BR112017025723B1 - COMPOSITIONS, SYSTEMS AND METHODS FOR SEQUENCING POLYNUCLEOTIDES USING CHAINS ANCHORED TO POLYMERASES ADJACENT TO NANOPORES - Google Patents

Abstract

COMPOSITIONS, SYSTEMS AND METHODS FOR SEQUENCING POLYNUCLEOTIDES USING CHAINS ANCHORED TO POLYMERASES ADJACENT TO NANOPORES. A composition includes a nanopore including first and second sides and an opening, each of the nucleotides including an elongated tag, and a first polynucleotide that is complementary to a second polynucleotide. A polymerase may be disposed adjacent to the first side of the nanopore and configured to add nucleotides to the first polynucleotide based on a sequence of the second polynucleotide. A permanent chain may include a head region anchored to the polymerase, a tail region, and an elongated body disposed between them, which occurs at the opening of the nanopore. A first portion may be disposed on the elongated body that binds to the elongated marker of a first nucleotide upon which the polymerase is acting. A reporter region can be disposed on the elongated body, which indicates when the first nucleotide is complementary or not complementary to a next nucleotide in the sequence of the second polynucleotide.

Description

CROSS REFERENCE TO RELATED ORDERS

[0001] This application claims the benefit of the following application, the contents of which, in full, are incorporated by reference in this document:

[0002] North American Provisional Patent Application No. 62/170,563, filed on June 3, 2015 and entitled “Compositions, Systems, and Methods for Sequencing Polynucleotides Using Tethers Anchored to Polymerases Adjacent to Nanopores”.

[0003] This request is related to the following requests, the contents of which are incorporated by reference in this document:

[0004] North American Provisional Patent Application No. 62/007,248, filed on June 3, 2014 and entitled “Compositions, Systems, and Methods for Detecting Events Using Tethers Anchored to or Adjacent to Nanopores”;

[0005] North American Provisional Patent Application No. 62/157,371, filed on May 5, 2015 and entitled “Compositions, Systems, and Methods for Detecting Events Using Tethers Anchored to or Adjacent to Nanopores”; It is

[0006] North American Patent Application No. 14/728,721, filed on June 2, 2015 and entitled “Compositions, Systems, and Methods for Detecting Events Using Tethers Anchored to or Adjacent to Nanopores”.

SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 25, 2016, has the name 12957-180-28_SL.txt and a size of 665 bytes.

FIELD OF INVENTION

[0008] This application refers, in general, to polynucleotide sequencing.

BACKGROUND OF THE INVENTION

[0009] A significant amount of academic and business time and energy has been invested in sequencing polynucleotides, such as DNA. For example, Olsen et al, “Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment),” JACS 135: 7855-7860 (2013), the content of which is incorporated by reference herein, discloses the bioconjugation of single molecules of the Klenow fragment (KF) of DNA polymerase I into electronic nanocircuits in order to allow electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Or, for example, Hurt et al, “Specific Nucleotide Binding and Rebinding to Individual DNA Polymerase Complexes Captured on a Nanopore,” JACS 131: 37723778 (2009), the contents of which, in full, are incorporated by reference herein, reveal the measurement of disruption time for DNA complexes with the KF at the top of a nanopore in an applied electric field. Or, for example, Kim et al, “Detecting single-abasic residues within a DNA strand immobilized in a biological nanopore using an integrated CMOS sensor,” Sens. Actuators B Chem. 177: 1075-1082 (2012), the content of which is incorporated, in full, by reference in this document, discloses the use of a current or flow measurement sensor in experiments involving DNA captured in an α-hemolysin nanopore. Or, for example, Garalde et al., “Distinct Complexes of DNA Polymerase I (Klenow Fragment) for Based and Sugar Discrimination during Nucleotide Substrate Selection,” J. Biol. Chem. 286: 14480-14492 (2011), the content of which is incorporated in its entirety by reference herein, reveals distinctive KF-DNA complexes based on their properties when they are captured in an electric field at the top of an α-hemolysin pore . Other references disclosing measurements involving α-hemolysin include the following, all by Howorka et al, the contents of which are incorporated, in full, by reference in this document: “Kinetics of duplex formation for individual DNA strands within a single protein nanopore,” PNAS 98 : 12996-13301 (2001); “Probing Distance and Electrical Potential within a Protein Pore with Tethered DNA,” Biophysical Journal 83: 3202-3210 (2002); and “Sequence-specific detection of individual DNA strands using engineered nanopores,” Nature Biotechnology 19: 636-639 (2001).

