Method and device for regulating activity of biomolecule

Abstract

The present invention relates to the field of sequencing. Specifically provided are a method for regulating the activity of a biomolecule, and a corresponding device or system. Further provided are methods for identifying or characterizing the biomolecule. The methods are suitable for detection and / or sequencing, in particular, nanopore sequencing.

Description

A method and apparatus for regulating the activity of biomolecules Technical Field

[0001] This invention relates to the field of sequencing. Specifically, this invention provides methods for regulating the activity of biomolecules, as well as corresponding devices or systems. This invention also provides methods for identifying or characterizing biomolecules, which are widely applicable in detection and / or sequencing, particularly nanopore sequencing. Background Technology

[0002] Currently, nanopore-based sequencing technology is considered the most promising among various nanopore sequencing technologies, and it has been successfully commercialized. The most widely used nanopore sequencers employ strand sequencing, which involves recording the blocking current signals generated by the continuous perforation of single-stranded DNA molecules, and then using deep learning algorithms to analyze these signals to identify the DNA molecule's base sequence.

[0003] One of the core key aspects of nanopore sequencing is the control of sequencing initiation and speed. Most existing nanopore sequencing technologies rely on motor proteins to drive the unwinding of double-stranded DNA to form single-stranded DNA. The current changes caused by different bases entering the nanopore distinguish these bases, thus obtaining the single-stranded DNA sequence. In current technologies, motor proteins play a crucial role. They not only unwind double-stranded DNA but also control the speed at which DNA molecules pass through the nanopore, ensuring that base recognition algorithms can effectively distinguish different sequences. Therefore, controlling motor proteins indirectly regulates sequencing.

[0004] Currently, in most commercially available sequencing methods, spacer regions are commonly used to inhibit the movement of motor proteins before sequencing begins. Specifically, during the construction of sequencing libraries, double-stranded library molecules are designed to contain spacer regions with a specially structured composition. Motor proteins bind to these spacer regions, so that when the motor proteins unwind the double-stranded library molecules, the spacer regions can effectively restrict the movement of the motor proteins along the library molecule chains, thereby preventing the target library from unwinding prematurely before being captured by the nanopores.

[0005] However, the above methods generally have one or more of the following drawbacks: (1) The library construction method is complex and requires the existence of spacer regions composed of special structures (such as iSp18) to restrict the movement of motor proteins. (2) There is a risk of random failure of library molecules in solution. Because the helicase bound to the library strand in the above methods is active, there is a certain probability that it will cross the spacer region and start sequencing directly in solution without being detected as an effective signal, thus becoming an invalid library. (3) Premature ATP consumption and sequencing speed reduction. When sequencing is not started, the motor protein is active and may idle at the initial binding site, thereby consuming ATP and reducing the sequencing speed during sequencing.

[0006] Therefore, a new method and apparatus are needed to achieve flexible and effective control of sequencing speed while ensuring sequencing efficiency and accuracy.

[0007] Summary of the Invention

[0008] This application utilizes the ion transport properties of nanopores to provide methods and apparatus for regulating the activity of biomolecules (e.g., motor proteins), enabling the successful initiation, shutdown, and control of the activity of biomolecules (e.g., putting them into an active or inactive state). Furthermore, this application also provides methods for identifying and / or characterizing biomolecules (particularly methods for sequencing biomolecules). Therefore, the methods of this application can detect the presence of target biomolecules or characterize the detected biomolecules, and regulate the sequencing process.

[0009] The methods and apparatus provided by this invention are not only applicable to DNA molecular detection or sequencing, but can also be widely applied to the detection and / or sequencing of biomolecules such as RNA, proteins, and polysaccharides, or to multi-omics detection and / or sequencing. Therefore, the methods and apparatus provided by this invention can be further applied to the start-up, shutdown, and rate control of any reaction requiring key substances.

[0010] Methods for regulating biomolecular activity

[0011] In one aspect, the present invention provides a method for regulating the activity of a first biomolecule, the method comprising:

[0012] (a) Provide:

[0013] (i) A membrane having one or more pores; the membrane separating a first fluid compartment and a second fluid compartment, the pores connecting the first fluid compartment and the second fluid compartment;

[0014] (ii) The first biomolecule located in the first fluid compartment, and

[0015] (iii) One or more first factors that can directly or indirectly regulate the activity of the first biomolecule (e.g., regulate the level of the activity of the first biomolecule).

[0016] Furthermore, the first factor can move between the first fluid chamber and the second fluid chamber through the orifice under the drive of its own concentration gradient and / or external forces (e.g., electric field force, magnetic field force and / or pressure);

[0017] (b) By regulating the movement of the first factor between the first fluid compartment and the second fluid compartment, the concentration of the first factor or the rate of concentration change in the first fluid compartment is altered (e.g., increased or decreased) to regulate the activity of the first biomolecule.

[0018] In the method of this application, depending on the different selections of the first biomolecule being regulated, the method can be applied to different detection and / or sequencing scenarios. Examples include RNA sequencing; DNA sequencing; sequencing of other nucleic acid molecules such as LNA, PNA, or XNA; amino acid sequencing of proteins or peptides; polysaccharide sequencing; haplotype analysis; and methylation detection.

[0019] In some embodiments, the activity of the first biomolecule can be modulated to place it in an active or inactive operating mode. For example, when the first fluid compartment contains all the necessary components for the first biomolecule to function (e.g., one or more first factors), the first biomolecule is in an active operating mode. Conversely, when the first fluid compartment does not contain all the necessary components for the first biomolecule to function (e.g., one or more first factors), the first biomolecule is in an inactive operating mode.

[0020] In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of proteins and / or nucleic acids. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of nucleic acid-binding proteins. For example, the activity of single-stranded DNA-binding proteins in binding to single-stranded DNA. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of motor proteins. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of helicases. For example, the activity of unwinding double-stranded DNA, or the activity of controlling DNA passage through nanopores. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of polymerases. For example, the activity of polymerizing and extending DNA, strand displacement activity, or exonuclease activity.

[0021] In some embodiments, the activity is related to the ability of a first biomolecule to regulate a second biomolecule.

[0022] In some embodiments, the activity is related to the ability of the first biomolecule to control at least a portion (e.g., a chain) of the second biomolecule to move relative to the pore.

[0023] In some embodiments, the activity of the first biomolecule is modulated to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore.

[0024] In some embodiments, after the membrane is provided in the method of this application, a first fluid chamber and a second fluid chamber are automatically formed on both sides of the membrane, separating the first fluid chamber and the second fluid chamber. Therefore, in some embodiments, the first fluid chamber and the second fluid chamber may not be provided in step (a). In some embodiments, the first fluid chamber and / or the second fluid chamber may not have defined boundaries; that is, they may be open spaces.

[0025] In some embodiments, in step (a), a first fluid compartment and a second fluid compartment are provided. In some embodiments, the first fluid compartment and / or the second fluid compartment may also have well-defined boundaries; that is, they may be enclosed spaces.

[0026] In some implementations, the method of this application also provides a chip.

[0027] In some embodiments, the chip includes an array of microcavities on a substrate (e.g., a wafer substrate or a PCB substrate). In some embodiments, after the membrane comes into contact with the microcavities, the interior of the microcavities can form a second fluid chamber with the membrane and can store a solution. In some embodiments, the upper part of the microcavities can be directly or indirectly tightly connected to the membrane and form a first fluid chamber.

[0028] In some embodiments, in step (a), a second biomolecule located in the first fluid compartment is also provided. In some embodiments, the first and second biomolecules exist in the form of a complex.

[0029] In the method of this application, the first biomolecule, the second biomolecule, and the pore can be contacted in any order. In some embodiments, the second biomolecule contacts the pore in the form of a complex with the first biomolecule. In some embodiments, when a voltage is applied across the pore, the complex containing the second biomolecule and the first biomolecule contacts the pore, and the second biomolecule is controlled to move relative to the pore (e.g., through the pore).

[0030] In some embodiments, in step (a), a second factor capable of regulating the first factor (e.g., regulating the concentration of the first factor) is also provided.

[0031] In some implementations, as described above, the method includes:

[0032] (a) Provide:

[0033] (i) a first fluid compartment and a second fluid compartment, and a membrane having one or more pores; the membrane separating the first fluid compartment and the second fluid compartment, the pores communicating with the first fluid compartment and the second fluid compartment;

[0034] (ii) A first biomolecule and a second biomolecule located in a first fluid compartment, wherein the first biomolecule is capable of controlling the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore; and

[0035] (iii) One or more first factors capable of directly or indirectly regulating the activity of the first biomolecule, wherein the activity is related to the ability of the first biomolecule to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore;

[0036] Furthermore, the first factor can move between the first fluid chamber and the second fluid chamber through the orifice under the drive of its own concentration gradient and / or external forces (e.g., electric field force, magnetic field force and / or pressure);

[0037] (b) By modulating the movement of the first factor between the first fluid compartment and the second fluid compartment, changing (e.g., increasing or decreasing) the concentration of the first factor or the rate of concentration change in the first fluid compartment, the activity of the first biomolecule is modulated, thereby controlling the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore.

[0038] Second biomolecular perforation

[0039] In some embodiments, the first biomolecule is able to control at least a portion (e.g., a chain) of the second biomolecule to pass through the pore.

[0040] In some embodiments, the first biomolecule is able to control at least a portion (e.g., a chain) of the second biomolecule to move through the aperture in a direction parallel (e.g., the same or opposite) to the applied electric and / or magnetic field forces (e.g., moving through the aperture from a low-potential chamber to a high-potential chamber).

[0041] In some embodiments, the first biomolecule is able to connect to the pore (e.g., non-covalently).

[0042] First biomolecule

[0043] In some embodiments, the first biomolecule is a protein (e.g., an enzyme).

[0044] In some embodiments, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof.

[0045] In some embodiments, the polymerase is a polymerase with chain displacement activity.

[0046] In some embodiments, the polymerase is selected from the following: Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof.

[0047] In this document, any suitable helicase may be used. The helicase may be selected from the SF1, SF2, SF3, SF4, SF5, or SF6 family. In some embodiments, the specific helicase is selected from the following: Dda, Hel308, RecD, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, gp41, T7gp4, BCH105, or any combination thereof. In some embodiments, the helicase is a wild-type protein of any of the above helicases, its homolog, or a mutant thereof.

[0048] Detailed information on helicase Hel308 can be found in WO 2013 / 057495, the entire contents of which are incorporated herein by reference. Detailed information on helicase RecD can be found in WO 2013 / 098562, the entire contents of which are incorporated herein by reference. Detailed information on helicase Dda can be found in WO2015 / 055981 and WO 2016 / 055777, the entire contents of which are incorporated herein by reference. Detailed information on helicase BCH105 can be found in PCT / CN2021 / 143662 and PCT / CN2022 / 144205, the entire contents of which are incorporated herein by reference.

[0049] In some embodiments, the helicase is further linked to an additional polypeptide selected from tags, restriction enzyme sites, signal peptides or lead peptides, detectable markers, or any combination thereof.

[0050] In some embodiments, the amino acid recognition protein is capable of specifically recognizing the terminal amino acid of a polypeptide or protein. Therefore, in some embodiments, when the first biomolecule is an amino acid recognition protein, the second biomolecule can be a polypeptide or protein. Specific information on amino acid recognition proteins can be found in PCT / CN2023 / 143631, the entire contents of which are incorporated herein by reference.

[0051] In some embodiments, the amino acid recognition protein is selected from any one or more of the following:

[0052] (i) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of ClpS family;

[0053] (ii)Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt;

[0054] (iii) human UBR1, human UBR2 or Saccha-romyces cerevisiae UBR1 of the UBR family;

[0055] (iiii) GID4 of H. sapiens or Saccharomyces cerevisiae:

[0056] (iiiiii) Drosophila melanogaster BIR2;

[0057] (iiiiii) H. sapiens N-meristoyltransferase NMT1.

[0058] Second biomolecule

[0059] In some embodiments, the second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof.

[0060] In some embodiments, the second biomolecule is selected from natural, synthetic, wholly or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof.

[0061] In some embodiments, the second biomolecule includes a adapter (e.g., a sequencing adapter).

[0062] In some embodiments, the second biomolecule is DNA containing a connector.

[0063] In some embodiments, the second biomolecule is a polypeptide containing a linker.

[0064] In some embodiments, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule.

[0065] In some implementations, the connector may or may not include one or more spacer zones.

[0066] In some implementations, the connector does not include a spacer area.

[0067] In some implementations, the spaced regions have one or more features selected from the following:

[0068] (1) Each of the one or more spacer regions independently comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), synthetic polymers with nucleotide side chains, or any combination thereof;

[0069] (2) Each of the one or more spacer regions independently contains one or more base-free nucleotides;

[0070] (3) Each of the one or more spacer regions independently contains one or more chemical groups that cause one or more biomolecules (e.g., helicases) to stop.

[0071] In some embodiments, the second biomolecule is a nucleic acid molecule (e.g., a chain-like nucleic acid molecule).

[0072] In some implementations, the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule.

[0073] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the helicase is able to separate the two strands of the double-stranded nucleic acid molecule to provide a single-stranded nucleic acid molecule and control the movement of the single-stranded nucleic acid molecule through the pore.

[0074] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor can directly or indirectly regulate the helicase's ability to unwind the double-stranded nucleic acid molecule.

[0075] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor can directly or indirectly regulate the helicase's ability to unwind the double-stranded nucleic acid molecule, thereby controlling at least a portion of the double-stranded nucleic acid molecule to pass through the pore.

[0076] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the helicase forms a complex with the double-stranded nucleic acid molecule through a linker contained in the double-stranded nucleic acid molecule.

[0077] The second biomolecule is non-perforated.

[0078] In some embodiments, the first biomolecule is able to control at least a portion of the second biomolecule to move relative to the pore without passing through it.

[0079] In some embodiments, the direction of movement of the second biomolecule is independent of the direction of the applied electric and / or magnetic field forces.

[0080] In some embodiments, the first biomolecule is capable of connecting to the pore (e.g., covalently).

[0081] First biomolecule

[0082] In some embodiments, the first biomolecule is a protein (e.g., an enzyme).

[0083] In some embodiments, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof.

[0084] In some embodiments, the polymerase may be a polymerase with or without chain displacement activity.

[0085] In some embodiments, the polymerase is selected from the following: Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof.

[0086] In this document, any suitable helicase may be used. The helicase may be selected from the SF1, SF2, SF3, SF4, SF5, or SF6 family. In some embodiments, the specific helicase is selected from the following: Dda, Hel308, RecD, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, gp41, T7gp4, BCH105, or any combination thereof. In some embodiments, the helicase is a wild-type protein of any of the above helicases, its homolog, or a mutant thereof.

[0087] Detailed information on helicase Hel308 can be found in WO 2013 / 057495, the entire contents of which are incorporated herein by reference. Detailed information on helicase RecD can be found in WO 2013 / 098562, the entire contents of which are incorporated herein by reference. Detailed information on helicase Dda can be found in WO2015 / 055981 and WO 2016 / 055777, the entire contents of which are incorporated herein by reference. Detailed information on helicase BCH105 can be found in PCT / CN2021 / 143662 and PCT / CN2022 / 144205, the entire contents of which are incorporated herein by reference.

[0088] In some embodiments, the helicase is further linked to an additional polypeptide selected from tags, restriction enzyme sites, signal peptides or lead peptides, detectable markers, or any combination thereof.

[0089] In some embodiments, the amino acid recognition protein is capable of specifically recognizing the terminal amino acid of a polypeptide or protein. Therefore, in some embodiments, when the first biomolecule is an amino acid recognition protein, the second biomolecule can be a polypeptide or protein. Specific information on amino acid recognition proteins can be found in PCT / CN2023 / 143631, the entire contents of which are incorporated herein by reference.

[0090] In some embodiments, the amino acid recognition protein is selected from any one or more of the following:

[0091] (i) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of ClpS family;

[0092] (ii)Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt;

[0093] (iii) human UBR1, human UBR2 or Saccha-romyces cerevisiae UBR1 of the UBR family;

[0094] (iiii) GID4 of H. sapiens or Saccharomyces cerevisiae:

[0095] (iiiiii) Drosophila melanogaster BIR2;

[0096] (iiiiii) H. sapiens N-meristoyltransferase NMT1.

