takes to advance the polymer by one monomer unit. The amplitude and duration of each voltage-controlled pulse and / or thermal-controlled pulse are also selected so as not to break or damage the film. Generally, in most embodiments, the voltage-controlled pulse is not constant, so the amplitude of the voltage-controlled pulse is greater than the amplitude of a constant drive voltage.

[0065] The relatively fragile diPhPC film can be destroyed by a constant voltage bias greater than approximately 180mV, but can withstand short, ~1 microsecond pulses with voltages up to approximately 500mV. Therefore, in this example, voltage-controlled and / or thermal-controlled pulses used to easily overcome the forces binding the clamp component to the polymer monomer units in the absence of ATP are specified to have a duration of less than approximately 1 microsecond and a voltage of less than approximately 500mV.

[0066] In this embodiment, clamping can be carried out as any of a broad range of highly processable DNA enzymes that bind to a single strand of DNA. For example, translocases such as helicases and polymerases can be used as clamping components. Among helicases, many are available that bind to unpaired single-stranded regions where the other regions are dsDNA. These are described by von Hippel et al., Cell, 104:177-190, 2001 (which is incorporated herein by reference in its entirety).

[0067] In this embodiment and various embodiments of this specification, it may be preferable to use only one clamp rather than several clamps attached to the DNA polymer. As a result, a helicase that binds to an unpaired single-stranded DNA region is a particularly well-suited clamp. This is because those skilled in the field of molecular biology understand, for example, how to prevent multiple helicases from binding to an ssDNA sample by temporarily converting most of the ssDNA to dsDNA.

[0068] SF1 family helicases, such as T4 Dda helicase, can be preferred clamp choices in many embodiments. This is because the modes of movement of T4 Dda helicases 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 these helicases make with the bound DNA are in contact with the DNA's sugar phosphate backbone. These are described in Saikrishnan et al., Cell, 137:849-859, 2009 (which is incorporated herein by reference in its entirety). As a result, it is understood herein that SF1 family helicases bind equally well to all nucleic acid base sequences they encounter on the DNA strand to which they bind.

[0069] Furthermore, members of this family of translocases are found in many organisms, and the force required to slide the binding helicase along DNA in the absence of ATP varies considerably depending on the organism from which the SF1 family helicase is purified. For example, it may be preferable to clamp the DNA, where binding is not overcome by a constant bias voltage of less than approximately 180 mV, but binding to the DNA strand can be overcome by a voltage pulse of more than approximately 200 mV, optionally coupled with a thermal pulse, both lasting for a duration of less than approximately 500 μs.

[0070] 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 in conjunction with, or instead of, a voltage control pulse. This laser raises the temperature of a volume of less than about 10 yoctoliters of ionic solution in close proximity to the clamp to which one or more gold, silver, or other light-absorbing nanoparticles are bonded, as described above. A constant background temperature of the medium is preferably maintained at a bias temperature of about 15°C in this embodiment, and then raised to about 50°C by laser light emitted in time to coincide with the voltage control pulse.

[0071] The duration between voltage and / or thermal control pulses is longer than the shortest time required for monomer identification at the desired level of accuracy, but the bias voltage V drive Shorter than the minimum time required to characterize the slide of a clamped monomer unit under the conditions. The minimum measurement time for this slide varies depending on the specific helicase selected. For example, when ATP is absent and the preferred T4 Dda helicase is used as the clamp, V drive At 120mV, it is observed that intervals between steps of less than 1 second are extremely rare. Therefore, the interval between voltage-controlled pulses can be set to 100 milliseconds or less, which is an easily achievable condition. This avoids undetected slides due to the driving voltage, thereby ensuring that the number of pulses applied to drive the entire length of the target DNA polymer through the nanopore is equal to the number of monomer subunits, i.e., the number of nucleic acid bases, within the polymer length.

[0072] Embodiments relating to exemplary parameters for determining chemical matters and monomer sequencing along the target polymer length In further embodiments, a nanopore system, clamp, and control pulse are selected to enable the determination of the chemical properties of the target polymer and the sequencing of monomer subunits along the target polymer. This embodiment considers a target polymer molecule DNA, whose nucleic acid bases are the monomer subunits of the polymer, as described above. Here, since more detailed information regarding the monomer subunits is required, the bias voltage for driving the polymer through the nanopore needs to be greater than the bias voltage in the previously described embodiments, in order to minimize the effects of Brownian motion, which would otherwise degrade the detailed information. As a result, some of the parameters that are the subject of this embodiment, as well as the parameters of other commonly used features of nanopore detection, differ from those in the previous embodiments.