[0010] North American Patent No. 8,652,779, to Turner et al., the contents of which are, in full, incorporated by reference in this document, discloses compositions and methods of nucleic acid sequencing using a single polymerase enzyme complex including an enzyme polymerase and a nucleic acid template bound near a nanopore, and the nucleotide analogues in solution. Nucleotide analogues include charge-blocking tags that are linked to the polyphosphate moiety of the nucleotide analogue, such that the charge-blocking tags are cleaved when the nucleotide analogue is incorporated into a growing nucleic acid. According to Turner, the charge-blocking marker is detected by the nanopore to determine the presence and identity of the incorporated nucleotide and thus determine the sequence of a template nucleic acid. U.S. Patent Publication No. 2014/0051069, to Jayasinghe et al., the entire contents of which are incorporated by reference herein, refers to constructs that include a transmembrane protein pore subunit and a handling enzyme of nucleic acid.

[0011] However, previously known compositions, systems and methods, such as those described by Olsen, Hurt, Kim, Garalde, Howorka, Turner and Jayasinghe, may not necessarily be sufficiently robust, reproducible or sensitive, and may not have a yield high enough for practical application, for example, as demanding commercial applications such as genome sequencing in clinical scenarios and others that demand highly accurate and inexpensive operations. Therefore, improved compositions, systems and methods for polynucleotide sequencing are needed.

SUMMARY

[0012] Embodiments of the present invention provide compositions, systems and methods for sequencing polynucleotides using chains anchored to polymerases adjacent to nanopores.

[0013] In one aspect, a composition includes a nanopore including a first side, a second side, and an opening extending through the first and second sides. The composition may also include a plurality of nucleotides, wherein each of the nucleotides includes an elongated tag. The composition may also include a first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide. The composition may also include a polymerase disposed adjacent the first side of the nanopore, with the polymerase configured to add nucleotides from the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide. The composition may also include a permanent chain including a head region, a tail region and an elongated body disposed therebetween, the head region being anchored to the polymerase, wherein the elongated body occurs at the opening of the nanopore. The composition may also include a first portion disposed in the elongated body, wherein the first portion is configured to bind to the elongated marker of a first nucleotide on which the polymerase is acting, as well as a reporter region disposed in the elongated body, in which the reporter region is configured to indicate when the first nucleotide is complementary or not complementary to a following nucleotide in the sequence of the second polynucleotide.

[0014] In some embodiments, the first portion is configured to generate a first polymerase-responsive signal state acting on the first nucleotide, the first nucleotide being identifiable based on the first signal state. In some embodiments, the polymerase that acts upon the first nucleotide includes the polymerase that binds to the first nucleotide. In some embodiments, the first signal state includes an electrical or optical signal.

[0015] In some embodiments, the reporter region is configured to generate a second signal state, which is detectable based on the second signal state, whether the first nucleotide is complementary or non-complementary to the following nucleotide of the second polynucleotide. In some embodiments, the reporter region is configured to generate the second state of the polymerase-responsive signal by successfully incorporating the first nucleotide into the first polynucleotide.

[0016] In some embodiments, the reporter region is configured to generate the second state of the signal responsive to polymerase-responsive pyrophosphate release, successfully incorporating the first nucleotide into the first polynucleotide. In some embodiments, the polymerase is modified so as to delay the release of the pyrophosphate responsive to the incorporation of the first nucleotide into the first polynucleotide. In some embodiments, the polymerase includes a polymerase Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5 , PR722, or modified recombinant L17. In some embodiments, the polymerase includes a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of: an amino acid substitution at position 484; an amino acid substitution at position 198 and an amino acid substitution at position 381. In some embodiments, the polymerase includes a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E375Y, K512Y, T368F , A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W and L381A.

[0017] In some embodiments, the reporter region is configured to generate the second state of the signal responsive to a conformational change of the polymerase. In some embodiments, a magnitude or duration of the conformational change of the polymerase being responsive to the first nucleotide being complementary or noncomplementary to the next nucleotide of the second polynucleotide, a magnitude or duration of time, or both, of the second state of the signal being based on the conformational change of the polymerase.

[0018] In some embodiments, the second state of the signal includes an electrical signal. In some embodiments, the second signal state includes an optical signal.

[0019] In some embodiments, the elongated marker includes a first nucleotide sequence, and the first portion includes a second nucleotide sequence that is complementary to the first nucleotide sequence. A system may include such a composition and may further include measurement circuits configured to measure a first current or flow state through the opening. In some embodiments, the first stream or flow is based on the elongated marker, the first nucleotide being identifiable based on the first stream or flow state. In some embodiments, the measurement circuit is further configured to measure a second current or flow through the opening. In some embodiments, the second current or flow state is based on a position of the reporter region within the opening, being determinable based on the second current or flow state, whether the first nucleotide is complementary or non-complementary to the following nucleotide in the second. polynucleotide.