[0097] Second biomolecule

[0098] In some embodiments, the second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof.

[0099] In some embodiments, the second biomolecule is selected from natural, synthetic, wholly or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof.

[0100] In some embodiments, the second biomolecule includes a adapter (e.g., a sequencing adapter).

[0101] In some embodiments, the second biomolecule is DNA containing a connector.

[0102] In some embodiments, the second biomolecule is a polypeptide containing a linker.

[0103] In some embodiments, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule.

[0104] In some implementations, the connector may or may not include one or more spacer zones.

[0105] In some implementations, the connector does not include a spacer area.

[0106] In some implementations, the spaced regions have one or more features selected from the following:

[0107] (1) Each of the one or more spacer regions independently comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), synthetic polymers with nucleotide side chains, or any combination thereof;

[0108] (2) Each of the one or more spacer regions independently contains one or more base-free nucleotides;

[0109] (3) Each of the one or more spacer regions independently contains one or more chemical groups that cause one or more biomolecules (e.g., helicases) to stop.

[0110] In some embodiments, the second biomolecule is a nucleic acid molecule (e.g., a circular nucleic acid molecule).

[0111] In some embodiments, the first biomolecule is a polymerase (e.g., DNA polymerase) and the second biomolecule is a circular nucleic acid molecule.

[0112] In some embodiments, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the polymerase can use the circular nucleic acid molecule as a template to extend primers that bind to the circular nucleic acid molecule and control the circular nucleic acid molecule to move relative to the pore without passing through the pore.

[0113] In some implementations, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor can directly or indirectly regulate the polymerase's ability to extend primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template.

[0114] In some embodiments, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor can directly or indirectly regulate the polymerase's ability to extend primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template, thereby controlling the relative movement of the circular nucleic acid molecule to the pore without passing through the pore.

[0115] In some embodiments, an electric field, a magnetic field, and / or pressure are provided across the membrane, causing the first factor to move through the pore between the first fluid compartment and the second fluid compartment under the drive of the electric field force, magnetic field force, and / or pressure.

[0116] In some implementations, the first factor moves along the direction of the driving force under the influence of electric field force, magnetic field force, and / or pressure.

[0117] In some embodiments, the first factor moves along any one or a combination of the following: (i) potential difference, (ii) concentration gradient, (iii) electrochemical gradient, and (iiii) the direction of applied pressure.

[0118] In some implementations, after an electric field and / or magnetic field is applied, the potential of the second fluid compartment is higher or lower than that of the first fluid compartment.

[0119] In some embodiments, after an electric field and / or magnetic field is applied, the potential of the second fluid chamber is higher than that of the first fluid chamber, and the first factor moves from the second fluid chamber to the first fluid chamber through the nanopore.

[0120] In some embodiments, a second electrode (e.g., a positive electrode) is provided in a second fluid chamber, and a first electrode (e.g., a negative electrode) is provided in a first fluid chamber, with a voltage applied across the first and second electrodes.

[0121] In some embodiments, the first fluid compartment and the second fluid compartment contain a solution.

[0122] First Factor and Second Factor

[0123] It is understood that the first factor in this application can be any key factor required for the function of the first biomolecule (e.g., a motor protein). And any factor capable of regulating the first factor can serve as the second factor.

[0124] Furthermore, in the method described herein, since the first factor and / or the second factor are capable of moving between the first fluid compartment and the second fluid compartment, the initially provided first factor and / or second factor are not limited to a specific compartment. In some embodiments, the first factor is located in both the first fluid compartment and / or the second fluid compartment. In some embodiments, the second factor is located in both the first fluid compartment and / or the second fluid compartment.

[0125] In some implementations, the first factor is an activator of a first biomolecule.

[0126] In some implementations, the first factor can positively regulate the activity of the first biomolecule (e.g., enhance the activity of the first biomolecule, enable the first biomolecule to start working, and / or improve the working efficiency of the first biomolecule).

[0127] In some implementations, the first factor is an ion in the solution.

[0128] In some implementations, the first factor is selected from metal ions, ATP, or any combination thereof.

[0129] In some embodiments, the metal ion is selected from Mn2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Or any combination thereof. In some embodiments, in step (a), the second factor is capable of modulating the first factor (e.g., modulating the concentration of the first factor).

[0130] In some implementations, the second factor negatively regulates the activity of the first biomolecule by modulating the first factor (e.g., reducing the activity of the first biomolecule, causing the first biomolecule to stop working, and / or reducing the working efficiency of the first biomolecule).

[0131] In some implementations, the second factor is a repressor, chelator, and / or inhibitor of the first factor.

[0132] In some implementations, when the first factor is a metal ion, the second factor is a chelating agent of the first factor.

[0133] In some implementations, the second factor is selected from EDTA, EGTA, DTPA, TTHA, NTA, or any combination thereof.

[0134] In some implementations, the activity of the first biomolecule is positively modulated by one or more methods selected from the following:

[0135] (1) To bring the first biomolecule into contact with the first factor or to increase the concentration of the first factor that is in contact with the first biomolecule;

[0136] (2) Increase the concentration of the first factor in the first fluid compartment or increase the rate of increase of the concentration of the first factor in the first fluid compartment;

[0137] (3) By (i) applying an electric field, magnetic field and / or pressure, or (ii) increasing the voltage of the electric field and / or magnetic field, or (iii) increasing the pressure, or (iiii) increasing the initial concentration of the first factor in the second fluid compartment, to increase the concentration of the first factor in the first fluid compartment or to increase the rate of increase of the concentration of the first factor in the first fluid compartment.

[0138] In some implementations, the activity of the first biomolecule is negatively regulated by one or more methods selected from the following:

[0139] (1) The first biomolecule comes into contact with a first factor at a lower concentration (e.g., below the concentration required for the first biomolecule under normal operating conditions) or the concentration of the first factor that comes into contact with the first biomolecule is reduced (e.g., the concentration is reduced to 0).

[0140] (2) Reduce the concentration of the first factor in the first fluid compartment or reduce the rate of increase of the concentration of the first factor in the first fluid compartment;

[0141] (3) By (i) removing the electric field, magnetic field and / or pressure applied across the membrane, or (ii) reducing the voltage of the electric field and / or magnetic field, or (iii) reducing the pressure, or (iiii) reducing the initial concentration of the first factor in the second fluid compartment, to reduce the concentration of the first factor in the first fluid compartment or reduce the rate of increase of the concentration of the first factor in the first fluid compartment.

[0142] (4) Make the first factor come into contact with the second factor or increase the concentration of the second factor that comes into contact with the first factor.

[0143] In some embodiments, in step (a) of the method:

[0144] (1) A first factor is provided in a second fluid compartment at a concentration sufficient to initiate the action of the first biomolecule, while the first fluid compartment does not provide the first factor. In some embodiments, the first fluid compartment may or may not provide a second factor.

[0145] (2) A first factor is provided in a second fluid compartment at a concentration sufficient to initiate the action of the first biomolecule, while a first factor is provided in a first fluid compartment at a concentration insufficient to initiate the action of the first biomolecule. In some embodiments, the first fluid compartment may or may not provide a second factor.

[0146] (3) A first factor is provided in the second fluid compartment at a concentration sufficient to initiate the action of the first biomolecule, and a first factor is provided in the first fluid compartment at a concentration sufficient to initiate the action of the first biomolecule. In some embodiments, a second factor is also provided in the first fluid compartment.

[0147] Regulating the activity of the first biomolecule through factor flow

[0148] In some embodiments, in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state.

[0149] When the first factor carries a positive charge (e.g., Mn) 2+ Mg2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid compartment is higher than that of the first fluid compartment. The first factor moves from the second fluid compartment to the first fluid compartment through the hole to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working.

[0150] In some embodiments, increasing the potential difference between the second fluid compartment and the first fluid compartment, and / or increasing the initial concentration of the first factor in the second fluid compartment, increases the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively modulating the activity of the first biomolecule (e.g., improving the working efficiency of the first biomolecule).

[0151] In some embodiments, reducing the potential difference between the second fluid compartment and the first fluid compartment, reducing the initial concentration of the first factor in the second fluid compartment, and / or providing the second factor in the first fluid compartment can reduce the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., causing the first biomolecule to stop working and / or reducing the working efficiency of the first biomolecule).

[0152] In some implementations, the second factor is a chelating agent for metal ions (e.g., EDTA, EGTA, DTPA, TTHA, NTA).

[0153] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, thus putting the first biomolecule into an active state;

[0154] When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid chamber is lower than that of the first fluid chamber. The first factor moves from the first fluid chamber to the second fluid chamber through the hole to reduce the concentration of the first factor in the first fluid chamber, thereby reducing the working efficiency of the first biomolecule.

[0155] In some implementations, the second fluid compartment may or may not provide the first factor.

[0156] In some embodiments, increasing the potential difference between the first fluid compartment and the second fluid compartment, decreasing the initial concentration of the first factor in the first fluid compartment, and / or providing the second factor in the first fluid compartment can increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule (e.g., further reducing the efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

[0157] In some implementations, the second factor is a chelating agent for metal ions (e.g., EDTA, EGTA, DTPA, TTHA, NTA).

[0158] In some embodiments, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, in order to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule (e.g., slowing down the rate at which the working efficiency of the first biomolecule decreases or increasing the working efficiency of the first biomolecule).

[0159] In some embodiments, in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state.

[0160] When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is higher than that of the second fluid compartment, the first factor moves from the second fluid compartment through the pore to the first fluid compartment to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working.

[0161] In some embodiments, the potential difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased to increase the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule (e.g., reducing the rate at which the efficiency of the first biomolecule decreases or increasing the efficiency of the first biomolecule).

[0162] In some embodiments, reducing the potential difference between the second fluid compartment and the first fluid compartment, and / or reducing the initial concentration of the first factor in the second fluid compartment, slows down the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., reducing the efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

[0163] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, thus putting the first biomolecule into an active state;

[0164] When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is lower than that of the second fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the pore to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the efficiency of the first biomolecule.

[0165] In some implementations, the second fluid compartment may or may not provide the first factor.

[0166] In some embodiments, the potential difference between the first fluid compartment and the second fluid compartment is increased, and / or the initial concentration of the first factor in the first fluid compartment is decreased, to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule (e.g., further reducing the efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

[0167] In some embodiments, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, in order to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule (e.g., slowing down the rate at which the working efficiency of the first biomolecule decreases or increasing the working efficiency of the first biomolecule).

[0168] In some embodiments, in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state.

[0169] When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid chamber is higher than that in the first fluid chamber, the first factor moves from the second fluid chamber to the first fluid chamber through the hole to increase the concentration of the first factor in the first fluid chamber, thereby activating the first biomolecule.

[0170] In some embodiments, increasing the pressure difference between the second fluid compartment and the first fluid compartment, and / or increasing the initial concentration of the first factor in the second fluid compartment, increases the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule (e.g., improving the working efficiency of the first biomolecule).

[0171] In some embodiments, reducing the pressure difference between the second fluid compartment and the first fluid compartment, and / or reducing the initial concentration of the first factor in the first fluid compartment, slows down the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., reducing the efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

[0172] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, thus putting the first biomolecule into an active state;

[0173] When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid chamber is lower than that in the first fluid chamber, the first factor moves from the first fluid chamber to the second fluid chamber through the hole to reduce the concentration of the first factor in the first fluid chamber, thereby reducing the working efficiency of the first biomolecule.

[0174] In some implementations, the second fluid compartment may or may not provide the first factor.

[0175] In some embodiments, the pressure difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, in order to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule (e.g., slowing down the rate at which the working efficiency of the first biomolecule decreases).

[0176] In some embodiments, the pressure difference between the first fluid compartment and the second fluid compartment is increased, and / or the initial concentration of the first factor in the first fluid compartment is reduced, to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule (e.g., further reducing the efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

[0177] In some embodiments, in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state.

[0178] When the first factor carries a positive charge, a negative charge, or no charge, and the concentration of the first factor in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the pore to increase the concentration of the first factor in the first fluid compartment, thereby activating the first biomolecule.

[0179] In some implementations, the initial concentration of the first factor in the second fluid compartment is increased to increase the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule.

[0180] In some implementations, the initial concentration of the first factor in the first fluid compartment is reduced to slow the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule.

[0181] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, and a second fluid compartment does not provide the first factor or provides a first factor at a concentration lower than that of the first factor in the first fluid compartment, so that the first biomolecule is in a working state;

[0182] When the first factor carries a positive charge, a negative charge, or no charge, and the concentration of the first factor in the second fluid compartment is lower than that in the first fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the hole to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the working efficiency of the first biomolecule.

[0183] In some implementations, the initial concentration of the first factor in the first fluid compartment is increased to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule.

[0184] In some implementations, the initial concentration of the first factor in the first fluid compartment is reduced to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule.

[0185] membrane

[0186] Membranes suitable for the methods of this application are well known in the art.

[0187] In some embodiments, the first and second factors are unable to move between the first and second fluid compartments through the membrane. In some embodiments, the orifice is located on and passes through the membrane. In some embodiments, the first and second factors are able to move between the first and second fluid compartments through the orifice.

[0188] In some embodiments, the membrane is an amphiphilic layer. The amphiphilic layer is formed from amphiphilic molecules, such as phospholipids that are both hydrophilic and lipophilic. The amphiphilic molecules can be synthetic or naturally occurring. The amphiphilic layer can also be chemically modified or functionalized to promote the coupling of biomolecules.

[0189] In some embodiments, the membrane is a phospholipid membrane (e.g., DOPE, DPPC, DPhPC, DOPC, or POPC). The art teaches the use of various phospholipid membranes, for example, disclosed in international applications PCT / GB08 / 000563 (published as WO2008 / 102121), PCT / GB08 / 004127 (published as WO2009 / 077734), and PCT / GB2006 / 001057 (published as WO2006 / 100484).

[0190] In some embodiments, the membrane is a polymer membrane (e.g., a diblock polymer, a triblock polymer (such as PMOXA-PDMS-PMOXA)).

[0191] In some embodiments, the membrane is a solid thin film.

[0192] Solid films can be formed from organic and inorganic materials, including but not limited to: microelectronic materials, insulating materials such as Si3N4, Al2O3 and SiO2, organic and inorganic polymers such as polyamides, plastics such as Teflon, or elastomers such as two-component addition-cured silicone rubber and glass.

[0193] Solid films can be formed from graphene. Suitable graphene films that can be used are disclosed in international application PCT / US2008 / 010637 (published as WO2009 / 035647).

[0194] The solid layer can be formed from BN.

[0195] In some embodiments, the membrane is a mixture of any two or more substances.

[0196] hole

[0197] It is understood that the pores applicable to this application are not limited to a specific structure, as long as the pores allow the first factor to flow through between the first fluid compartment and the second fluid compartment. In some embodiments, the pores allow a first biomolecule to pass through. In some embodiments, the pores may be biological or artificial.

[0198] In some embodiments, the opening diameter of the pore is 0.1 nm-10 μmm (e.g., 0.1-1 nm, 1-10 nm, 10-100 nm, 100-300 nm, 300-500 nm, 500-800 nm, 800-1000 nm, 1 μm-5 μm, 5 μm-10 μm).

[0199] Biopore

[0200] In some embodiments, the pore is a biological pore, such as a biological protein pore or a polynucleotide pore.

[0201] In some embodiments, the pores are biological protein pores.

[0202] In some embodiments, the bioprotein pore may be a monomer or an oligomer. In some embodiments, the pore consists of a plurality of repeating subunits. In some embodiments, the bioprotein pore contains channels through which ions can flow.

[0203] In some embodiments, the bioprotein pore contains one or more positively charged amino acids, such as arginine and / or lysine. These amino acids facilitate the interaction between the pore and a second biomolecule (e.g., a nucleic acid molecule).