[0073] This embodiment provides a bias drive voltage V that is larger than the bias drive voltage of the previous embodiment. drive V is required, therefore, drive +Vpulse Since the amplitude of the synthesis voltage is correspondingly larger than that of the previous embodiment, a film that is more fragile and stronger than those used in the previous embodiment is preferred here. Suitable films include solid films such as graphene films, silicon nitride films, or other suitable solid films, or amphiphilic triblock copolymer films, such as poly(dimethylsiloxane)-block-poly(2-methyloxazoline)-poly(dimethylsiloxane) or mycolic acid films, as described in Zhao et al., Science, 279: 548-552, 1998 (which is incorporated herein by reference in its entirety), and as described in Langford et al., J. Lipid Res., 52: 272-277, 2011 (which is incorporated herein by reference in its entirety), they can all form a stronger ion-impermeable film than diPhPC lipid bilayers.

[0074] In the support structure, membranes extending over relatively small openings, such as support structure openings less than 20 microns in diameter, are also preferred. This is because they are significantly stronger than membranes formed over conventional support structure openings with diameters of 10 microns to 50 microns. Selected diameters, for example, nanopores with a diameter greater than 1 nm or less than approximately 2 nm, can penetrate straight through the membrane and penetrate membranes such as graphene membranes. Alternatively, bio-nanopores such as variants of CsgG porin are preferred protein nanopores provided for the membrane. Both of these can distinguish between DNA nucleic acid bases more easily than α-hemolysin, as described by Howarra in US20180148481 (which is incorporated herein by reference in its entirety).

[0075] Here, the bias drive voltage V is approximately 140mV to 250mV. driveIt is preferable to minimize Brownian motion and minimize the time each consecutive monomer unit spends outside the high-resistance region, where it is the most sensitive narrow opening of the nanopore, thereby minimizing the number of monomers contributing to each ion current measurement. This condition can be understood by referring to Figures 14A-14C with respect to an example of a drive voltage of about 120 mV. When a drive voltage of 120 mV 38 is applied to the nanopore 20, one monomer unit 42 is positioned in the highly sensitive region 44 of the nanopore, and as shown in Figure 14B, Brownian motion may momentarily displace the monomer unit from the highly sensitive region. Applying a larger bias drive voltage, exceeding 125 mV, allows the subunit 42 to return to the highly sensitive region more quickly, as shown in Figure 14C, thereby improving monomer identification. Therefore, it may be preferable to use a relatively high bias drive voltage exceeding about 120 mV or to use a variable voltage to counteract Brownian motion.

[0076] In this embodiment, to apply a 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 a volume of less than about 10 yoctoliters of ionic solution in close proximity to a clamp, which has gold nanoparticles bonded to the clamp or nanopore, in the manner described above. A constant background temperature of the solution is preferably maintained at about 10°C and raised to about 50°C by the laser light. The laser light is controlled in the manner described above to coincide with a voltage-controlled pulse.

[0077] With the relatively rigid films selected here, a constant voltage bias exceeding approximately 500 mV can damage the films across the support structure aperture diameter of less than approximately 10 microns. However, voltage-controlled pulses with pulse durations between approximately 1 microsecond and 3 microseconds and a magnitude of less than approximately 900 mV are acceptable. Therefore, in the absence of ATP, the voltage-controlled pulses used to easily overcome the forces binding the clamp components to the monomer units of the polymer are preferably those with a duration shorter than approximately 3 microseconds and a voltage magnitude less than approximately 900 mV.

[0078] In this embodiment, SF1 family helicases are preferred. This is because members of this family of translocases are found in many organisms, and the force required to slide the helicase bound along DNA in the absence of ATP varies considerably depending on the organism from which the SF1 family helicases are purified. In this example, a clamp is preferred in which binding to the DNA strand can be overcome by a control pulse duration of less than approximately 500 ns and a magnitude of less than approximately 900 mV, but binding to the DNA cannot be overcome by a bias drive voltage of less than approximately 500 mV.