[0020] In some embodiments, the first portion and a second portion of the chain are configured to hybridize to each other to form a hairpin structure. In some embodiments, the compositions further include a voltage source configured to apply a voltage to the first and second sides. In some embodiments, the first portion and the second portion of the chain are configured to dehybridize each other in response to voltage in a two-step process.

[0021] In another aspect, a method may include providing a nanopore, including a first side, a second side, and an opening extending through the first and second sides. The method may further include providing a plurality of nucleotides, each of the nucleotides including an elongated tag. The method may further include providing a first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide. The method may further include providing a polymerase disposed adjacent the first side of the nanopore, with the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide, wherein the polymerase is anchored to a permanent chain including a head region, a tail region, and an elongated body arranged between them, with the elongated body occurring at the opening of the nanopore. The method may further include determining that a first nucleotide is acting on the polymerase based on binding of the elongated tag to a first portion disposed on the elongated body. The method may further include a reporter region disposed on the elongated body, indicating when the first nucleotide is complementary or not complementary to a next nucleotide in the sequence of the second polynucleotide.

[0022] In some embodiments, said determination includes generating a first state of the signal responsive to the polymerase acting on the first nucleotide and identifying the first nucleotide based on the first state of the signal. In some embodiments, the polymerase that acts upon the first nucleotide includes the polymerase that binds to the first nucleotide. In some embodiments, the first signal state includes an electrical or optical signal.

[0023] In some embodiments, said indication includes detecting a second signal state and determining, based on the second signal state, that the first nucleotide is complementary or not complementary to the following nucleotide of the second polynucleotide. In some embodiments, the method includes generating the second state of the polymerase-responsive signal by incorporating the first nucleotide into the first polynucleotide.

[0024] In some embodiments, the method includes generating the second state of the signal responsive to polymerase-responsive pyrophosphate release by incorporating the first nucleotide into the first polynucleotide. In some embodiments, the polymerase is modified so as to delay the release of the pyrophosphate responsive to the incorporation of the first nucleotide into the first polynucleotide. In some embodiments, the polymerase includes a polymerase Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5 , PR722, or modified recombinant L17. In some embodiments, the polymerase includes a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of: an amino acid substitution at position 484; an amino acid substitution at position 198 and an amino acid substitution at position 381. In some embodiments, the polymerase includes a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E375Y, K512Y, T368F , A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W and L381A.

[0025] In some embodiments, the method includes generating the second state of the signal responsive to a conformational change of the polymerase. In some embodiments, a magnitude or a time duration of the conformational change of the polymerase being responsive to the first nucleotide being complementary or noncomplementary to the next nucleotide of the polynucleotide, a magnitude or a time duration, or both, of the second state of the signal being based in the conformational change of the polymerase.

[0026] In some embodiments, the second state of the signal includes an electrical signal. In some embodiments, the second signal state includes an optical signal.

[0027] In some embodiments, the elongated marker includes a first nucleotide sequence, and the first portion includes a second nucleotide sequence that is complementary to the first nucleotide sequence. In some embodiments, said determination further includes measuring a first current or flow state through the opening. In some embodiments, the first current or flow state is based on the elongated marker, wherein said determination further includes identifying the first nucleotide based on the first current or flow state. In some embodiments, said indication includes moving the reporter region within the polymerase-responsive opening acting on the first nucleotide. In some embodiments, said indication further includes measuring a second current or flow state through the opening. In some embodiments, the second current or flow state is based on a position of the reporter region within the opening, said indication further including determining, based on the second current or flow state, whether the first nucleotide is complementary or is not complementary to the following nucleotide in the second polynucleotide.

[0028] In some embodiments, the first portion and a second portion of the chain hybridize with each other to form a hairpin structure. Some embodiments further include applying a voltage across the first and second sides. In some embodiments, the first portion and the second portion of the current dehybridize from each other in response to voltage in a two-step process.

[0029] In one embodiment, the method includes determining that a subsequent nucleotide is acting on the polymerase based on binding of the elongated marker to the first portion disposed in the elongated body; and with a reporter region disposed in the elongated body, indicating when the subsequent nucleotide is complementary or not complementary to a nucleotide that is subsequent to the next nucleotide in the sequence of the second polynucleotide. Some embodiments include repeating such steps for a plurality of subsequent nucleotides that are complementary or noncomplementary to a plurality of nucleotides that are subsequent to the next nucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figures 1A to 1D schematically illustrate compositions for sequencing polynucleotides using a chain anchored to a polymerase adjacent to a nanopore, according to some embodiments of the present invention.

[0031] Figure 2A schematically illustrates a system including a measurement circuit configured to measure the movement of at least one reporter region within the opening of a nanopore, in accordance with some embodiments of the present invention.

[0032] Figure 2B schematically illustrates a plan view of a system including a measurement circuit configured to measure the movement of respective reporter regions within respective openings of a nanopore array, in accordance with some embodiments of the present invention.