[0204] In some embodiments, the biological protein pores are derived from any one or more of the following, or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria spp. autotransporter lipoprotein (NalP), WZA, GspD, BCP34, and BCP58. Specific information on BCP34 and BCP58 can be found in PCT / CN2022 / 143298 and PCT / CN2022 / 143060, the entire contents of which are incorporated herein by reference.

[0205] Furthermore, methods for inserting or placing biological protein pores into membranes are known in the art, see, for example, the description in international patent application PCT / GB2006 / 001057 (published as WO2006 / 100484).

[0206] Solid hole

[0207] In some implementations, the hole is a solid-state hole.

[0208] In some embodiments, the pores are solid nanopores.

[0209] In some embodiments, the solid nanopores are selected from SiNx, SiO2, graphene, Al2O3, boron nitride (BN), HfO2, or any combination thereof.

[0210] In some embodiments, the pores are modified, for example, chemically modified.

[0211] In some implementations, ions can pass through pores and generate an electric current after an electric field and / or magnetic field is applied across the membrane.

[0212] In some embodiments, the pore is capable of connecting and / or binding to a first biomolecule (e.g., covalently).

[0213] Methods for identifying or characterizing second biomolecules

[0214] In a second aspect, this application provides a method for identifying or characterizing a second biomolecule, the method comprising: regulating the activity of a first biomolecule by the method described in the first aspect, wherein, in a state of regulated activity, the first biomolecule controls at least a portion (e.g., a chain) of the second biomolecule to move relative to the pore, and generating one or more measurements; wherein the measurements can be used to identify or characterize the second biomolecule.

[0215] In some embodiments, the method is performed by the steps described in (a) to (c) below:

[0216] (a) Provide:

[0217] (i) A membrane having one or more pores; the membrane separating a first fluid compartment and a second fluid compartment, the pores connecting the first fluid compartment and the second fluid compartment;

[0218] (ii) A first biomolecule and a second biomolecule located in a first fluid compartment, wherein the first biomolecule is capable of controlling the relative movement of at least a portion (e.g., a chain) of the second biomolecule relative to the pore; and, as the at least portion of the second biomolecule moves relative to the pore, one or more measurements are generated;

[0219] (iii) One or more first factors capable of directly or indirectly regulating the activity of the first biomolecule, wherein the activity is related to the ability of the first biomolecule to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore; and,

[0220] (b) The first factor is moved through a pore between the first fluid compartment and the second fluid compartment under the drive of its own concentration gradient and / or external force, and the concentration of the first factor in the first fluid compartment is changed to regulate the activity of the first biomolecule.

[0221] (c) In a state of regulated activity, the first biomolecule controls the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore and acquires one or more measurements for identifying or characterizing the second biomolecule.

[0222] As used herein, the term "measured value" refers to a measurable or quantifiable numerical value or property of a second biomolecule. For different biomolecules, the measured value may be the same or different numerical value or property.

[0223] In some embodiments, the “measured value” refers to a measurable parameter (e.g., ion current, light signal) generated under the influence (e.g., interaction) of at least a portion of the second biomolecule as it moves relative to the nanopore.

[0224] In some embodiments, the measured value may be the blocking current or its variation through the nanopore, the tunneling current or its variation across the nanopore, conductance, electrochemical current through the transverse electrode, resistance, impedance, potential, or the time or velocity of the relative movement of the second biomolecule with respect to the nanopore.

[0225] Therefore, in some embodiments, step (c) of the method includes: measuring the changing current value through the pore during relative movement of the second biomolecule with respect to the pore, and thereby identifying or characterizing the second biomolecule.

[0226] In some embodiments, after the membrane is provided, a first fluid chamber and a second fluid chamber are automatically formed on both sides of the membrane, separating the first and second fluid chambers. Therefore, in some embodiments, the first and second fluid chambers may not be provided in step (a). In some embodiments, the first and / or second fluid chambers may not have defined boundaries; that is, they may be open spaces.

[0227] In some embodiments, in step (a), a first fluid compartment and a second fluid compartment are provided. In some embodiments, the first fluid compartment and / or the second fluid compartment may also have well-defined boundaries; that is, they may be enclosed spaces.

[0228] In some implementations, the method of this application also provides a chip.

[0229] In some embodiments, the chip includes an array of microcavities on a substrate (e.g., a wafer substrate or a PCB substrate). In some embodiments, after the membrane comes into contact with the microcavities, the interior of the microcavities can form a second fluid chamber with the membrane and can store a solution. In some embodiments, the upper part of the microcavities can be directly or indirectly tightly connected to the membrane and form a first fluid chamber.

[0230] In some embodiments, in step (a), the first biomolecule and the second biomolecule are present in the form of a complex.

[0231] In the method of this application, the first biomolecule, the second biomolecule, and the pore can be contacted in any order. In some embodiments, the second biomolecule contacts the pore in the form of a complex with the first biomolecule. In some embodiments, when a voltage is applied across the pore, the complex containing the second biomolecule and the first biomolecule contacts the pore, and the second biomolecule is controlled to move relative to the pore (e.g., through the pore).

[0232] In some embodiments, in step (a), a second factor capable of regulating the first factor (e.g., regulating the concentration of the first factor) is also provided.

[0233] In some embodiments, the provided first factor and / or second factor are located in a first fluid compartment and / or a second fluid compartment.

[0234] In some embodiments, in step (b), an external force (e.g., electric force, magnetic force, and / or pressure) is applied to cause the first factor to move through the orifice between the first fluid chamber and the second fluid chamber under the drive of the external force.

[0235] Second biomolecular perforation

[0236] In some embodiments, the first biomolecule controls at least a portion (e.g., a chain) of the second biomolecule to pass through the pore and obtain one or more measurements.

[0237] In some embodiments, the first biomolecule controls at least a portion of the second biomolecule to move through the aperture in a direction parallel (e.g., the same or opposite) to the applied electric and / or magnetic field forces (e.g., moving through the aperture from a low-potential chamber to a high-potential chamber), and obtains one or more measurements.

[0238] In some embodiments, as at least a portion of the second biomolecule passes through the pore, a changing current is generated in the pore, and a measurement is obtained by measuring the changing current.

[0239] In some embodiments, as at least a portion of the second biomolecule passes through the pore, a varying blocking ion current is generated in the pore, and a measurement is obtained by measuring the varying blocking ion current.

[0240] In some embodiments, when the second biomolecule is a nucleic acid molecule, as different nucleotides of the nucleic acid molecule pass through the pore, ion currents corresponding to the changes in different nucleotides are generated in the pore. The nucleic acid molecule is identified or characterized by detecting the changes in the ion currents to identify or characterize the different nucleotides.

[0241] First biomolecule

[0242] In some embodiments, the first biomolecule is a protein (e.g., an enzyme).

[0243] In some embodiments, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof.

[0244] In some embodiments, the polymerase is a polymerase with chain displacement activity.

[0245] In some embodiments, the polymerase is selected from the following: Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof.

[0246] In this paper, any suitable helicase may be used. The helicase may be selected from the SF1, SF2, SF3, SF4, SF5 or SF6 family.

[0247] In some embodiments, the specific helicase is selected from the following: Dda, Hel308, RecD, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, gp41, T7gp4, BCH105, or any combination thereof. In some embodiments, the helicase is a wild-type protein of any of the above helicases, or a homology thereof, or a mutant thereof.

[0248] Detailed information on helicase Hel308 can be found in WO 2013 / 057495, the entire contents of which are incorporated herein by reference. Detailed information on helicase RecD can be found in WO 2013 / 098562, the entire contents of which are incorporated herein by reference. Detailed information on helicase Dda can be found in WO2015 / 055981 and WO 2016 / 055777, the entire contents of which are incorporated herein by reference. Detailed information on helicase BCH105 can be found in PCT / CN2021 / 143662 and PCT / CN2022 / 144205, the entire contents of which are incorporated herein by reference.

[0249] In some embodiments, the helicase is further linked to an additional polypeptide selected from tags, restriction enzyme sites, signal peptides or lead peptides, detectable markers, or any combination thereof.

[0250] In some embodiments, the amino acid recognition protein is capable of specifically recognizing the terminal amino acid of a polypeptide or protein. Therefore, in some embodiments, when the first biomolecule is an amino acid recognition protein, the second biomolecule can be a polypeptide or protein. Specific information on amino acid recognition proteins can be found in PCT / CN2023 / 143631, the entire contents of which are incorporated herein by reference.

[0251] In some embodiments, the amino acid recognition protein is selected from any one or more of the following:

[0252] (i) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of ClpS family;

[0253] (ii)Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt;

[0254] (iii) human UBR1, human UBR2 or Saccha-romyces cerevisiae UBR1 of the UBR family;

[0255] (iiii) GID4 of H. sapiens or Saccharomyces cerevisiae:

[0256] (iiiiii) Drosophila melanogaster BIR2;

[0257] (iiiiii) H. sapiens N-meristoyltransferase NMT1.

[0258] Second biomolecule

[0259] In some embodiments, the second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof.

[0260] In some embodiments, the second biomolecule is selected from natural, synthetic, wholly or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof.

[0261] In some embodiments, the second biomolecule includes a adapter (e.g., a sequencing adapter).

[0262] In some embodiments, the second biomolecule is DNA containing a connector.

[0263] In some embodiments, the second biomolecule is a polypeptide containing a linker.

[0264] In some embodiments, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule.

[0265] In some implementations, the connector may or may not include one or more spacer zones.

[0266] In some implementations, the connector does not include a spacer area.

[0267] In some implementations, the spaced regions have one or more features selected from the following:

[0268] (1) Each of the one or more spacer regions independently comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), synthetic polymers with nucleotide side chains, or any combination thereof;

[0269] (2) Each of the one or more spacer regions independently contains one or more base-free nucleotides;

[0270] (3) Each of the one or more spacer regions independently contains one or more chemical groups that cause one or more biomolecules (e.g., helicases) to stop.

[0271] In some embodiments, the second biomolecule is a nucleic acid molecule (e.g., a chain-like nucleic acid molecule).

[0272] In some implementations, the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule.

[0273] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the helicase separates the two strands of the double-stranded nucleic acid molecule to provide a single-stranded nucleic acid molecule and controls the movement of the single-stranded nucleic acid molecule through the pore.

[0274] In some implementations, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor directly or indirectly regulates the helicase to unwind the double-stranded nucleic acid molecule.

[0275] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor directly or indirectly regulates the helicase to unwind the double-stranded nucleic acid molecule, thereby controlling at least a portion of the double-stranded nucleic acid molecule to pass through the pore.

[0276] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is DNA containing a sequencing adapter, the helicase forms a complex with the DNA through the sequencing adapter.

[0277] In some embodiments, when the first biomolecule is a helicase and the second biomolecule is DNA containing a sequencing adapter, the helicase binds to the sequencing adapter of the DNA and forms a complex with the DNA.

[0278] The second biomolecule is non-perforated.

[0279] In some embodiments, the first biomolecule controls at least a portion of the second biomolecule to move relative to the pore without passing through it, and obtains one or more measurements.

[0280] In some embodiments, the direction of movement of the second biomolecule is independent of the direction of the applied electric and / or magnetic field forces.

[0281] In some embodiments, the first biomolecule is connected to the pore (e.g., covalently).

[0282] In some embodiments, as at least a portion of the second biomolecule moves relative to the pore, a changing current is generated in the pore, and a measurement value is obtained by measuring the changing current.

[0283] In some embodiments, as at least a portion of the second biomolecule moves relative to the pore, a varying blocking ion current is generated in the pore, and a measurement is obtained by measuring the varying blocking ion current.

[0284] In some embodiments, when the second biomolecule is a nucleic acid molecule, as different nucleotides of the nucleic acid molecule move relative to the pore, ion currents corresponding to the changes in different nucleotides are generated in the pore. By detecting the changes in the ion currents, different nucleotides are identified or characterized, thereby identifying or characterizing the nucleic acid molecule.

[0285] In this type of implementation, although the second biomolecule does not pass through the pore, as the second biomolecule moves relative to the pore, the first biomolecule or other molecules above the pore interfere with and change the ionic current in the pore. Therefore, as the second biomolecule moves relative to the pore, a correspondingly changing ionic current is generated in the pore.

[0286] In some implementations, the other molecules may be modified or unmodified dNTPs.

[0287] First biomolecule

[0288] In some embodiments, the first biomolecule is a protein (e.g., an enzyme).

[0289] In some embodiments, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof.

[0290] In some embodiments, the polymerase may be a polymerase with or without chain displacement activity.

[0291] In some embodiments, the polymerase is selected from the following: Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof.

[0292] In this document, any suitable helicase may be used. The helicase may be selected from the SF1, SF2, SF3, SF4, SF5, or SF6 family. In some embodiments, the specific helicase is selected from the following: Dda, Hel308, RecD, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, gp41, T7gp4, BCH105, or any combination thereof. In some embodiments, the helicase is a wild-type protein of any of the above helicases, its homolog, or a mutant thereof.

[0293] Detailed information on helicase Hel308 can be found in WO 2013 / 057495, the entire contents of which are incorporated herein by reference. Detailed information on helicase RecD can be found in WO 2013 / 098562, the entire contents of which are incorporated herein by reference. Detailed information on helicase Dda can be found in WO2015 / 055981 and WO 2016 / 055777, the entire contents of which are incorporated herein by reference. Detailed information on helicase BCH105 can be found in PCT / CN2021 / 143662 and PCT / CN2022 / 144205, the entire contents of which are incorporated herein by reference.

[0294] In some embodiments, the helicase is further linked to an additional polypeptide selected from tags, restriction enzyme sites, signal peptides or lead peptides, detectable markers, or any combination thereof.

[0295] In some embodiments, the amino acid recognition protein is capable of specifically recognizing the terminal amino acid of a polypeptide or protein. Therefore, in some embodiments, when the first biomolecule is an amino acid recognition protein, the second biomolecule can be a polypeptide or protein. Specific information on amino acid recognition proteins can be found in PCT / CN2023 / 143631, the entire contents of which are incorporated herein by reference.

[0296] In some embodiments, the amino acid recognition protein is selected from any one or more of the following:

[0297] (i) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of ClpS family;

[0298] (ii)Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt;

[0299] (iii) human UBR1, human UBR2 or Saccha-romyces cerevisiae UBR1 of the UBR family;

[0300] (iiii) GID4 of H. sapiens or Saccharomyces cerevisiae:

[0301] (iiiiii) Drosophila melanogaster BIR2;

[0302] (iiiiii) H. sapiens N-meristoyltransferase NMT1.

[0303] Second biomolecule

[0304] In some embodiments, the second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof.

[0305] In some embodiments, the second biomolecule is selected from natural, synthetic, wholly or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof.

[0306] In some embodiments, the second biomolecule includes a adapter (e.g., a sequencing adapter).

[0307] In some embodiments, the second biomolecule is DNA containing a connector.

[0308] In some embodiments, the second biomolecule is a polypeptide containing a linker.

[0309] In some embodiments, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule.

[0310] In some implementations, the connector may or may not include one or more spacer zones.

[0311] In some implementations, the connector does not include a spacer area.

[0312] In some implementations, the spaced regions have one or more features selected from the following:

[0313] (1) Each of the one or more spacer regions independently comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), synthetic polymers with nucleotide side chains, or any combination thereof;

[0314] (2) Each of the one or more spacer regions independently contains one or more base-free nucleotides;

[0315] (3) Each of the one or more spacer regions independently contains one or more chemical groups that cause one or more biomolecules (e.g., helicases) to stop.

[0316] In some embodiments, the second biomolecule is a nucleic acid molecule (e.g., a circular nucleic acid molecule).

[0317] In some embodiments, the first biomolecule is a polymerase (e.g., DNA polymerase) and the second biomolecule is a circular nucleic acid molecule.

[0318] In some embodiments, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the polymerase uses the circular nucleic acid molecule as a template to extend primers that bind to the circular nucleic acid molecule and controls the circular nucleic acid molecule to move relative to the pore without passing through the pore.

[0319] In some implementations, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor directly or indirectly regulates the polymerase to extend the primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template.