[0079] Similar to the previous embodiment, the duration between control pulses is selected here to be longer than the shortest time required for monomer identification at the desired level of accuracy, but shorter than the shortest time known to characterize the clamp slide under the condition of bias drive voltage only. This shortest measurement time for the slide varies depending on the specific helicase selected. In the absence of ATP, using the preferred SF1 family helicase as the clamp, the average time between slides exceeds approximately 100 milliseconds and decreases exponentially, thus V driveUnder conditions of only a bias drive voltage of 300mV, a slide from one nucleic acid base to the next occurs very rarely over a span of approximately 15ms. Therefore, by setting the interval of the voltage control pulses to occur for a duration that can be easily implemented, for example between approximately 5ms and 10ms, probabilistic sliding is minimized, and a deterministic step is provided to ensure that each consecutive nucleic acid base in the DNA sequence is reliably detected and identified by its current signature in the nanopore.

[0080] In the experimental example, an MspA nanopore was subjected to a diphytanoylphosphatidylcholine membrane and positioned in the nanopore system as shown in Figure 2. DNA equipped with a Dda helicase clamp was loaded into the cis reservoir system in an ionic solution of 1 M KCl, 25 mM phosphate buffer, 2 mM ADPNP, and 2 mM MgCl2 at pH 8.0. The solution was maintained at 37°C. The bias drive voltage applied to the nanopore was 160 mV. Voltage-controlled pulses were applied using the voltage pulse control system shown in Figure 5 and the control method shown in Figure 3. In the first experiment, the duration of the voltage-controlled pulse was 500 microseconds, and in the second experiment, the duration of the voltage-controlled pulse was 1 millisecond. In both experiments, the amplitude of the voltage-controlled pulse was 300 mV. The nanopore current was measured and filtered at 2 kHz.

[0081] Figures 15A and 15B are plots of nanopore current measured as a function of time when a voltage-controlled pulse is applied, showing the applied voltage across the nanopore as a function of time for voltage-controlled pulses with durations of 500 microseconds and 1 millisecond, respectively. The measured nanopore ion current is shown as the upper line, with its axis on the left side of the plot, and the applied voltage pulse is shown as the lower line, with its axis label on the right side of the plot. As shown in both data plots, the measured change in the ion current through the nanopore is due to the applied voltage-controlled pulse V pulse This coincided in time with a single applied voltage controlled pulse V. pulseIn response, the helicase clamp shows that it stepped one monomer along the DNA chain length. In the plot in Figure 15B, the overshoot of the capacitive current spike shown in the applied pulse is a result of improper compensation by the electronics during the voltage-controlled pulse with a relatively long duration of 1 millisecond.

[0082] This description, the embodiments provided herein, and experimental examples demonstrate that the nanopore system, clamp control, and methodology provided herein achieve a deterministic rather than probabilistic step in the movement of target polymer molecules through nanopores, and that this enables the characterization of linearly connected, continuous polymer subunits of the target polymer having nanopores. This makes accurate evaluation of the target polymer, which has been difficult until now, possible.

[0083] While preferred embodiments have been described in detail herein, it will be apparent to those skilled in the art that various modifications, additions, substitutions, etc., can be made without departing from the spirit of the invention, and it will be understood that these are therefore considered to be within the scope of the invention as defined in the appended claims.

Claims

1. A nanopore system for controlling the movement of target polymer molecules through nanopores, The target polymer molecule is selected from nucleic acid polymer molecules and protein polymer molecules and comprises a plurality of consecutive polymer subunits along the length of the target polymer molecule. The nanopore system includes a first fluid reservoir and a second fluid reservoir, a clamp, an electrical circuit, a pulse generator, and a control device. The first fluid reservoir and the second fluid reservoir are fluidly connected by nanopores arranged as flow paths between the first fluid reservoir and the second fluid reservoir, The clamp is selected from enzyme clamps, biomolecular clamps, biochemical complex clamps, and protein clamps, and is provided in the first fluid reservoir, and the clamp has an outer diameter larger than the diameter of the nanopore, and is fitted to contact the nanopore and to reversibly bond to a plurality of consecutive polymer subunits of a target polymer molecule in the ionic solution in the first fluid reservoir. The electrical circuit has an electrode in the first fluid reservoir and an electrode in the second fluid reservoir to apply a voltage bias to the nanopore between the first and second fluid reservoirs in order to induce the migration of the target polymer molecules to the nanopore. The pulse generator is connected to the electrical circuit and operates to apply control pulses to the nanopore between the first fluid reservoir and the second fluid reservoir, stepping the clamp along successive polymer subunits of the target polymer molecule toward the nanopore for each polymer subunit, thereby stepping the target polymer molecule further toward the nanopore, the nanopore system does not include energy for the clamp and an energy source for the clamp, and does not supply energy to step the clamp along successive polymer units. The control device is connected to the electrical circuit and operates to control the application of the control pulse to the nanopore. Nanopore system.