[0033] Figure 3 illustrates a method for sequencing polynucleotides using a chain anchored to a polymerase adjacent to a nanopore, according to some embodiments of the present invention.

[0034] Figures 4A to 4E schematically illustrate an exemplary use of a composition for sequencing a polynucleotide using a chain anchored to a polymerase adjacent to a nanopore, according to some embodiments of the present invention.

[0035] Figure 4F schematically illustrates an exemplary signal that can be generated during the use of the composition described in figures 4A to 4E, according to some embodiments of the present invention.

[0036] Figures 5A to 5D schematically illustrate an exemplary use of a composition for sequencing a polynucleotide using a chain anchored to a polymerase adjacent to a nanopore, according to some embodiments of the present invention.

[0037] Figure 6 schematically illustrates an exemplary signal that can be generated during the use of the composition described in figures 5A to 5D, according to some embodiments of the present invention.

[0038] Figures 7A to 7B schematically illustrate exemplary conformational changes of a polymerase.

[0039] Figures 7C and 7D schematically illustrate a composition including a chain anchored to a polymerase adjacent to a nanopore and configured for use in sequencing a polynucleotide, in accordance with some embodiments of the present invention.

[0040] Figure 8 illustrates exemplary reaction parameters, for example, rate constants and inactivity times, for reaction schemes in which a nucleotide respectively under the action of a polymerase is a match or not (adapted from Johnson, “The kinetic and chemical mechanism of high-fidelity DNA polymerases,” Biochim Biophys Acta 1804(5): 1041-1048 (2010), the entire contents of which are incorporated by reference herein).

[0041] Figure 9A schematically illustrates an exemplary nucleotide including an elongated marker, including a portion that interacts with a portion of the chain described in Figures 1A to 1D during use in sequencing a polynucleotide, in accordance with some embodiments of the present invention.

[0042] Figures 9B and 9C schematically illustrate exemplary nucleotides, including elongated markers including respective portions that can interact with an exemplary chain during use in sequencing a polynucleotide, in accordance with some embodiments of the present invention.

[0043] Figure 9D schematically illustrates an exemplary chain and portions that can interact with the chain during use in sequencing a polynucleotide, according to some embodiments of the present invention. The figure discloses SEQ ID NO: 1.

[0044] Figures 10A and 10B schematically illustrate the movement of an exemplary chain responsive to hybridization with a portion of an elongated marker of an exemplary nucleotide during use in sequencing a polynucleotide, in accordance with some embodiments of the present invention.

[0045] Figures 11A to 11C schematically illustrate exemplary structures for use in modifying a kinetic constant in a reaction scheme that can be used in sequencing a polynucleotide, according to some embodiments of the present invention.

[0046] Figures 12A and 12B illustrate exemplary structures for use in modifying a kinetic constant in a reaction scheme, in which a nucleotide is used by a polymerase, according to some embodiments of the present invention.

DETAILED DESCRIPTION

[0047] Embodiments of the present invention provide compositions, systems and methods for sequencing polynucleotides using chains anchored to polymerases adjacent to nanopores.

[0048] More specifically, the present compositions, systems and methods can be suitably used to sequence polynucleotides in a manner that is robust, reproducible, sensitive and has a high throughput. For example, the present compositions may include a nanopore and a permanent chain that is anchored to a polymerase that is disposed adjacent to the nanopore. The nanopore may include first and second sides and an opening extending through the first and second sides. The permanent current may include head and tail regions and an elongated body disposed therebetween. In particular embodiments, the chain head region is anchored to the polymerase, which is disposed adjacent to the first side of the nanopore, and the elongated body occurs at the opening of the nanopore. Furthermore, the present compositions may include a plurality of nucleotides, each of which includes an elongated tag. The elongated marker chain, or both, may include one or more attributes that facilitate sequencing of a polynucleotide, for example, a template polynucleotide. For example, the chain or elongated tag, or both, may include one or more attributes that provide a first signal state based on the polymerase acting on a nucleotide, and may include one or more attributes that provide a second signal state based on the polymerase acting on a nucleotide. at that nucleotide, being complementary or non-complementary to a next nucleotide in a sequence of the polynucleotide being sequenced. In some embodiments, the identity of the nucleotide under the action of the polymerase can be determined based on the first state of the signal. Furthermore, in some embodiments, the complementarity of that nucleotide to a following nucleotide in a sequence of the polynucleotide being sequenced can be determined based on the second state of the signal, which can help distinguish whether that nucleotide is complementary to the polynucleotide being sequenced. or if, instead, the polymerase temporarily acted on a nucleotide that was not complementary to the next nucleotide in the sequence of the polynucleotide being sequenced, but without adding that nucleotide to the growing complementary polynucleotide. Thus, the second signal state may be useful for assigning bases to chromatogram peaks during a nucleic acid sequencing application, for example, by providing an error checking function. It should be understood that such first and second states of the signal, which may respectively represent the identities of the nucleotides or whether such nucleotides are a match or not, need not necessarily occur at different times from each other, and in fact, may occur at the same time. from each other. For example, a measured signal (e.g., optical or electrical) may include a composite of more than one signal state (e.g., two signal states), wherein each of the signal states provides information about one or more of a nucleotide identity or whether such nucleotide is a match or not.