[0320] In some embodiments, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor directly or indirectly regulates the polymerase to extend the primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template, thereby controlling the circular nucleic acid molecule to move relative to the pore without passing through the pore.

[0321] In some embodiments, the measurement can characterize one or more of the following features of the second biomolecule: the nucleotide sequence of the nucleic acid molecule, whether the nucleotides of the nucleic acid molecule are modified (e.g., methylation, hydroxymethylation), the amino acid sequence of the polypeptide, the polysaccharide sequence, or any combination thereof.

[0322] The method described in this application can be applied to sequencing technologies, such as nanopore sequencing.

[0323] Therefore, in some embodiments, in step (a), reagents for sequencing (e.g., inorganic salts and / or buffers) are also provided. In some embodiments, the reagents for sequencing are present in a first fluid compartment and / or a second fluid compartment.

[0324] In some embodiments, the reagent may be an inorganic salt. For example, potassium chloride (KCl), sodium chloride (NaCl), lithium chloride, and / or calcium chloride (CaCl2). The salt concentration may be saturated. In some embodiments, the inorganic salt is selected from sodium chloride, potassium chloride, lithium chloride, or any combination thereof.

[0325] In some embodiments, the reagent may be a buffer solution. For example, HEPES and / or Tris-HCl buffer. The pH of the buffer solution may be selected from 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8 or 7.5 to 8.5. In some embodiments, the buffer solution is selected from HEPES, Tris-HCl, or any combination thereof.

[0326] In some embodiments, the method of this application also provides a dNTP substrate. In some embodiments, when the first biomolecule is a polymerase, a primer and dNTP are also provided in step (a) for the polymerase to extend the primer that binds to a second biomolecule (e.g., a circular nucleic acid molecule).

[0327] In some implementations, when the first factor is a metal ion, ATP is also provided in step (a).

[0328] In some embodiments, when the first biomolecule is a polymerase, primers and dNTPs are also provided in step (a) for the polymerase to extend the primers that bind to the second biomolecule (e.g., a circular nucleic acid molecule).

[0329] In some implementations, the membrane is as defined in the first aspect.

[0330] In some implementations, the aperture is as defined in the first aspect.

[0331] In some implementations, the first factor is as defined in the first aspect.

[0332] In some implementations, the second factor is as defined in the first aspect.

[0333] In some embodiments, in step (a) of the method:

[0334] (1) A first factor is provided in a second fluid compartment at a concentration sufficient to initiate the action of the first biomolecule, while the first fluid compartment does not provide the first factor; in some embodiments, the first fluid compartment may or may not provide the second factor.

[0335] (2) A first factor is provided in the second fluid compartment at a concentration sufficient to initiate the action of the first biomolecule, and the first fluid compartment provides a first factor at a concentration insufficient to initiate the action of the first biomolecule; in some embodiments, the first fluid compartment may or may not provide the second factor; or

[0336] (3) A first factor is provided in the second fluid compartment at a concentration sufficient to enable the first biomolecule to start working; in some embodiments, the first fluid compartment is also provided with a second factor.

[0337] Regulating the movement of second biomolecules through factor flow

[0338] In some embodiments, in step (a) of the method, a first factor is provided in the second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated.

[0339] When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid compartment is higher than that of the first fluid compartment. The first factor moves from the second fluid compartment to the first fluid compartment through the orifice to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working and generate one or more measurements.

[0340] In some embodiments, increasing the potential difference between the second fluid compartment and the first fluid compartment, and / or increasing the initial concentration of the first factor in the second fluid compartment, increases the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively modulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0341] In some implementations, the first fluid compartment may or may not provide a second factor.

[0342] In some embodiments, reducing the potential difference between the second fluid compartment and the first fluid compartment, reducing the initial concentration of the first factor in the second fluid compartment, and / or providing the second factor in the first fluid compartment slows the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively modulating the activity of the first biomolecule and reducing the migration velocity of the second biomolecule relative to the pore. In some embodiments, the migration velocity of the second biomolecule relative to the pore is reduced until it ceases to migrate.

[0343] In some embodiments, the second factor is selected from metal ion chelators (e.g., EDTA, EGTA, DTPA, TTHA, NTA).

[0344] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, so that the first biomolecule is in an active state, at which time one or more measurements (e.g., varying ion current) are generated;

[0345] When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid compartment is lower than that of the first fluid compartment. The first factor moves from the first fluid compartment to the second fluid compartment through the pore, thereby reducing the concentration of the first factor in the first fluid compartment. This reduces the efficiency of the first biomolecule and decreases the migration speed of the second biomolecule relative to the pore. In some embodiments, the migration speed of the second biomolecule relative to the pore is reduced until it stops moving.

[0346] In some implementations, the second fluid compartment may or may not provide the first factor.

[0347] In some embodiments, increasing the potential difference between the first and second fluid compartments, decreasing the initial concentration of the first factor in the first fluid compartment, and / or providing a second factor in the first fluid compartment increases the rate at which the concentration of the first factor decreases in the first fluid compartment, thereby negatively modulating the activity of the first biomolecule and reducing the migration velocity of the second biomolecule relative to the pore. In some embodiments, the migration velocity of the second biomolecule relative to the pore is reduced until it ceases to migrate.

[0348] In some embodiments, the second factor is selected from metal ion chelators (e.g., EDTA, EGTA, DTPA, TTHA, NTA).

[0349] In some embodiments, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0350] In some embodiments, in step (a) of the method, a first factor is provided in the second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated.

[0351] When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is higher than that of the second fluid compartment, the first factor moves from the second fluid compartment through the pore to the first fluid compartment to increase the concentration of the first factor in the first fluid compartment, thereby initiating the first biomolecule to work and generating one or more measurements.

[0352] In some embodiments, increasing the potential difference between the second fluid compartment and the first fluid compartment, and / or increasing the initial concentration of the first factor in the second fluid compartment, increases the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively modulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0353] In some embodiments, reducing the potential difference between the second fluid compartment and the first fluid compartment, and / or reducing the initial concentration of the first factor in the second fluid compartment, slows the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively modulating the activity of the first biomolecule and reducing the migration velocity of the second biomolecule relative to the pore. In some embodiments, the migration velocity of the second biomolecule relative to the pore is reduced until it ceases to migrate.

[0354] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, so that the first biomolecule is in an active state, at which point one or more measurements (e.g., varying ion current) will be generated;

[0355] When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is lower than that of the second fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the pore to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the efficiency of the first biomolecule and reducing the movement speed of the second biomolecule relative to the pore.

[0356] In some implementations, the second fluid compartment may or may not provide the first factor.

[0357] In some embodiments, increasing the potential difference between the first fluid compartment and the second fluid compartment, and / or decreasing the initial concentration of the first factor in the first fluid compartment, increases the rate at which the concentration of the first factor decreases in the first fluid compartment, thereby negatively modulating the activity of the first biomolecule and reducing the migration velocity of the second biomolecule relative to the pore. In some embodiments, the migration velocity of the second biomolecule relative to the pore is reduced until it ceases to migrate.

[0358] In some embodiments, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0359] In some embodiments, in step (a) of the method, a first factor is provided in the second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated.

[0360] When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the orifice to increase the concentration of the first factor in the first fluid compartment, thereby activating the first biomolecule and generating one or more measurements.

[0361] In some embodiments, increasing the pressure difference between the second fluid compartment and the first fluid compartment, and / or increasing the initial concentration of the first factor in the second fluid compartment, increases the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively modulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0362] In some embodiments, reducing the pressure difference between the second fluid compartment and the first fluid compartment, and / or reducing the initial concentration of the first factor in the first fluid compartment, slows the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively modulating the activity of the first biomolecule and reducing the migration velocity of the second biomolecule relative to the pore. In some embodiments, the migration velocity of the second biomolecule relative to the pore is reduced until it ceases to migrate.

[0363] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, so that the first biomolecule is in an active state, at which time one or more measurements (e.g., varying ion current) are generated;

[0364] When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid chamber is lower than that in the first fluid chamber, the first factor moves from the first fluid chamber to the second fluid chamber through the orifice to reduce the concentration of the first factor in the first fluid chamber. This reduces the efficiency of the first biomolecule and decreases the migration speed of the second biomolecule relative to the orifice. In some embodiments, the migration speed of the second biomolecule relative to the orifice is reduced until it stops moving.

[0365] In some implementations, the second fluid compartment may or may not provide the first factor.

[0366] In some embodiments, the pressure difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased to slow down the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0367] In some embodiments, increasing the pressure difference between the first fluid compartment and the second fluid compartment, and / or decreasing the initial concentration of the first factor in the first fluid compartment, increases the rate at which the concentration of the first factor decreases in the first fluid compartment, thereby negatively modulating the activity of the first biomolecule and reducing the migration velocity of the second biomolecule relative to the pore. In some embodiments, the migration velocity of the second biomolecule relative to the pore is reduced until it ceases to migrate.

[0368] In some embodiments, in step (a) of the method, a first factor is provided in the second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated.

[0369] When the first factor is positively charged, negatively charged, or uncharged, and the concentration of the first factor in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the orifice to increase the concentration of the first factor in the first fluid compartment. When the concentration of the first factor in the first fluid compartment is sufficient to enable the first biomolecule to start working, one or more measurements are generated.

[0370] In some embodiments, the initial concentration of the first factor in the second fluid compartment is increased to increase the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0371] In some embodiments, the initial concentration of the first factor in the first fluid compartment is reduced to slow the rate at which the concentration of the first factor in the first fluid compartment increases, thereby negatively regulating the activity of the first biomolecule and reducing the migration rate of the second biomolecule relative to the pore.

[0372] In some embodiments, in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, and a second fluid compartment does not provide the first factor or provides a first factor at a concentration lower than that of the first factor in the first fluid compartment, so that the first biomolecule is in a working state and one or more measurements are generated (e.g., varying ion current).

[0373] When the first factor carries a positive charge, a negative charge, or no charge, and the concentration of the first factor in the second fluid compartment is lower than that in the first fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the hole to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the working efficiency of the first biomolecule.

[0374] In some embodiments, the initial concentration of the first factor in the first fluid compartment is increased to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

[0375] In some embodiments, the initial concentration of the first factor in the first fluid compartment is reduced to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule and reducing the migration rate of the second biomolecule relative to the pore.

[0376] Regulation of sequencing

[0377] In some embodiments, the identification or characterization of the second biomolecule includes sequencing the second biomolecule.

[0378] In some embodiments, the sequence information of the second biomolecule (e.g., the nucleotide sequence of the nucleic acid molecule, whether the nucleotides of the nucleic acid molecule are modified (e.g., methylation modification, hydroxymethylation modification), the amino acid sequence of the polypeptide, and the polysaccharide sequence) is obtained by the method described above.

[0379] In some embodiments, when the second biomolecule is a nucleic acid molecule, as different nucleotides of the nucleic acid molecule move relative to the pore (e.g., through the pore), ion currents corresponding to the changes in the different nucleotides are generated in the pore. By detecting the multiple changes in ion currents, multiple nucleotides are identified and / or analyzed, thereby sequencing the nucleic acid molecule.

[0380] In some implementations, sequencing is regulated by modulating the activity of a first biomolecule to alter the relative movement of at least a portion (e.g., a strand) of a second biomolecule with respect to the pore.

[0381] In some implementations, sequencing is initiated by modulating the activity of a first biomolecule to control at least a portion of a second biomolecule to begin relative movement with respect to the pore.

[0382] In some implementations, sequencing is stopped by modulating the activity of a first biomolecule to control at least a portion of a second biomolecule to cease relative movement with respect to the pore.

[0383] In some embodiments, the sequencing rate is altered (e.g., increased or decreased) by modulating the activity of the first biomolecule to change (e.g., increase or decrease) the rate at which at least a portion of the second biomolecule moves relative to the pore.

[0384] Device or system

[0385] In a third aspect, this application provides an apparatus or system comprising:

[0386] (a) A reaction module, which includes:

[0387] (i) A membrane having one or more pores; the membrane separating a first fluid compartment and a second fluid compartment, the pores connecting the first fluid compartment and the second fluid compartment;

[0388] (ii) A first factor capable of moving between a first fluid compartment and a second fluid compartment, the first factor being capable of directly or indirectly regulating the activity of a first biomolecule (e.g., regulating the level of activity of the first biomolecule).

[0389] Furthermore, the first factor can move between the first fluid chamber and the second fluid chamber through the orifice under the drive of its own concentration gradient and / or external forces (e.g., electric field force, magnetic field force and / or pressure);

[0390] (b) A control module capable of regulating the movement of the first factor through the orifice between the first fluid compartment and the second fluid compartment.

[0391] In the apparatus or system of this application, depending on the different selections of the first biomolecule being regulated, the apparatus or system of this application can be applied to different detection and / or sequencing scenarios. For example, RNA sequencing; DNA sequencing; sequencing of other nucleic acid molecules such as LNA, PNA, or XNA; amino acid sequencing of proteins or peptides; haplotype analysis; methylation detection and / or mapping.

[0392] In some embodiments, the activity of the first biomolecule can be modulated to place it in an active or inactive operating mode. For example, when the first fluid compartment contains all the necessary components for the first biomolecule to function (e.g., one or more first factors), the first biomolecule is in an active operating mode. Conversely, when the first fluid compartment does not contain all the necessary components for the first biomolecule to function (e.g., one or more first factors), the first biomolecule is in an inactive operating mode.

[0393] In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of proteins and / or nucleic acids. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of nucleic acid-binding proteins. For example, the activity of single-stranded DNA-binding proteins in binding to single-stranded DNA. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of motor proteins. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of helicases. For example, the activity of unwinding double-stranded DNA, or the activity of controlling DNA passage through nanopores. In some embodiments, the "activity" of the first biomolecule includes, but is not limited to, one or more activities of polymerases. For example, the activity of polymerizing and extending DNA, strand displacement activity, or exonuclease activity.

[0394] In some embodiments, the activity is related to the ability of a first biomolecule to regulate a second biomolecule.

[0395] In some embodiments, the activity is related to the ability of the first biomolecule to control at least a portion (e.g., a chain) of the second biomolecule to move relative to the pore.

[0396] In some embodiments, by modulating the activity of the first biomolecule, at least a portion (e.g., a chain) of the second biomolecule is controlled to move relative to the pore, and one or more measurements are generated.

[0397] In some embodiments, the device or system further includes a detection and / or analysis module capable of detecting and / or analyzing one or more measurements resulting from the relative movement of at least a portion (e.g., a chain) of a second biomolecule with respect to the pore.

[0398] In some implementations, the second biomolecule is identified or characterized by detecting and / or analyzing the measured values.

[0399] In some implementations, the second biomolecule is sequenced by detecting and / or analyzing the measured values ​​(e.g., changing ion currents).

[0400] In some embodiments, the measurement characterizes one or more of the following features of the second biomolecule: the nucleotide sequence of the nucleic acid molecule, whether the nucleotides of the nucleic acid molecule are modified (e.g., methylation, hydroxymethylation), the amino acid sequence of the polypeptide, the polysaccharide sequence, or any combination thereof.

[0401] In some embodiments, the device or system further includes a solid support capable of supporting the membrane.

[0402] In some embodiments, the solid support, after carrying the membrane, is able to form and / or provide a second fluid compartment and / or a first fluid compartment together with the membrane.

[0403] In some embodiments, the apparatus or system further includes a first fluid compartment and a second fluid compartment located in the reaction module.

[0404] In some embodiments, the device or system further includes a second factor located in the reaction module, which is capable of regulating the first factor (e.g., regulating the concentration of the first factor).

[0405] In some embodiments, the first factor and / or the second factor are located in a first fluid compartment and / or a second fluid compartment.

[0406] In some embodiments, the device or system further includes a first biomolecule located in a first fluid compartment. In some embodiments, the first biomolecule is as defined in the first aspect.

[0407] In some embodiments, the device or system further includes a second biomolecule located in the first fluid compartment. In some embodiments, the second biomolecule is as defined in the first aspect.

[0408] In some embodiments, the device or system further comprises a complex formed upon contact between a first biomolecule and a second biomolecule.