2. A nanopore system for characterizing a target polymer molecule, wherein the target polymer molecule is selected from nucleic acid polymer molecules and protein polymer molecules, and comprises a plurality of continuous polymer subunits along the length of the target polymer molecule. The nanopore system includes a first fluid reservoir and a second fluid reservoir, a clamp, an electrical circuit, a pulse generator, and a control device. The first fluid reservoir and the second fluid reservoir are fluidly connected by nanopores arranged as flow paths between the first fluid reservoir and the second fluid reservoir, The clamp is selected from enzyme clamps, biomolecular clamps, biochemical complex clamps, and protein clamps, and is provided in the first fluid reservoir, and the clamp has an outer diameter larger than the diameter of the nanopore, and is fitted to contact the nanopore and to reversibly bond to a plurality of consecutive polymer subunits of a target polymer molecule in the ionic solution in the first fluid reservoir. The electrical circuit has an electrode in the first fluid reservoir and an electrode in the second fluid reservoir to apply a voltage bias to the nanopore between the first and second fluid reservoirs in order to induce the migration of the target polymer molecules to the nanopore. The pulse generator is connected to the electrical circuit and operates to apply control pulses to the nanopore between the first fluid reservoir and the second fluid reservoir, stepping the clamp along successive polymer subunits of the target polymer molecule toward the nanopore for each polymer subunit, thereby stepping the target polymer molecule toward the nanopore, the system does not include energy for the clamp and an energy source for the clamp, and does not supply energy to step the clamp along successive polymer units. The control device is connected to the electrical circuit and operates to control the collection of electrical indicators of the polymer subunits of the target polymer molecule while the polymer subunits step through the nanopore. Nanopore system.

3. A nanopore system according to claim 1, wherein the clamp comprises a helicase.

4. A nanopore system according to claim 1, wherein the clamp comprises an SF1 family helicase.

5. A nanopore system according to claim 1, wherein the clamp comprises T4 Dda helicase.

6. A nanopore system according to claim 1, wherein the clamp comprises polymerase.

7. A nanopore system according to claim 1, wherein the nanopore includes a bio-nanopore, and the bio-nanopore is arranged in a membrane selected from a triblock copolymer membrane, a mycolic acid membrane, a lipid bilayer membrane, and a tetraether lipid membrane.

8. A nanopore system according to claim 7, wherein the membrane comprises a diphytanoylphosphatidylcholine membrane (diPhPC).

9. A nanopore system according to claim 7, wherein the nanopore is selected from CsgG bacterial porin nanopores and Mycobacterium smegmatis porin A (MspA) nanopores.

10. A nanopore system according to claim 7, wherein the biological nanopore is a protein channel.

11. A nanopore system according to claim 1, wherein the diameter of the nanopore is between 1 nm and 2 nm.

12. A nanopore system according to claim 1, further comprising a current amplifier in the circuit, wherein the current amplifier operates to generate an analogous electrical index of the polymer subunit of the target polymer while the polymer subunit steps through the nanopore.

13. A nanopore system according to claim 12, further comprising an analog-to-digital converter, the analog-to-digital converter being connected to the current amplifier by the circuit, and digitizing the analog electrical indicators of the polymer subunits of the target polymer molecule.

14. A nanopore system according to claim 13, wherein the control device is connected to the analog-to-digital converter by the electrical circuit and controls the recording of digital indicators of polymer subunits of the target polymer molecule.

15. A nanopore system according to claim 1, wherein the target polymer molecule is a double-stranded nucleic acid polymer molecule, the double-stranded nucleic acid polymer molecule has a single-stranded region along the length of the nucleic acid polymer molecule, and the clamp is adapted to reversibly bond to a plurality of consecutive polymer subunits located along the single-stranded region of the length of the nucleic acid polymer molecule.

16. A nanopore system according to claim 1, wherein the target polymer molecule is a single-stranded nucleic acid polymer molecule.

17. A nanopore system according to claim 1, wherein the target polymer molecule is a polymer molecule selected from DNA, RNA, and PNA.

18. A nanopore system according to claim 1, wherein the nanopore is a solid material that is atomically thin.

19. A nanopore system according to claim 1, wherein the continuous plurality of polymer subunits, wherein the continuous plurality of polymer subunits, wherein the clamp is adapted to be reversibly bonded thereto, comprises 1 to 20 polymer subunits.

20. A nanopore system according to claim 1, wherein the ionic solution contains ADPNPs.

Images