[0049] Previously known methods for sequencing by synthesis (SBS) have been developed. For example, a single-stranded DNA (ssDNA) can pass through a biological nanopore, such as a protein nanopore, which is embedded in a barrier such as a lipid bilayer, responsive to an electrical potential being applied across the nanopore. In what may be referred to as “strand” sequencing, since ssDNA nucleotides pass through a pore blockage, and combinations of these nucleotides can create unique current or flow blocks corresponding to the identities of the nucleotides in the particular combinations that pass through. through constriction. These strands being sequenced are not permanently bound to the pore or polymerase. Rather, these strands translocate through the pore so that the effective position of the strand changes relative to the pore. However, the extremely fast translocation rate of ssDNA (~1 nt/μsec), as well as the native resolution of the constriction that encompasses a combination of nucleotides, rather than a single nucleotide, can make accurate measurement of such current blocks difficult. or flow on a nucleotide by nucleotide basis. Enzyme “motors” were used to slow translocation speed to a rate more compatible with data acquisition (milliseconds per nucleotide). However, such motors, when used in strand sequencing setups, can introduce error modes such as skips, slips, or swaps, which can inhibit reliable detection of nucleotides in ssDNA. These and other motor-independent error modes that can occur during strand sequencing may result from the “flexibility” or elasticity of the ssDNA that resides between the motor and the nanopore constriction. Such flexibility may be a function of the ssDNA sequence and may result in different currents or fluxes for the same combination of nucleotides transiting the constriction if different instances of that combination are respectively surrounded by different ssDNA sequences. Furthermore, because the constriction can be relatively small, e.g., about 2 nt, and Brownian motion is always present, the “reading head” of the pore can be an effective size of 4 nucleotides, e.g. The constriction reads a combination of about 4 nucleotides at a time, thus making it more difficult to uniquely identify each nucleotide, since there are 44 (256) streams or streams that need to be differentiated from each other.

[0050] Therefore, there is still a need for improvements in SBS, for example, for cheap, accurate, long-read and high-throughput compositions, systems and methods for SBS. SBS using nanopores, e.g., biological nanopores, represents a potential solution to this need due to the nanoscale reproducibility and ease of production of these proteins. Taken together, it is expected that an approach that lacks is driving (e.g., using nucleic enzymes as a detector that is coupled to a nanopore rather than a motor that modulates the passage of a target strand through a nanopore), more tolerant to Brownian motion, and which has a resolution of a single nucleotide, considerably advances the field of nanopore DNA sequencing.

[0051] Firstly, some terms used in the present invention will be briefly explained. Next, some exemplary compositions, exemplary systems including measurement circuits (e.g., electrical or optical measurement circuits) that can be used with the present compositions, exemplary methods that can be used with the present compositions, and some specific examples of compositions that can be used during such methods will be described.

Exemplary terms

[0052] As used in the present invention, the term “pore” is intended to mean a structure that includes an opening that allows molecules to pass through them from a first side of the pore to a second side of the pore. That is, the opening extends through the first and second sides of the pore. Molecules that can pass through a pore opening can include, for example, ions or water-soluble molecules such as amino acids, proteins, nucleotides and nucleic acids. The pore can be arranged within a barrier. When at least a portion of a pore opening has a width of 100 nm or less, e.g., 10 nm or less, or 2 nm or less, the pore may be, but need not necessarily be, referred to as a “nanopore.” ”. Optionally, a portion of the opening may be narrower than one or both of the first and second sides of the pore, in which case that portion of the opening may be referred to as a "constriction". Alternatively or in addition, the opening of a pore, or the constriction of a pore (if present), or both, may be greater than 0.1 nm, 0.5 nm, 1 nm, 10 nm or greater. A pore may include multiple constrictions, for example, at least two, three, four, five or more than five constrictions.

[0053] As used in the present invention, a “barrier” is intended to mean a structure that normally inhibits the passage of molecules from one side of the barrier to the other side of the barrier. Molecules for which passage is inhibited may include, for example, ions or water-soluble molecules such as amino acids, proteins, nucleotides and nucleic acids. A pore can be arranged within a barrier, and opening the pore can allow molecules from one side of the barrier to pass to the other side of the barrier. Barriers include membranes of biological origin and non-biological barriers such as solid-state membranes.