[0409] In some embodiments, the apparatus or system further includes: the first factor as defined in the first aspect.

[0410] In some embodiments, the apparatus or system further includes: the second factor as defined in the first aspect.

[0411] In some embodiments, the device or system further includes the membrane as defined in the first aspect.

[0412] In some embodiments, the device or system further includes the aperture as defined in the first aspect.

[0413] In some embodiments, the device or system further includes: the control module includes components (e.g., electrodes) that provide electric force, magnetic force, and / or pressure.

[0414] In some embodiments, the device or system further includes: the solid support comprising a microcavity.

[0415] In some embodiments, after the solid support supports the membrane, the area below the membrane and the interior of the microcavity together form a second fluid chamber, and the area above the membrane forms a first fluid chamber.

[0416] In some implementations, the solid support is a chip.

[0417] Terminology Definition

[0418] In this text, the term "biomolecule" refers to molecules relevant to biological systems. This term includes, but is not limited to, nucleic acids (e.g., DNA, RNA), polypeptides, proteins, and any complexes thereof. The biomolecules may be naturally occurring or engineered or synthetic.

[0419] In this text, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" have the same meaning and can be used interchangeably. They refer to biomolecules composed of monomeric nucleotide chains (polymers or oligomers). The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It should also be understood that the method of this invention, in addition to detecting and identifying natural nucleic acids, can also be used to detect and identify non-natural nucleic acids, such as peptide nucleic acids (PNA), morpholinonucleotides, locked nucleic acids (LNA), gamma-hydroxynucleotides (GNA), and threononucleotides (TNA), etc.

[0420] In various embodiments of the present invention, nucleic acids can be derived from a variety of sources, such as bacteria, viruses, humans and animals, plants and fungi, etc. The nucleic acids can also be derived from pathogens. In the present invention, nucleic acids can be genomic, extrachromosomal, or synthetic. It is understood that in the embodiments shown herein, "DNA" is merely an example, and other nucleic acids, such as RNA or all of the above, can be used.

[0421] Furthermore, the term "nucleic acid molecule" herein includes polymeric forms of nucleotides of any length, including but not limited to ribonucleotides or deoxyribonucleotides. In some embodiments, these terms may include triple-stranded, double-stranded, and single-stranded DNA, PNA, and triple-stranded, double-stranded, and single-stranded RNA. They also include modified polynucleotides (such as methylation) as well as unmodified forms. More specifically, the term "nucleic acid molecule" may include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide (e.g., N-glycosides or C-glycosides of purine or pyrimidine bases).

[0422] In this text, the terms "polypeptide" and "peptide" have the same meaning and are used interchangeably. They refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers, where one or more amino acid residues can be synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, and to polymers of naturally occurring amino acids. Polypeptides can also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipolysis, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. Peptides can be prepared using recombinant technologies, such as by expressing recombinant or synthetic polynucleotides.

[0423] In this text, the term "protein" is used to describe folded polypeptides having secondary or tertiary structures. Proteins can consist of a single polypeptide or can comprise multiple polypeptides assembled to form a multimer. The multimer can be a homooligomer or a heterooligomer. Proteins can be naturally occurring or wild-type proteins, or modified or non-natural proteins. Proteins can differ from wild-type proteins, for example, through the addition, substitution, or deletion of one or more amino acids.

[0424] In this text, the term "motor protein" generally refers to a protein that can bind directly or indirectly to nucleic acids and drive the relative movement of nucleic acid molecules with nanopores through the hydrolysis of energy-supplying molecules. Examples of motor proteins include, but are not limited to, helicases, polymerases, ligases, reverse transcriptases, or exonucleases.

[0425] In this text, the term "helicase" generally refers to an enzyme that binds to single-stranded nucleic acids and breaks the hydrogen bonds between the two strands of the nucleic acid molecule by hydrolyzing the high-energy phosphate bonds of ATP molecules, thereby breaking the hydrogen bonds between the two strands of the nucleic acid molecule and forming a single-stranded nucleic acid molecule. Examples of helicases include, but are not limited to, helicases that move along the 3' to 5' direction of the nucleic acid (such as helicase Rep and its mutants) or helicases that move along the 5' to 3' direction of the nucleic acid (such as helicase Dda and its mutants).

[0426] In this text, the term "polymerase" generally refers to any enzyme capable of catalyzing the polymerization of bases. Examples of polymerases include, but are not limited to, nucleic acid polymerases, transcriptases, reverse transcriptases, or terminal transferases. For example, a polymerase may be Bst DNA polymerase or phi29 DNA polymerase, but is not limited thereto. Examples of polymerases are known in the art, and those skilled in the art can select a suitable polymerase according to actual needs.

[0427] Beneficial effects of the invention

[0428] This application utilizes the ion transport properties of nanopores to provide methods and apparatus for regulating the activity of biomolecules (e.g., motor proteins), enabling the successful initiation, shutdown, and control of the activity of biomolecules (e.g., putting them into an active or inactive state). Furthermore, this application also provides methods for identifying and / or characterizing biomolecules (particularly methods for sequencing biomolecules). Therefore, the methods of this application can detect the presence of biomolecules or characterize detected biomolecules and regulate sequencing. In summary, the methods and apparatus of this application can be applied to drug development, medicine, diagnostics, life science research, and environmental monitoring.

[0429] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0430] Figure 1 shows two specific configurations of the nanopore sequencing device of this application, wherein Figure 1A and Figure 1B show two morphologies of the pore (i.e., biological nanopores and solid nanopores), respectively.

[0431] Figure 2 is a schematic diagram of the sequencing initiation method in Example 2.

[0432] Figure 3 is a schematic diagram of magnesium ion transport in Example 2.

[0433] Figure 4 is a schematic diagram of a nanopore sequencing speed control method shown in Example 4.

[0434] Figure 5 shows the magnesium ion concentration gradient distribution in Example 5, where the potential difference is 180mV.

[0435] Figure 6 shows the magnesium ion concentration gradient distribution in Example 5, where the potential difference is 0 mV.

[0436] Figure 7 shows the results of magnesium ion concentration gradient distribution under the conditions of applying an electric field and not applying an electric field in Example 5.

[0437] Figure 8 shows the potassium ion concentration gradient distribution under the condition of potential difference in Example 6.

[0438] Figure 9 shows the results of the chloride ion concentration gradient distribution under the condition of potential difference in Example 6.

[0439] Figure 10 shows the sequencing signal detected by nanopore DNA sequencing performed by the method of the present invention in Example 7.

[0440] Figure 11 illustrates the results of controlling the nanopore sequencing speed by regulating the factors required for motor proteins between the two compartments using the method of the present invention in Example 8. Higher voltage results in more magnesium ions flowing from the trans compartment into the cis compartment. As the magnesium ion concentration near the nanopore in the cis compartment increases, the library molecules bound to motor proteins in the cis compartment are unwound more quickly, thereby increasing the sequencing rate.

[0441] Sequence information

[0442] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0443] Table 1: Sequence Description Example

[0444] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0445] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Susubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommendations. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0446] Example 1

[0447] This embodiment demonstrates a device that can be used for nanopore sequencing.

[0448] The device comprises a system with a membrane, nanopores, and a carrier chip. Biomolecules (e.g., motor proteins) and their essential components (such as cofactors and ATP) are separated on either side of the nanopore into a cis compartment (cis compartment, also referred to as the first fluid compartment) and a trans compartment (trans compartment, also referred to as the second fluid compartment). During sequencing, the essential components move from the trans compartment through the nanopores to the cis compartment via electrophoresis, diffusion, and electroosmosis, where they further bind and react with motor proteins and other molecules, thereby activating the motor proteins and initiating sequencing.

[0449] The device comprises the following parts:

[0450] a. Nanopores. Nanopores can be biological nanopores as shown in Figure 1A, such as pore proteins like MspA, α-Haemolysin, CsgG, and Aerolysin, or their mutants or fusions; or solid nanopores as shown in Figure 1B, such as SiNx (silicon nitride), SiO2, graphene, Al2O3, boron nitride (BN), and HfO2. Nanopores allow key substances in the trans compartment to be transported to the cis compartment via basic transport methods such as electrophoresis, diffusion, or electroosmosis.

[0451] b. Membranes supporting nanopores. These membranes can be phospholipid membranes (e.g., DOPE, DPPC, DPhPC, DOPC, POPC, etc.), polymer membranes (e.g., diblock polymers, triblock polymers (e.g., PMOXA-PDMS-PMOXA), solid films (SiNx films, SiO2 films, Al2O3 films, graphene films, BN films, etc.), or mixed membranes of any two or more substances (e.g., mixed membranes of phospholipids and block polymers, mixed membranes of phospholipids and solid films, etc.).

[0452] c. Chip supporting the membrane-pore system. The core structure of this chip typically consists of an array of microcavities fabricated on a wafer substrate or PCB substrate using specialized MEMS processes. The interior of these microcavities serves as the trans compartment for nanopore sequencing, storing the solutions necessary for sequencing. The upper part of the microcavity can be directly or indirectly tightly connected to the membrane-pore system, forming a relatively independent compartment.

[0453] d. Electrode System. The electrode system typically consists of a microelectrode within the microcavity and an external electrode, with the external electrode usually shared by multiple channels. Electrode materials are typically Ag / AgCl, Au, Pt, Pd, Ru, Ir, Ni, TiN, ITO, glassy carbon, graphene, etc. The microelectrode and external electrode materials can be the same or different. Composite materials can also be used.

[0454] e. Corresponding circuit control and signal acquisition system. The circuit system typically has voltage / current control and signal detection functions. For a typical nanopore sensor system, the operating voltage is usually DC or AC, with a voltage amplitude between 100mV and 300mV. The nanopore system has a relatively large impedance (usually around 0.5-1GΩ), while the corresponding characteristic current signal amplitude is usually around 100pA. Therefore, the circuit system usually has one or more operational amplifiers to amplify the picoampere-level raw current signal, and then measure the amplified signal. The amplified signal can be a current signal or a voltage signal.

[0455] f. Asymmetric solution systems. One or more key substances for initiating sequencing (e.g., the cofactor Mg2+ for activating helicase) are missing on one side of the CIS or Trans compartment. 2+ Or (such as ATP), or the concentration of the key substance is too low to effectively initiate sequencing; while the other compartment contains a certain amount of one or more of the key substance. Nanopores allow the key substance in one compartment to be transported to the other compartment via basic transport mechanisms such as electrophoresis, diffusion, or electroosmosis. This key substance can be a cofactor that activates helicase activity, such as monovalent, divalent, and trivalent metal ions (Mg). 2+ Mn 2+ Fe 2+ Fe 3+ Co 2+ Ni 2+ Zn 2+ Cu 2+ Cu + Ca 2+ It can also be other key substances, such as ATP, EDTA, a certain inhibitor or deinhibitor, etc.

[0456] d. Test molecule. The test molecule is usually a library complex, which typically contains an adapter, target molecule, and helicase. The target molecule can be a nucleic acid molecule, protein molecule, target peptide chain, polysaccharide molecule, etc. The test molecule may or may not contain a spacer region that restricts the movement of motor proteins.

[0457] The sequencing device was constructed according to the method disclosed in the literature Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, TJ, Montemagno, C., and Guo, P. (2009). Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nat Nanotechnol 4, 765-772 (PMID:19893523 PMCID:PMC2777743 DOI:10.1038 / nnano.2009.259). The solutions in the two chambers on both sides of the membrane were asymmetric solution systems.

[0458] Example 2

[0459] This embodiment demonstrates a method for controlling the start and stop of nanopore sequencing.

[0460] As shown in Figure 2, the cofactor Mg required for the function of nucleic acid helicase is stored in the trans compartment. 2+ The solution pre-filled in the CIS compartment does not contain Mg. 2+ Mg in the CIS compartment without voltage applied 2+ The concentration was too low to allow the library molecules in the CIS compartment to unwind properly. However, after applying a bias voltage of 180 mV, Mg... 2+ Due to the ion electrophoresis effect of the electric field, Mg is rapidly transported from the trans chamber through the nanopores to the cis chamber, further forming Mg near the nanopores. 2+ Concentration gradient field (a schematic diagram of magnesium ion transport is shown in Figure 3). The concentration gradient field is shown in Figure 2b; the concentration is highest near the nanopore and decreases rapidly away from the nanopore. Therefore, when library molecules are captured by the nanopore, the high concentration of Mg near the pore contributes to the high concentration of magnesium ions. 2+ The library molecules bind to helicase, thereby activating the helicase's activity and initiating the sequencing of the library molecules.

[0461] Example 3

[0462] This embodiment demonstrates a method for controlling the speed of nanopore sequencing.

[0463] This method involves adjusting the initial concentration of one or more key substances (e.g., cofactor Mg) in the trans compartment. 2+ This further influences the final concentration and concentration distribution of key substances in the CIS compartment, ultimately controlling enzyme activity and sequencing speed. For example, it can adjust Mg... 2+The initial concentration (0.1 mM-300 mM) in the trans chamber was controlled to further regulate Mg. 2+ Final concentration and concentration distribution in the CIS compartment.

[0464] Example 4

[0465] This embodiment demonstrates another method for controlling the speed of nanopore sequencing.

[0466] Storing one or more key substances (such as cofactor metal ions Mg) in the trans compartment 2+ Zn 2+ Ca 2+ For example, magnesium ions (at a concentration of 20 mM), while the CIS compartment stores inhibitors of this key substance (e.g., EDTA, where EDTA can react with Mg). 2+ Chelation, thereby reducing Mg 2+ By adjusting the types and concentrations of key substances in the trans compartment and the types and concentrations of inhibitors in the CIS compartment, the final concentration and concentration distribution of the key substance in the CIS compartment can be affected, thereby ultimately controlling enzyme activity and sequencing speed.

[0467] As shown in Figure 4, EDTA was not added to the CIS compartment shown in Figure 4A, while EDTA was added to the CIS compartment shown in Figure 4B. A brief comparison of the effects of EDTA on Mg near the nanopores is also presented. 2+ Concentration gradient distribution. As shown in Figure 4B, when EDTA is present in the CIS compartment, it reacts with Mg. 2+ Chelation occurs, forming a chelate, ethylenediaminetetraacetic acid magnesium sodium (EDTA-Mg). This allows free Mg to... 2+ As the concentration decreases, the Mg concentration further shrinks. 2+ Distribution range.

[0468] Example 5

[0469] This example demonstrates Mg 2+ As a key substance and its concentration gradient distribution.

[0470] In this embodiment, a model was established in COMSOL software to analyze the key substances (in this embodiment, helicase cofactor Mg) in the cis and trans compartments on both sides of the nanopore. 2+ Taking the concentration of a sample as an example, theoretical simulation analysis is performed to intuitively demonstrate the concentration gradient distribution of key substances after they are transported from one side of the compartment to the other through nanopores.

[0471] The system model parameters are set as follows: the nanopore opening diameter is 2 nm, the length is 10 nm, the width of both the upper and lower chambers is 100 nm, and the height is 52.5 nm. The upper chamber is filled with an aqueous solution of 0.5 M KCl, and the lower chamber is filled with an aqueous solution of 0.5 M KCl and 20 mM MgCl2. This embodiment mainly considers two modes of material transport: free diffusion of ions and ion electrophoresis. In this embodiment, the effect of electroosmosis is not considered, therefore the charge on the inner side of the nanopore is not set and is assumed to be neutral. Then, different electric fields are applied to the system, and the steady-state Mg... 2+ Distribution of concentration gradient.

[0472] Condition 1: The lower compartment is given a boundary condition potential of 0.18V, the upper compartment is grounded at 0V, and there is an applied potential difference of approximately 0.18V on both sides of the hole;

[0473] Under these conditions, Mg 2+ The concentration gradient distribution of Mg is mainly determined by the combined effects of ion free diffusion and ion electrophoresis. The simulation results are shown in Figure 5. After steady-state, Mg... 2+ The concentration gradient distribution is as follows:

[0474] As shown in Figure 5, Mg 2+ The concentration of Mg in the lower chamber passes through the nanopores to reach the upper chamber. The concentration is high near the nanopores, then decreases rapidly with increasing distance from the nanopores, reaching its lowest point (close to 0 mM) at the sidewall (far end) of the chamber. This concentration gradient distribution is directly influenced by the Mg concentration in the lower chamber. 2+ The effect of initial concentration.