[0054] As used in the present invention, “chain” is intended to mean an elongated member having a head region, a tail region and an elongated body therebetween. A chain includes a molecule. A chain may be, but need not necessarily be, in an elongated state, for example, it may include an elongated molecule. For example, an elongated body of a chain may have secondary or tertiary configurations, such as clips, bends, helical configurations, or the like. The chains may include polymers such as polynucleotides or synthetic polymers. The currents may have lengths (e.g., measured in an extended or maximally stretched state) ranging, for example, from about 5 nm to about 500 nm, e.g., from about 10 nm to about 100 nm. The chains may have widths ranging, for example, from about 1 nm to about 50 nm, for example, from about 2 nm to about 20 nm. Currents can be linear or branched. A current may be considered “permanent” when it is not removed from a defined composition under the conditions under which the composition is used, for example, in a detection method. A chain that is used in a cyclic or repeated reaction can also be considered “permanent” when there is no effective change in the position of the chain from one cycle to the next or from one reaction to a repeat reaction. It will be understood that the position of a permanent current may change during an individual cycle or reaction, even if there is no actual change in position across the cycles or reactions.

[0055] As used in the present invention, a “head region” of a chain is intended to mean a functional group of the chain that is linked to another member. Such a bond may be formed through a chemical bond, for example, through a covalent bond, hydrogen bond, ionic bond, dipole-dipole bond, London dispersion forces or by any combination of these. In one embodiment, such a link may be formed by hybridizing a first oligonucleotide from the head region to a second oligonucleotide from another member. Alternatively, such a bond may be formed using physical or biological interactions, for example, an interaction between a first protein structure of the head region and a second protein structure of the other member that inhibits detachment of the head region of the other member. Exemplary members to which a head region of a chain may bind include a pore, e.g., the first or second side of the pore, a barrier in which the pore is disposed, and a molecule, such as a protein, disposed on the first or second side of the pore. on the second side of the pore. If the chain head region is connected to another member that is disposed on the first or second side of the pore, the chain head region can be considered adjacent to the pore. The head region may be, but does not necessarily need to be, located at the end of the chain.

[0056] As used in the present invention, "anchored" means a link between a first member and a second member that is permanent, for example, is sufficiently stable to be useful for sequencing a polynucleotide or, for example, is mobile, but it undergoes no effective movement under the conditions in which the attached limbs are used. In some embodiments, such permanent bonding is typically irreversible under conditions where the bonded members are used, for example, in a detection method. In other embodiments, such permanent binding is reversible, but persists for at least the period of time in which it is used to sequence a polynucleotide. For example, a chain may be permanently attached to or adjacent to a polymerase during use of the chain to sequence a polynucleotide, and may be subsequently removable or replaceable with another chain. Covalent bonds are just one example of a bond that can be appropriately used to anchor a first member to a second member. Other examples include duplexes between oligonucleotides, peptide-peptide interactions, and streptavidin-biotin or streptavidin-desthiobiotin.

[0057] As used herein, a “tail region” of a chain is intended to mean a portion of the chain that is disposed distally from the head region. The tail region may freely extend outside the head region, for example, it may not be attached to any other limb. The tail region may alternatively be linked. Such a bond may be formed through a chemical bond, for example, through a covalent bond, hydrogen bond, ionic bond, dipole-dipole bond, London dispersion forces or by any combination of these. In one embodiment, such a link may be formed by hybridizing a first oligonucleotide from the tail region to a second nucleotide from another member. Alternatively, such a bond may be formed using physical or biological interactions, for example, an interaction between a first protein structure of the tail region and a second protein structure of the other member that inhibits binding of the tail region of the other member. Any member to which the tail region is attached may be, but is not necessarily, the same member to which the head region is attached. The tail region may be, but is not necessarily, located at the end of the chain.

[0058] As used herein, an “elongate body” is intended to mean a portion of a member, such as a chain, that is sufficiently long and narrow to be disposed within at least a portion of a pore opening. When an elongated body is linked to a nucleotide being used, such an elongated body may be referred to as an “elongated marker” so as to facilitate the distinction of an elongated body from a chain. An elongated body may be formed from any suitable material of biological origin or non-biological origin, or a combination thereof. In one example, the elongate body includes a polymer. Polymers can be biological or synthetic polymers. Exemplary biological polymers that can be conveniently included in the elongated body include polynucleotides, polypeptides, polysaccharides, polynucleotide analogs, and polypeptide analogs. Exemplary polynucleotides and polynucleotide analogs suitable for use in an elongated body include DNA, enantiomeric DNA, RNA, PNA (peptide nucleic acid), morpholinos, and LNA (locked nucleic acid). Exemplary synthetic polypeptides may include charged amino acids as well as hydrophilic and neutral residues. Exemplary synthetic polymers that may suitably be included in an elongated body include PEG (polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinyl alcohol), PE (polyethylene), LDPE (low density polyethylene), HDPE (high density polyethylene), polypropylene, PVC (polyvinyl chloride), PS (polystyrene), NYLON (aliphatic polyamides), TEFLON® (tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes, polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters), poly(alkyl methacrylates) and other polymeric chemical and biological linkers, as described in Hermanson, Bioconjugate Techniques, third edition, Academic Press, London (2013). Furthermore, an elongate body may optionally include a portion that can interact with another portion. Such moieties may include biological polymers of DNA, RNA, PNA, LNA, morpholinos or enantiomeric DNA, as an example. The elongated body regions can be charged or neutral depending on the specific implementation of the reporter readout.