[0475] Condition 2: The boundary condition potential applied to the lower chamber is 0V, and the upper chamber is grounded with a potential of 0V. There is no external potential difference across the nanopore. Under this condition, because there is no external potential difference across the nanopore, there is no significant field-guided ion electrophoresis behavior. Mg 2+ The concentration gradient distribution of Mg is mainly determined by the free diffusion of ions. The simulation results are shown in Figure 6. After steady-state, Mg... 2+ The concentration gradient distribution is as follows:

[0476] As shown in Figure 6, some Mg 2+ These Mg molecules diffuse freely from the lower chamber through the nanopores to reach the upper chamber. 2+ All of them are concentrated in the nanopores, causing Mg near the nanopores to 2+ The concentration is high, but Mg 2+ The concentration decreases rapidly with increasing distance from the nanopore. The concentration gradient distribution is directly influenced by the Mg in the lower chamber. 2+ The effect of initial concentration.

[0477] To more intuitively compare the effects of these two transport behaviors on Mg2+ The effect of concentration gradient distribution is shown in Figure 7A. Taking the center point at the outlet of the nanopores in the upper chamber as the origin and the horizontal direction as the X-axis, conditions 1 (with electric field) and 2 (without electric field) are compared. 2+ The concentration distribution along the X-axis is shown in Figure 7B.

[0478] As shown in Figure 7B, when an electric field is applied, Mg 2+ The highest concentration of Mg was reached at the nanopores, approximately 15.5 mM, after which it rapidly decreased with increasing X, eventually approaching zero at the far end. Without an applied electric field, Mg... 2+ The highest concentration of Mg was reached at the nanopores, approximately 3.5 mM, after which it rapidly decreased with increasing X, eventually approaching zero at the distal end. This demonstrates that when an electric field is applied, Mg... 2+ It can be efficiently transported to the upper chamber via ion electrophoresis through nanopores. Without an applied electric field, Mg can only be transported through diffusion. 2+ Most of them are concentrated near the nanopores, thus enabling sequencing to be maintained for a certain period of time. However, due to Mg... 2+ The concentration of [agent] decreases rapidly with increasing distance from the nanopore, so diffusion alone may not be able to sustain sequencing for a long time.

[0479] Example 6

[0480] Based on the conditions of Example 5, this example investigates the concentration distribution of potassium ions and chloride ions in the two chambers of the nanopore when an electric field is applied.

[0481] As shown in Figure 8, the initial potassium ion concentration in both chambers was 0.5 M. When 0.18 V was applied, the potassium ion concentration in the lower chamber remained basically unchanged, while the potassium ion concentration in the upper chamber changed to some extent due to the nanopore transport effect.

[0482] As shown in Figure 9, the initial chloride ion concentrations in the upper and lower chambers are 0.5 M and 0.52 M, respectively. When 0.18 V is applied, the chloride ion concentration in the lower chamber remains basically unchanged, while the chloride ion concentration in the upper chamber changes to some extent due to the transport effect of the nanopores.

[0483] Example 7

[0484] This embodiment demonstrates successful DNA nanopore sequencing using the method described in this invention.

[0485] In this embodiment, the 2020SEP library molecule to be sequenced was first constructed. This library molecule is a complex composed of adapters (SEQ NO:1-2), target nucleic acid molecules (SEQ NO:3), and a motor protein. The nucleic acid molecule has three highly repetitive poly regions that generate specific electrical signals when passing through a nanopore. The construction method of the 2020SEP library molecule is as follows: the sense and antisense strands of the Y-shaped adapter without spacers are annealed, then ligated with the 2020SEP fragment using DNA ligase, and simultaneously helicase is added to obtain the library. Helicase BCH105 (patent application PCT / CN2021 / 143662) was used as the motor protein. This helicase is the SF1B helicase with high sequence novelty from the Deep-Sea Metagenomic Database (DS-RGC), which can unwind double-stranded DNA in the 5'-3' direction and has good nanopore sequencing performance. In addition, BCP34, a porin extracted from the deep sea (patent application PCT / CN2022 / 143298), was used as a biological protein pore, which has good base signal resolution ability.

[0486] Using the helicase and porin described above, a sequencing device was constructed according to the method in Example 1, and the 2020SEP library molecules were sequenced. Magnesium-free sequencing buffer 1 (0.5M KCl, 20mM ATP, pH 8.0) was added to the cis compartment; magnesium-containing sequencing buffer 2 (0.5M KCl, 20mM MgCl2, pH 8.0) was added to the trans compartment. Both the trans and cis compartments used silver / silver chloride electrodes. Then, the 2020SEP library molecules were added to the cis compartment. After applying a sequencing voltage of 0.18V, the sequencing signal shown in Figure 10 was observed. The current signal was approximately 210 pA when no nucleic acid molecules passed through the nanopores, and the current value dropped to approximately 50 pA when DNA molecules passed through the nanopores. As the helicase successfully initiated sequencing after binding magnesium ions, the step current shown in Figure 10 was observed, corresponding to the signal formed by the perforation of different bases in the nucleic acid single strand. Figure 10 shows a complete 2020 SEP sequencing signal, which took approximately 30 seconds. The three dashed boxes indicate three characteristic peaks formed by polyregion perforations. The lower image in Figure 10 shows a magnified view of the first polyregion characteristic signal. As can be seen from the sequencing signal graph in Figure 10, the method of this invention can successfully sequence the library to be sequenced.

[0487] Furthermore, the sequence obtained from sequencing was compared with the known target nucleic acid molecule sequences of the test library, and the results showed that the two sequences were almost completely identical, which further proves that the method of the present invention can be successfully used for the detection of target molecules.

[0488] Example 8

[0489] This embodiment demonstrates several methods for controlling the speed of nanopore sequencing.

[0490] Based on the sequencing system of Example 7, the transport of magnesium ions was regulated by adjusting the voltage applied to the system. The higher the voltage, the stronger the ion transport activity, resulting in more magnesium ions transported from the trans compartment to the CIS compartment. Consequently, the concentration of magnesium ions near the nanopores of the CIS compartment increases, allowing library molecules bound to motor proteins in the CIS compartment to bind to magnesium ions more quickly and unwind successfully, thus improving the sequencing rate.

[0491] As shown in Figure 11, voltages U = 0.18V, 0.2V, and 0.23V were applied across the nanopore, using a 2020SEP library with a length of approximately 1496bp. Sequencing speed was estimated by dividing the library length by the sequencing time. Calculations showed that when the voltages across the nanopore were 0.18V, 0.2V, and 0.23V, the sequencing speeds were 35.8bp / s, 52.3bp / s, and 84.4bp / s, respectively. This demonstrates that by adjusting the system voltage, the concentration of magnesium ions near the nanopore in the CIS compartment can be adjusted, thereby controlling the rate of binding of motor proteins to magnesium ions and regulating the nanopore sequencing speed. The higher the voltage applied across the nanopore, the faster the sequencing speed.

[0492] Furthermore, while keeping the voltage constant, the concentration gradient distribution of magnesium ions near the nanopores of the CIS compartment can be controlled by adjusting the concentration of magnesium ions in the trans compartment or the concentration of magnesium ion inhibitors (such as EDTA) in the CIS compartment, thereby regulating the sequencing speed of the entire system.

[0493] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for regulating the activity of a first biomolecule, the method comprising: (a) Provide: (i) A membrane having one or more pores; The membrane separates the first fluid chamber and the second fluid chamber, and the pores connect the first fluid chamber and the second fluid chamber; (ii) The first biomolecule located in the first fluid compartment, and (iii) One or more first factors that can directly or indirectly regulate the activity of the first biomolecule (e.g., regulate the level of the activity of the first biomolecule). Furthermore, the first factor can move between the first fluid chamber and the second fluid chamber through the orifice under the drive of its own concentration gradient and / or external forces (e.g., electric field force, magnetic field force and / or pressure); (b) By regulating the movement of the first factor between the first fluid compartment and the second fluid compartment, the concentration of the first factor or the rate of concentration change in the first fluid compartment is altered (e.g., increased or decreased) to regulate the activity of the first biomolecule.

2. The method of claim 1, wherein, The activity is related to the ability of the first biomolecule to regulate the second biomolecule; Preferably, the activity is related to the ability of the first biomolecule to control at least a portion (e.g., a chain) of the second biomolecule to move relative to the pore; Preferably, the activity of the first biomolecule is modulated to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore.

3. The method of claim 1 or 2, having one or more features selected from the following: (1) In step (a), a first fluid compartment and a second fluid compartment are also provided; (2) In step (a), a second biomolecule located in the first fluid compartment is also provided; preferably, the first biomolecule and the second biomolecule exist in the form of a complex; (3) In step (a), a second factor is also provided that can regulate the first factor (e.g., regulate the concentration of the first factor).

4. The method according to any one of claims 1-3, the method comprising: (a) Provide: (i) a first fluid compartment and a second fluid compartment, and a membrane having one or more pores; The membrane separates the first fluid chamber and the second fluid chamber, and the pores connect the first fluid chamber and the second fluid chamber; (ii) A first biomolecule and a second biomolecule located in a first fluid compartment, wherein the first biomolecule is capable of controlling the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore; and (iii) One or more first factors capable of directly or indirectly regulating the activity of the first biomolecule, wherein the activity is related to the ability of the first biomolecule to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore; Furthermore, the first factor can move between the first fluid chamber and the second fluid chamber through the orifice under the drive of its own concentration gradient and / or external forces (e.g., electric field force, magnetic field force and / or pressure); (b) By modulating the movement of the first factor between the first fluid compartment and the second fluid compartment, changing (e.g., increasing or decreasing) the concentration of the first factor or the rate of concentration change in the first fluid compartment, the activity of the first biomolecule is modulated, thereby controlling the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore.

5. The method according to any one of claims 2-4, wherein the first biomolecule is capable of controlling at least a portion (e.g., a chain) of the second biomolecule to pass through the pore; Preferably, the first biomolecule is capable of controlling at least a portion (e.g., a chain) of the second biomolecule to move through the aperture in a direction parallel (e.g., the same or opposite) to the applied electric and / or magnetic field forces (e.g., moving through the aperture from a low-potential compartment to a high-potential compartment).

6. The method of claim 5, wherein, The first biomolecule is a protein (e.g., an enzyme); Preferably, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof; Preferably, the polymerase is a polymerase with chain displacement activity.

7. The method of claim 5 or 6, wherein, The second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof; Preferably, the second biomolecule is selected from natural, synthetic, or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof; Preferably, the second biomolecule includes a adapter (e.g., a sequencing adapter); Preferably, the second biomolecule is DNA containing a linker; Preferably, the second biomolecule is a polypeptide containing a linker; Preferably, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule; Preferably, the connector may or may not include one or more spacer areas; Preferably, the connector does not include a spacer area.

8. The method according to any one of claims 5-7, wherein, The second biomolecule is a nucleic acid molecule (e.g., a chain-like nucleic acid molecule); Preferably, the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the helicase can separate the two strands of the double-stranded nucleic acid molecule to provide a single-stranded nucleic acid molecule and control the movement of the single-stranded nucleic acid molecule through the pore; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor can directly or indirectly regulate the helicase's ability to unwind the double-stranded nucleic acid molecule; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor can directly or indirectly regulate the helicase's ability to unwind the double-stranded nucleic acid molecule, thereby controlling at least a portion of the double-stranded nucleic acid molecule to pass through the pore; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the helicase forms a complex with the double-stranded nucleic acid molecule through the linker contained in the double-stranded nucleic acid molecule.

9. The method according to any one of claims 2-4, wherein the first biomolecule is capable of controlling at least a portion of the second biomolecule to move relative to the pore without passing through the pore; Preferably, the direction of movement of the second biomolecule is independent of the direction of the applied electric and / or magnetic field forces; Preferably, the first biomolecule is capable of connecting to the pore (e.g., covalently).

10. The method of claim 9, wherein, The first biomolecule is a protein (e.g., an enzyme); Preferably, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof.

11. The method of claim 10, wherein, The second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof; Preferably, the second biomolecule is selected from natural, synthetic, or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof; Preferably, the second biomolecule includes a adapter (e.g., a sequencing adapter); Preferably, the second biomolecule is DNA containing a linker; Preferably, the second biomolecule is a polypeptide containing a linker; Preferably, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule; Preferably, the connector may or may not include one or more spacer areas; Preferably, the connector does not include a spacer area.

12. The method according to any one of claims 9-11, wherein, The second biomolecule is a nucleic acid molecule (e.g., a circular nucleic acid molecule); Preferably, the first biomolecule is a polymerase (e.g., DNA polymerase) and the second biomolecule is a circular nucleic acid molecule; Preferably, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the polymerase can use the circular nucleic acid molecule as a template to extend the primers that bind to the circular nucleic acid molecule, and control the circular nucleic acid molecule to move relative to the pore without passing through the pore; Preferably, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor can directly or indirectly regulate the polymerase's ability to extend primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template. Preferably, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor can directly or indirectly regulate the polymerase's ability to extend primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template, thereby controlling the circular nucleic acid molecule to move relative to the pore without passing through the pore.

13. The method of claim 6 or 10, wherein it has one or more features selected from the following: (1) The polymerase is selected from the following: Bst DNA polymerase, SD DNA polymerase, phi29 DNA polymerase, Bsu Large Fragment DNA polymerase, Klenow Fragment DNA polymerase, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, E. coli RNA polymerase, or any combination thereof; (2) The helicase has one or more of the following characteristics: (i) The helicase is selected from the following: Dda, Hel308, RecD, UvrD, Rep, RecQ, PcrA, eIF4A, NS3, gp41, T7gp4, BCH105, or any combination thereof; (ii) The helicase is any wild-type of any protein in (1), or a homology thereof or a mutant thereof; (iii) The helicase is also attached to another polypeptide, which is selected from tags, restriction sites, signal peptides or lead peptides, detectable markers, or any combination thereof. (3) The amino acid recognition protein can specifically recognize the terminal amino acid of a polypeptide or protein; (4) The amino acid recognition protein is selected from any one or more of the following: (i) Agrobacterium tumefaciens ClpS1, Agrobacterium tumefaciens ClpS2, Synechococcus elongatus ClpS1, Synechococcus elongatus ClpS2, Thermosynechococcus elongatus ClpS, Escherichia coli ClpS or Plasmodium falciparum ClpS of ClpS family; (ii)Vibrio vulnificus Aspartate / glutamate leucyltransferase Bpt; (iii) human UBR1, human UBR2 or Saccha-romyces cerevisiae UBR1 of the UBR family; (iiii) GID4 of H. sapiens or Saccharomyces cerevisiae: (iiiiii) Drosophila melanogaster BIR2; (iiiiii) H. sapiens N-meristoyltransferase NMT1.

14. The method of any one of claims 1-13, wherein an electric field, a magnetic field and / or pressure are provided across the membrane, and the first factor is moved through the pore between the first fluid chamber and the second fluid chamber under the drive of the electric field force, the magnetic field force and / or pressure; Preferably, the first factor moves along the direction of the driving force under the drive of electric field force, magnetic field force and / or pressure; Preferably, the first factor moves along any one or a combination of the following (i) to (iiii): (i) potential difference, (ii) concentration gradient, (iii) electrochemical gradient, and (iiii) the direction of applied pressure; Preferably, after applying an electric field and / or magnetic field, the potential of the second fluid chamber is higher or lower than that of the first fluid chamber; Preferably, after an electric field and / or magnetic field is applied, the potential of the second fluid chamber is higher than that of the first fluid chamber, and the first factor moves from the second fluid chamber to the first fluid chamber through the nanopore; Preferably, a second electrode (e.g., a positive electrode) is provided in the second fluid chamber, and a first electrode (e.g., a negative electrode) is provided in the first fluid chamber, and a voltage is applied across the first and second electrodes; Preferably, the first fluid compartment and the second fluid compartment contain a solution.