[0059] As used in the present invention, a “reporter region” is intended to mean a portion that is, upon change, detectable using a suitable detection method or system. Such change may include, but is not limited to, a movement. The movements may be approximately 10 nm or less, or approximately 5 nm or less, or approximately 2 nm or less, or approximately 1 nm or less, or approximately 0.5 nm or less, or approximately 0. 2 nm or less, or even approximately 0.1 nm or less, and can be detected using the reporter region and a suitable detection method or system. The portion may have a detectable physical, chemical, electrical, optical or biological property or other suitable flow blocking property. For example, the portion may have an optical property that facilitates optical detection or characterization. Optical properties include fluorescence and the generation of a Raman signal. In an illustrative example, the moiety is a fluorescence resonance energy transfer (FRET) donor or acceptor that interacts with a corresponding FRET acceptor or donor so as to emit light of a specific wavelength that can be detected. The donor and acceptor can be considered partners in a FRET pair. Or, for example, the portion may have an electrical or flow blocking property. Electrical or flow blocking properties include electrostatic charge, for example, a positive charge or a negative charge. Or, for example, the portion may have a physical property. Physical properties include portion volume and shape. In an illustrative example, movement of the portion within the opening causes a measurable change in current or flow through the opening, or a constriction thereof, by modulating a flow or blocking current through the opening or constriction. Or, for example, the moiety may have a chemical or biological property that facilitates chemical or biological detection. Chemical or biological properties include the presence of a chemical or biological group, for example, a radioactive group or a group with enzymatic activity. One or more electrical, physical, chemical, biological, or flow-blocking properties may provide a measurable change in current through an opening or constriction, or an optical signal. In an illustrative example, movement of the portion within an opening causes a measurable change in a current through an opening or constriction, or causes a measurable change in the flow of molecules through an opening or construction, such change in flow being able to be electrically, chemically, biologically or optically detectable. A non-basic nucleotide is a non-limiting example of a moiety whose movement can cause a measurable change in a current through an opening or constriction or a measurable change in the flow of molecules through an opening or constriction.

[0060] As used herein, the “motion” or “motion” may be translational, rotational or conformational, or a combination thereof.

[0061] As used in the present invention, “action” of a polymerase on a nucleotide may include the entry of a nucleotide into an active site of the polymerase. The action of a polymerase on a nucleotide may also include, but is not limited to, the polymerase causing a chemical change to the nucleotide or a portion of that nucleotide. Chemical changes may include the polymerase removing a portion of the nucleotide, the polymerase adding the nucleotide to another molecule, the polymerase binding or releasing, the polymerase modifying the nucleotide or a portion thereof, and the polymerase forming or cleaving a chemical bond, e.g. example, during the synthesis of a polynucleotide, and the like. For example, the action of a polymerase on a nucleotide may include adding the nucleotide to a polynucleotide. The action of a polymerase on a nucleotide may optionally include the movement and chemical change of the polymerase, the nucleotide, or both. As purely illustrative and non-limiting examples, the action of a polymerase on a nucleotide may include one or more of: a polymerase testing a nucleotide, the polymerase rejecting a nucleotide if the nucleotide is incompatible with the next nucleotide in a polynucleotide being sequenced , the polymerase that removes a nucleotide from a polynucleotide using exonuclease activity, and the polymerase that removes a nucleotide from a polynucleotide using pyrophosphorylase. Figure 8 illustrates exemplary reaction parameters, e.g., rate constants and inactivity times, for reaction schemes in which a nucleotide respectively under the action of a polymerase is a match or not (adapted from Johnson, “The kinetic and chemical mechanism of high-fidelity DNA polymerases,” Biochim Biophys Acta 1804(5): 1041-1048 (2010), the entire contents of which are incorporated by reference herein). Polymerases, such as T7 Pol, typically distinguish between matching and mismatching nucleotides based on a combination of increased binding affinity for the correct matching nucleotide (e.g., 10-fold preference over a mismatched nucleotide), greatly reducing the catalytic rate for the incompatible nucleotide (e.g., approximately 1000 times slower for the incompatible) and a fairly high dissociation rate for the incompatible nucleotide from a closed catalytic state (e.g., approximately 300 times faster for an incompatible one) .