15. The method of any one of claims 1-14, wherein the first factor is an activator of a first biomolecule; Preferably, the first factor can positively regulate the activity of the first biomolecule (e.g., enhance the activity of the first biomolecule, enable the first biomolecule to start working and / or improve the working efficiency of the first biomolecule). Preferably, the first factor is an ion in the solution; Preferably, the first factor is selected from metal ions, ATP, or any combination thereof; Preferably, the metal ion is selected from Mn. 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ , or any combination thereof.

16. The method of any one of claims 1-15, wherein the second factor negatively regulates the activity of the first biomolecule by regulating the first factor (e.g., reducing the activity of the first biomolecule, causing the first biomolecule to stop working and / or reducing the working efficiency of the first biomolecule). Preferably, the second factor is a repressor protein, chelator, and / or inhibitor of the first factor; Preferably, when the first factor is a metal ion, the second factor is a chelating agent for the first factor; Preferably, the second factor is selected from EDTA, EGTA, DTPA, TTHA, NTA, or any combination thereof.

17. The method of claim 15 or 16, wherein the activity of the first biomolecule is positively modulated by one or more methods selected from the following: (1) To bring the first biomolecule into contact with the first factor or to increase the concentration of the first factor in contact with the first biomolecule; (2) Increase the concentration of the first factor in the first fluid compartment or increase the rate of increase of the concentration of the first factor in the first fluid compartment; (3) By (i) applying an electric field, magnetic field and / or pressure, or (ii) increasing the voltage of the electric field and / or magnetic field, or (iii) increasing the pressure, or (iiii) increasing the initial concentration of the first factor in the second fluid compartment, to increase the concentration of the first factor in the first fluid compartment or to increase the rate of increase of the concentration of the first factor in the first fluid compartment.

18. The method of any one of claims 15-17, wherein the activity of the first biomolecule is negatively regulated by one or more methods selected from the following: (1) The first biomolecule comes into contact with a first factor at a lower concentration (e.g., below the concentration required for the first biomolecule under normal operating conditions) or the concentration of the first factor that comes into contact with the first biomolecule is reduced (e.g., the concentration is reduced to 0). (2) Reduce the concentration of the first factor in the first fluid compartment or reduce the rate of increase of the concentration of the first factor in the first fluid compartment; (3) By (i) removing the electric field, magnetic field and / or pressure applied across the membrane, or (ii) reducing the voltage of the electric field and / or magnetic field, or (iii) reducing the pressure, or (iiii) reducing the initial concentration of the first factor in the second fluid compartment, to reduce the concentration of the first factor in the first fluid compartment or reduce the rate of increase of the concentration of the first factor in the first fluid compartment. (4) Make the first factor come into contact with the second factor or increase the concentration of the second factor that comes into contact with the first factor.

19. The method of any one of claims 1-18, wherein in step (a) of the method: (1) A first factor is provided in the second fluid compartment at a concentration sufficient to enable the first biomolecule to start working, while the first fluid compartment does not provide the first factor; Preferably, the first fluid compartment may or may not provide the second factor; (2) Provide a first factor at a concentration sufficient to activate the first biomolecule in the second fluid compartment. A first factor is provided at a concentration insufficient to initiate the function of the first biomolecule; Preferably, the first fluid compartment may or may not provide the second factor; (3) Provide a first factor in the second fluid compartment at a concentration sufficient to activate the first biomolecule; provide a first factor in the first fluid compartment at a concentration sufficient to activate the first biomolecule. Preferably, the first fluid compartment also provides a second factor.

20. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, thereby the first biomolecule is in a non-active state; When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid compartment is higher than that of the first fluid compartment. The first factor moves from the second fluid compartment to the first fluid compartment through the hole to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working. Preferably, the potential difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased, so as to increase the rate of increase of the concentration of the first factor in the first fluid compartment, thereby positively regulating the activity of the first biomolecule (e.g., improving the working efficiency of the first biomolecule). Preferably, reducing the potential difference between the second fluid compartment and the first fluid compartment, reducing the initial concentration of the first factor in the second fluid compartment, and / or providing the second factor in the first fluid compartment can reduce the rate of increase in the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., causing the first biomolecule to stop working and / or reducing the working efficiency of the first biomolecule). Preferably, the second factor is a chelating agent for metal ions (e.g., EDTA, EGTA, DTPA, TTHA, NTA).

21. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, thereby activating the first biomolecule; When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid chamber is lower than that of the first fluid chamber. The first factor moves from the first fluid chamber to the second fluid chamber through the hole to reduce the concentration of the first factor in the first fluid chamber, thereby reducing the working efficiency of the first biomolecule. Preferably, the second fluid compartment may or may not provide the first factor; Preferably, increasing the potential difference between the first fluid compartment and the second fluid compartment, decreasing the initial concentration of the first factor in the first fluid compartment, and / or providing a second factor in the first fluid compartment increases the rate at which the concentration of the first factor decreases in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., further reducing the working efficiency of the first biomolecule and / or causing the first biomolecule to stop). Stop working); Preferably, the second factor is a chelating agent for metal ions (e.g., EDTA, EGTA, DTPA, TTHA, NTA); Preferably, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, so as to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule (e.g., reducing the rate at which the working efficiency of the first biomolecule decreases or increasing the working efficiency of the first biomolecule).

22. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, thereby the first biomolecule is in a non-active state; When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is higher than that of the second fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the pore to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working. Preferably, the potential difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased, so as to increase the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule (e.g., reducing the rate at which the working efficiency of the first biomolecule decreases or increasing the working efficiency of the first biomolecule). Preferably, the potential difference between the second fluid compartment and the first fluid compartment is reduced, and / or the initial concentration of the first factor in the second fluid compartment is reduced, so as to slow down the rate of increase of the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., reducing the working efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

23. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, thereby activating the first biomolecule; When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is lower than that of the second fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the pore to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the efficiency of the first biomolecule. Preferably, the second fluid compartment may or may not provide the first factor; Preferably, the potential difference between the first fluid compartment and the second fluid compartment is increased, and / or the initial concentration of the first factor in the first fluid compartment is reduced, so as to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule (e.g., further reducing the working efficiency of the first biomolecule and / or causing the first biomolecule to stop working). Preferably, reducing the potential difference between the first fluid compartment and the second fluid compartment, and / or increasing the initial concentration of the first factor in the first fluid compartment, slows down the rate of decrease in the concentration of the first factor in the first fluid compartment, thereby positively regulating the activity of the first biomolecule. (For example, slowing down the rate at which the efficiency of the first biomolecule decreases or improving the efficiency of the first biomolecule).

24. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, thereby the first biomolecule is in a non-active state; When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the hole to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working. Preferably, the pressure difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased, so as to increase the rate of increase of the concentration of the first factor in the first fluid compartment, thereby positively regulating the activity of the first biomolecule (e.g., improving the working efficiency of the first biomolecule). Preferably, the pressure difference between the second fluid compartment and the first fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is reduced, so as to slow down the rate of increase of the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule (e.g., reducing the working efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

25. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, thereby activating the first biomolecule; When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid chamber is lower than that in the first fluid chamber, the first factor moves from the first fluid chamber to the second fluid chamber through the hole to reduce the concentration of the first factor in the first fluid chamber, thereby reducing the working efficiency of the first biomolecule. Preferably, the second fluid compartment may or may not provide the first factor; Preferably, the pressure difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, so as to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule (e.g., reducing the rate at which the working efficiency of the first biomolecule decreases). Preferably, the pressure difference between the first fluid compartment and the second fluid compartment is increased, and / or the initial concentration of the first factor in the first fluid compartment is reduced, so as to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule (e.g., further reducing the working efficiency of the first biomolecule and / or causing the first biomolecule to stop working).

26. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, thereby the first biomolecule is in a non-active state; When the first factor carries a positive charge, a negative charge, or no charge, and the concentration of the first factor in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the hole to increase the concentration of the first factor in the first fluid compartment, thereby activating the first biomolecule. Preferably, the initial concentration of the first factor in the second fluid compartment is increased to increase the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule; Preferably, the initial concentration of the first factor in the first fluid compartment is reduced to slow down the rate of increase of the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule.

27. The method of any one of claims 1-19, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to activate the first biomolecule, and a second fluid compartment provides no first factor or provides a first factor at a concentration lower than that of the first factor in the first fluid compartment, thereby activating the first biomolecule; When the first factor carries a positive charge, a negative charge, or no charge, and the concentration of the first factor in the second fluid compartment is lower than that in the first fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the hole to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the working efficiency of the first biomolecule. Preferably, the initial concentration of the first factor in the first fluid compartment is increased to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule; Preferably, the initial concentration of the first factor in the first fluid compartment is reduced to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule.

28. The method of any one of claims 1-27, wherein the membrane has one or more features selected from the following: (1) The first factor and the second factor cannot move between the first fluid compartment and the second fluid compartment through the membrane; (2) The hole is located on the membrane and passes through the membrane; Preferably, the first factor and the second factor are capable of moving between the first fluid chamber and the second fluid chamber through the orifice; (3) The membrane is an amphiphilic layer; (4) The membrane is a phospholipid membrane (e.g., DOPE, DPPC, DPhPC, DOPC or POPC); (5) The membrane is a polymer membrane (e.g., a diblock polymer, a triblock polymer (such as PMOXA-PDMS-PMOXA)); (6) The film is a solid film (e.g., SiNx film, SiO2 film, Al2O3 film, graphene film, BN film); (7) The membrane is a mixture of any two or more substances.

29. The method of any one of claims 1-28, wherein the aperture has one or more features selected from the following: (1) The opening diameter of the hole is 0.1nm-10μmm (e.g., 0.1-1nm, 1-10nm, 10-100nm, 10-100nm, 100-300nm, 300-500nm, 500-800nm, 800-1000nm, 1μm-5μm, 5μm-10μm); (2) The pores are biological protein pores; Preferably, the biological protein pores are derived from any one or more of the following or their homologs or mutants: hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, α-Haemolysin, CsgG, Aerolysin, cytolysin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria spp. autotransporter lipoprotein (NalP), WZA, GspD, BCP34, and BCP58; (3) The pores are solid nanopores; Preferably, the solid nanopores are selected from SiNx, SiO2, graphene, Al2O3, boron nitride (BN), HfO2, or any combination thereof; (4) The pores are modified, for example, chemically modified; (5) After an electric field and / or magnetic field is applied across the membrane, ions can pass through the pores and generate an electric current; (6) The pore is capable of connecting and / or binding to a first biomolecule (e.g., covalent connection).

30. A method for identifying or characterizing a second biomolecule, the method comprising: The activity of a first biomolecule is regulated by the method of any one of claims 1-29, wherein the first biomolecule, in a state of regulated activity, controls at least a portion (e.g., a chain) of the second biomolecule to move relative to the pore and generates one or more measurements; wherein the measurements can be used to identify or characterize the second biomolecule.

31. The method of claim 30, wherein the method is performed by the steps described in (a) to (c) below: (a) Provide: (i) A membrane having one or more pores; the membrane separating a first fluid compartment and a second fluid compartment, the pores connecting the first fluid compartment and the second fluid compartment; (ii) The first and second biomolecules located in the first fluid compartment, wherein... The first biomolecule is able to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore; and, as the at least portion of the second biomolecule moves relative to the pore, one or more measurements can be generated. (iii) One or more first factors capable of directly or indirectly regulating the activity of the first biomolecule, wherein the activity is related to the ability of the first biomolecule to control the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore; and, (b) The first factor is moved through a pore between the first fluid compartment and the second fluid compartment under the drive of its own concentration gradient and / or external force, and the concentration of the first factor in the first fluid compartment is changed to regulate the activity of the first biomolecule. (c) In a state of regulated activity, the first biomolecule controls the relative movement of at least a portion (e.g., a chain) of the second biomolecule with respect to the pore and acquires one or more measurements for identifying or characterizing the second biomolecule.

32. The method of claim 31, wherein it has one or more features selected from the following: (1) In step (a), a first fluid compartment and a second fluid compartment are also provided; (2) In step (a), the first biomolecule and the second biomolecule exist in the form of a complex; (3) In step (a), a second factor is also provided that can regulate the first factor (e.g., regulate the concentration of the first factor); Preferably, the provided first factor and / or second factor are located in the first fluid compartment and / or the second fluid compartment; (4) In step (b), an external force (e.g., electric force, magnetic force and / or pressure) is applied to cause the first factor to move through the hole between the first fluid chamber and the second fluid chamber under the drive of the external force.

33. The method of claim 31 or 32, wherein the first biomolecule controls at least a portion (e.g., a chain) of the second biomolecule to pass through the pore, and one or more measurements are obtained; Preferably, the first biomolecule controls at least a portion of the second biomolecule to move through the aperture in a direction parallel (e.g., the same or opposite) to the applied electric and / or magnetic field forces (e.g., moving through the aperture from a low-potential chamber to a high-potential chamber), and obtains one or more measurements. Preferably, as at least a portion of the second biomolecule passes through the pore, a changing current is generated in the pore, and a measurement value is obtained by measuring the changing current; Preferably, as at least a portion of the second biomolecule passes through the pore, a varying blocking ion current is generated in the pore, and a measurement value is obtained by measuring the varying blocking ion current; Preferably, when the second biomolecule is a nucleic acid molecule, as different nucleotides of the nucleic acid molecule pass through the pore, ion currents corresponding to the changes in different nucleotides are generated in the pore. By detecting the changes in the ion currents, different nucleotides are identified or characterized, thereby identifying or characterizing the nucleic acid molecule.

34. The method of claim 33, wherein, The first biomolecule is a protein (e.g., an enzyme); Preferably, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), and amino acid recognition proteins. Different proteins, or any combination thereof; Preferably, the polymerase is a polymerase with chain displacement activity.

35. The method of claim 33 or 34, wherein, The second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof; Preferably, the second biomolecule is selected from natural, synthetic, or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof; Preferably, the second biomolecule includes a adapter (e.g., a sequencing adapter); Preferably, the second biomolecule is DNA containing a linker; Preferably, the second biomolecule is a polypeptide containing a linker; Preferably, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule; Preferably, the connector may or may not include one or more spacer areas; Preferably, the connector does not include a spacer area.

36. The method of any one of claims 33-35, wherein, The second biomolecule is a nucleic acid molecule (e.g., a chain-like nucleic acid molecule); Preferably, the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the helicase separates the two strands of the double-stranded nucleic acid molecule to provide a single-stranded nucleic acid molecule and controls the movement of the single-stranded nucleic acid molecule through the pore; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor directly or indirectly regulates the helicase to unwind the double-stranded nucleic acid molecule; Preferably, when the first biomolecule is a helicase and the second biomolecule is a double-stranded nucleic acid molecule, the first factor directly or indirectly regulates the helicase to unwind the double-stranded nucleic acid molecule, thereby controlling at least a portion of the double-stranded nucleic acid molecule to pass through the pore; Preferably, when the first biomolecule is a helicase and the second biomolecule is DNA containing a sequencing adapter, the helicase forms a complex with the DNA through the sequencing adapter of the DNA. Preferably, when the first biomolecule is a helicase and the second biomolecule is DNA containing a sequencing adapter, the helicase binds to the sequencing adapter of the DNA and forms a complex with the DNA.

37. The method of claim 31 or 32, wherein the first biomolecule controls at least a portion of the second biomolecule to move relative to the pore without passing through the pore, and obtains one or more measurements; Preferably, the direction of movement of the second biomolecule is independent of the direction of the applied electric and / or magnetic field forces; Preferably, the first biomolecule is connected to the pore (e.g., covalently). Preferably, as at least a portion of the second biomolecule moves relative to the pore, a changing current is generated in the pore, and a measurement value is obtained by measuring the changing current; Preferably, as at least a portion of the second biomolecule moves relative to the pore, a varying blocking ion current is generated in the pore, and a measurement value is obtained by measuring the varying blocking ion current. Preferably, when the second biomolecule is a nucleic acid molecule, as different nucleotides of the nucleic acid molecule move relative to the pore, ion currents corresponding to the changes in different nucleotides are generated in the pore. By detecting the changes in the ion currents, different nucleotides are identified or characterized, thereby identifying or characterizing the nucleic acid molecule.

38. The method of claim 37, wherein, The first biomolecule is a protein (e.g., an enzyme); Preferably, the first biomolecule is selected from polymerases (e.g., DNA polymerase, RNA polymerase), exonucleases, helicases, topoisomerases, reverse transcriptases, helicase repressor proteins (e.g., proteins that inhibit helicase unwinding), amino acid recognition proteins, or any combination thereof.

39. The method of claim 37 or 38, wherein, The second biomolecule is selected from nucleic acid molecules (e.g., DNA, RNA), polypeptides, proteins, polysaccharides, or any combination thereof; Preferably, the second biomolecule is selected from natural, synthetic, or partially modified (e.g., methylated, hydroxymethylated) biomolecules, or any combination thereof; Preferably, the second biomolecule includes a adapter (e.g., a sequencing adapter); Preferably, the second biomolecule is DNA containing a linker; Preferably, the second biomolecule is a polypeptide containing a linker; Preferably, the first biomolecule forms a complex with the second biomolecule through a linker of the second biomolecule; Preferably, the connector may or may not include one or more spacer areas; Preferably, the connector does not include a spacer area.

40. The method of any one of claims 37-39, wherein, The second biomolecule is a nucleic acid molecule (e.g., a circular nucleic acid molecule); Preferably, the first biomolecule is a polymerase (e.g., DNA polymerase), and the second biomolecule is a circular nucleic acid molecule; Preferably, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the polymerase is in a circular form. The nucleic acid molecule serves as a template, extends and binds to the circular nucleic acid molecule, and controls the relative movement of the circular nucleic acid molecule to the pore without passing through it; Preferably, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor directly or indirectly regulates the polymerase to extend the primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template. Preferably, when the first biomolecule is a polymerase and the second biomolecule is a circular nucleic acid molecule, the first factor directly or indirectly regulates the polymerase to extend the primers that bind to the circular nucleic acid molecule using the circular nucleic acid molecule as a template, thereby controlling the circular nucleic acid molecule to move relative to the pore without passing through the pore.

41. The method according to any one of claims 37-40, wherein the method has one or more features selected from the following: (1) The measured value can characterize one or more of the following features of the second biomolecule: the nucleotide sequence of the nucleic acid molecule, whether the nucleotide of the nucleic acid molecule is modified (e.g., methylation modification, hydroxymethylation modification), the amino acid sequence of the polypeptide, the polysaccharide sequence, or any combination thereof; (2) In step (a), reagents (e.g., inorganic salts and / or buffers) for sequencing are also provided; Preferably, the inorganic salt is selected from sodium chloride, potassium chloride, lithium chloride, or any combination thereof; Preferably, the buffer solution is selected from HEPES, Tris-HCl, or any combination thereof; (3) When the first factor is a metal ion, ATP is also provided in step (a); (4) When the first biomolecule is a polymerase, primers and dNTPs are also provided in step (a) for the polymerase to extend the primers that bind to the second biomolecule (e.g., a circular nucleic acid molecule); (5) The first factor is as defined in any one of claims 15-27; (6) The second factor as defined in any one of claims 15-27; (7) The membrane as defined in claim 28; (8) Hole as defined in claim 29.

42. The method of any one of claims 30-41, wherein in step (a) of the method: (1) A first factor is provided in the second fluid compartment at a concentration sufficient to enable the first biomolecule to start working, while the first fluid compartment does not provide the first factor; Preferably, the first fluid compartment may or may not provide the second factor; (2) A first factor is provided in the second fluid compartment at a concentration sufficient to enable the first biomolecule to start working, and a first factor is provided in the first fluid compartment at a concentration insufficient to enable the first biomolecule to start working. Preferably, the first fluid compartment may or may not provide the second factor; or (3) Provide a first factor in the second fluid compartment at a concentration sufficient to activate the first biomolecule; provide a first factor in the first fluid compartment at a concentration sufficient to activate the first biomolecule. Preferably, the first fluid compartment also provides a second factor.

43. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated. When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid compartment is higher than that of the first fluid compartment. The first factor moves from the second fluid compartment to the first fluid compartment through the hole to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working and generate one or more measurement values. Preferably, the potential difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased, so as to increase the rate of increase of the concentration of the first factor in the first fluid compartment, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore. Preferably, the first fluid compartment may or may not provide the second factor; Preferably, reducing the potential difference between the second fluid compartment and the first fluid compartment, reducing the initial concentration of the first factor in the second fluid compartment, and / or providing the second factor in the first fluid compartment reduces the rate of increase of the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore; more preferably, reducing the migration speed of the second biomolecule relative to the pore until it stops moving. Preferably, the second factor is selected from metal ion chelating agents (e.g., EDTA, EGTA, DTPA, TTHA, NTA).

44. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, thereby putting the first biomolecule into a working state and generating one or more measurements (e.g., varying ion currents); When the first factor carries a positive charge (e.g., Mn) 2+ Mg 2+ Co 2+ Zn 2+ Cu 2+ Cu + Ni 2+ Fe 2+ Fe 3+ Ca 2+ Furthermore, the potential of the second fluid chamber is lower than that of the first fluid chamber. The first factor moves from the first fluid chamber to the second fluid chamber through the pore to reduce the concentration of the first factor in the first fluid chamber, thereby reducing the working efficiency of the first biomolecule and reducing the movement speed of the second biomolecule relative to the pore; more preferably, the movement speed of the second biomolecule relative to the pore is reduced until it stops moving. Preferably, the second fluid compartment may or may not provide the first factor; Preferably, increasing the potential difference between the first fluid compartment and the second fluid compartment, decreasing the initial concentration of the first factor in the first fluid compartment, and / or providing the second factor in the first fluid compartment increases the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore; more preferably, reducing the migration speed of the second biomolecule relative to the pore until it stops moving. Preferably, the second factor is selected from metal ion chelating agents (e.g., EDTA, EGTA, DTPA, TTHA, NTA); Preferably, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, so as to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

45. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated. When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is higher than that of the second fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the pore to increase the concentration of the first factor in the first fluid compartment, thereby initiating the first biomolecule to work and generating one or more measurements. Preferably, the potential difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased, so as to increase the rate of increase of the concentration of the first factor in the first fluid compartment, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore. Preferably, the potential difference between the second fluid compartment and the first fluid compartment is reduced, and / or the initial concentration of the first factor in the second fluid compartment is reduced, so as to reduce the rate of increase of the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore; more preferably, the migration speed of the second biomolecule relative to the pore is reduced until it stops moving.

46. ​​The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, so that the first biomolecule is in a working state and one or more measurements (e.g., varying ion current) will be generated; When the first factor carries a negative charge (e.g., ATP) and the potential of the first fluid compartment is lower than that of the second fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the pore to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the efficiency of the first biomolecule and reducing the movement speed of the second biomolecule relative to the pore. Preferably, the second fluid compartment may or may not provide the first factor; Preferably, the potential difference between the first fluid compartment and the second fluid compartment is increased, and / or the initial concentration of the first factor in the first fluid compartment is decreased, so as to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore; more preferably, the migration speed of the second biomolecule relative to the pore is reduced until it stops moving. Preferably, the potential difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, so as to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore.

47. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated. When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the orifice to increase the concentration of the first factor in the first fluid compartment, thereby enabling the first biomolecule to start working and generate one or more measurements. Preferably, the pressure difference between the second fluid compartment and the first fluid compartment is increased, and / or the initial concentration of the first factor in the second fluid compartment is increased, so as to increase the rate of increase of the concentration of the first factor in the first fluid compartment, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore. Preferably, the pressure difference between the second fluid compartment and the first fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is reduced, so as to reduce the rate of increase of the concentration of the first factor in the first fluid compartment, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore; more preferably, the migration speed of the second biomolecule relative to the pore is reduced until it stops moving.

48. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to start working, thereby putting the first biomolecule into a working state and generating one or more measurements (e.g., varying ion currents); When the first factor carries a positive charge, a negative charge, or no charge, and the pressure in the second fluid chamber is lower than that in the first fluid chamber, the first factor moves from the first fluid chamber to the second fluid chamber through the pore, thereby reducing the concentration of the first factor in the first fluid chamber. This reduces the efficiency of the first biomolecule and decreases the migration speed of the second biomolecule relative to the pore; more preferably, it reduces the concentration of the second biomolecule. The movement speed of molecules relative to the pores until they stop moving; Preferably, the second fluid compartment may or may not provide the first factor; Preferably, the pressure difference between the first fluid compartment and the second fluid compartment is reduced, and / or the initial concentration of the first factor in the first fluid compartment is increased, so as to reduce the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore. Preferably, the pressure difference between the first fluid compartment and the second fluid compartment is increased, and / or the initial concentration of the first factor in the first fluid compartment is decreased, so as to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore; more preferably, the migration speed of the second biomolecule relative to the pore is reduced until it stops moving.

49. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a second fluid compartment at a concentration sufficient to activate the first biomolecule, and the first fluid compartment does not provide the first factor or provides a first factor at a concentration insufficient to activate the first biomolecule, so that the first biomolecule is in a non-active state and no measurement (e.g., changing ion current) is generated. When the first factor is positively charged, negatively charged, or uncharged, and the concentration of the first factor in the second fluid compartment is higher than that in the first fluid compartment, the first factor moves from the second fluid compartment to the first fluid compartment through the orifice to increase the concentration of the first factor in the first fluid compartment. When the concentration of the first factor in the first fluid compartment is sufficient to enable the first biomolecule to start working, one or more measurements are generated. Preferably, the initial concentration of the first factor in the second fluid compartment is increased to increase the rate at which the concentration of the first factor in the first fluid compartment increases, thereby positively regulating the activity of the first biomolecule and increasing the migration speed of the second biomolecule relative to the pore. More preferably, the initial concentration of the first factor in the first fluid compartment is reduced to slow down the rate at which the concentration of the first factor in the first fluid compartment increases, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore.

50. The method of any one of claims 30-42, wherein in step (a) of the method, a first factor is provided in a first fluid compartment at a concentration sufficient to enable the first biomolecule to function, and a second fluid compartment provides no first factor or provides a first factor at a concentration lower than that of the first factor in the first fluid compartment, thereby enabling the first biomolecule to function and generating one or more measurements (e.g., varying ion currents). When the first factor carries a positive charge, a negative charge, or no charge, and the concentration of the first factor in the second fluid compartment is lower than that in the first fluid compartment, the first factor moves from the first fluid compartment to the second fluid compartment through the hole to reduce the concentration of the first factor in the first fluid compartment, thereby reducing the working efficiency of the first biomolecule. Preferably, the initial concentration of the first factor in the first fluid compartment is increased to reduce the decrease in the concentration of the first factor in the first fluid compartment. The speed of movement of the second biomolecule relative to the pore is increased, thereby positively regulating the activity of the first biomolecule and increasing the movement speed of the second biomolecule relative to the pore. More preferably, the initial concentration of the first factor in the first fluid compartment is reduced to increase the rate at which the concentration of the first factor in the first fluid compartment decreases, thereby negatively regulating the activity of the first biomolecule and reducing the migration speed of the second biomolecule relative to the pore.

51. The method of any one of claims 30-50, wherein, The identification or characterization of the second biomolecule includes sequencing the second biomolecule; Preferably, the sequence information of the second biomolecule (e.g., the nucleotide sequence of the nucleic acid molecule, whether the nucleotides of the nucleic acid molecule are modified (e.g., methylation modification, hydroxymethylation modification), the amino acid sequence of the polypeptide, and the polysaccharide sequence) is obtained by the method of any one of claims 30-50. Preferably, when the second biomolecule is a nucleic acid molecule, as different nucleotides of the nucleic acid molecule move relative to the pore (e.g., through the pore), ion currents corresponding to the changes in different nucleotides are generated in the pore. By detecting multiple changes in ion currents, multiple nucleotides are identified and / or analyzed, thereby sequencing the nucleic acid molecule.

52. The method of claim 51, wherein, Sequencing is regulated by modulating the activity of the first biomolecule to alter the relative movement of at least a portion (e.g., a strand) of the second biomolecule with respect to the pore. Preferably, sequencing is initiated by modulating the activity of the first biomolecule to control at least a portion of the second biomolecule to begin relative movement with respect to the pore; Preferably, sequencing is stopped by regulating the activity of the first biomolecule to control at least a portion of the second biomolecule to stop relative movement with respect to the pore; Preferably, the sequencing rate is altered (e.g., increased or decreased) by modulating the activity of the first biomolecule to change (e.g., increase or decrease) the rate at which at least a portion of the second biomolecule moves relative to the pore.

53. An apparatus or system comprising: (a) A reaction module, which includes: (i) A membrane having one or more pores; the membrane separating a first fluid compartment and a second fluid compartment, the pores connecting the first fluid compartment and the second fluid compartment; (ii) A first factor capable of moving between a first fluid compartment and a second fluid compartment, the first factor being capable of directly or indirectly regulating the activity of a first biomolecule (e.g., regulating the level of activity of the first biomolecule). Furthermore, the first factor can move between the first fluid chamber and the second fluid chamber through the orifice under the drive of its own concentration gradient and / or external forces (e.g., electric field force, magnetic field force and / or pressure); (b) A control module capable of regulating the movement of the first factor through the orifice between the first fluid compartment and the second fluid compartment.

54. The apparatus or system of claim 53, wherein, The activity is related to the ability of the first biomolecule to regulate the second biomolecule; Preferably, the activity is related to the ability of the first biomolecule to control at least a portion (e.g., a chain) of the second biomolecule to move relative to the pore; Preferably, by modulating the activity of the first biomolecule, at least a portion (e.g., a chain) of the second biomolecule is controlled to move relative to the pore, and one or more measurements are generated.

55. The apparatus or system of claim 53 or 54, further comprising one or more of the following: (1) A detection and / or analysis module capable of detecting and / or analyzing one or more measurements resulting from the relative movement of at least a portion (e.g., a chain) of a second biomolecule with respect to the pore; Preferably, the second biomolecule is identified or characterized by detecting and / or analyzing the measured values; Preferably, the second biomolecule is sequenced by detecting and / or analyzing the measured values ​​(e.g., changing ion currents); Preferably, the measured value characterizes one or more of the following features of the second biomolecule: the nucleotide sequence of the nucleic acid molecule, whether the nucleotides of the nucleic acid molecule are modified (e.g., methylation modification, hydroxymethylation modification), the amino acid sequence of the polypeptide, the polysaccharide sequence, or any combination thereof; (2) A solid support capable of supporting the membrane; Preferably, after the solid support carries the membrane, it can form and / or provide a second fluid compartment and / or a first fluid compartment together with the membrane; (3) The first fluid compartment and the second fluid compartment located in the reaction module; (4) A second factor located in the reaction module, which can regulate the first factor (e.g., regulate the concentration of the first factor); Preferably, the first factor and / or the second factor are located in the first fluid compartment and / or the second fluid compartment.

56. The apparatus or system of any one of claims 53-55, further comprising one or more of the following: (1) A first biomolecule located in the first fluid compartment; preferably, the first biomolecule is as defined in any one of claims 2-13; (2) A second biomolecule located in the first fluid compartment; preferably, the second biomolecule is as defined in any one of claims 2-13; (3) The complex formed after (1) and (2) come into contact; (4) The first factor is as defined in any one of claims 15-27; (5) The second factor is as defined in any one of claims 15-27; (6) The membrane as defined in claim 28; (7) The hole is as defined in claim 29; (8) The control module includes components (e.g., electrodes) that provide electric force, magnetic force and / or pressure; (9) The solid support contains microcavities; Preferably, after the solid support supports the membrane, the area below the membrane and the interior of the microcavity together form a second fluid chamber, and the area above the membrane forms a first fluid chamber. Preferably, the solid support is a chip.

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