[0062] As used in the present invention, a “conformational change” is intended to mean a change in the shape of a molecule (for example, a change in the relative atomic coordinates of a molecule). Such a conformational change may include a portion of a molecule moving relative to another part of the molecule. The chemical reactivity of a portion of the molecule may change in response to the relative movement of that portion, or of another portion of the molecule. A molecule can undergo a conformational change responsive to a stimulus. Such stimulation may include, but is not limited to, changes in or forces applied to the molecule, interactions with other molecules or environmental factors. The changes or forces applied to the molecule may include a physical force applied to the molecule or a portion thereof, an electric field applied to the molecule, or a chemical reaction with the molecule or a portion thereof, or a combination thereof, e.g. , binding to a substrate, catalysis and/or release of a product. Interactions with other molecules may include the presence of another molecule, a concentration of another molecule, an action by or on another molecule, or a combination thereof. An exemplary interaction with another molecule includes the hybridization of two oligonucleotides, or a polymerase acting on a nucleotide. Environmental factors may include a change in pH or a change in temperature, or a combination thereof.

[0063] As used in the present invention, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one phosphate group and, optionally, also includes a nucleobase. A nucleotide that does not have a nucleobase can be referred to as “abbasic”. Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, nucleotides with modified sugar and phosphate backbones, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP ), cytidine diphosphate (CDP) cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP) and deoxyuridine triphosphate (dUTP).

[0064] The term “nucleotide” is also intended to encompass any nucleotide analogue that is a type of nucleotide that includes a modified nucleobase, sugar and/or phosphate moiety compared to nucleotides that occur in nature. Exemplary modified nucleobases that may be included in a polynucleotide, with a native or analogous structure, include inosine, xatanine, hypoxatanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6 - methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6- azouracil, 6-azocytosine, 6-azothymine , 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-akinoadenine or guanine, 5-halouracil or substituted cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-aza-adenine, 7-deazaguanine, 7-deaza-adenine, 3-deazaguanine, 3-deaza-adenine or similar. As is known in the art, certain nucleotide analogs cannot be incorporated into a polynucleotide, for example, nucleotide analogs such as adenosine 5'-phosphosulfate.

[0065] Exemplary nucleotides modified at the phosphate moiety include, for example, the nucleotide analogs described by Lee et al., “Synthesis and reactivity of novel Y-phosphate modified ATP analogues,” Bioorganic & Medicinal Chemistry Letters 19: 3804-3807 ( 2009); Kumar et al, “PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis,” Scientific Reports 2: 684 (2012); Kumar et al., “Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases,” Nucleosides, Nucleotides, and Nucleic Acids 24: 401-408 (2005), and Mulder et al., “Nucleotide modification at the Y-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase,” Nucleic Acids Research 33: 4865-4873 (2005), the entire contents of which are incorporated by reference in the present invention. Lee et al. describes certain exemplary Y-phosphate modified ATP analogs with the following structures:

[0066] Kumar et al. (2012) reveals PEG-coumarin tags with different lengths that can be attached to the phosphate terminus of dNTP or NTP (dNTP/NTP) or to the phosphate terminus of tetraphosphate nucleotides (dN4P/N4P). Exemplary lengths include, for example, coumarin-PEG16-dN4P/N4P, coumarin-PEG20-dN4P/N4P, coumarin-PEG24-dN4P/N4P, and coumarin-PEG36-dN4P/N4P. Kumar et al. (2005) discloses tetra- and pentaphosphate-modified nucleotides, including dyes linked with or without ligands. As described in Kumar et al. (2005) exemplary unliganded dyes include DDAO, RESORUFIN, COUMARINS, alkyl-XANTHHENES, nitrophenol, hydroxy-indole, ELF and BBT; exemplary dyes linked via linkers include R110, REG, TAMRA, ROX, Cy dyes, and ET dyes; and exemplary ligands include diaminopropane, diaminoheptane, diaminododecane, EEE, PAP, diaminocyclohexane, diaminoxylene and pentallysine. Mulder et al. reveals chemically modified nucleotides, including 1-aminonaphthalene-5-sulfonate (ANS) linked to the Y-phosphate of a nucleotide, e.g., Y-P-aminonaphthalene-deoxy 5-sulfonate, or ribonucleotides (dNTP or NTP), such as ANS-ATP, ANS -CTP, ANS-GTP and ANS-TTP and/or the deoxy forms of these or other nucleotides.

[0067] As used in the present invention, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are linked together. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogues. A polynucleotide may be a single-stranded sequence of nucleotides, such as single-stranded RNA or DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, or may include a mixture of single-stranded and double-stranded sequences. of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA (dsDNA) and amplification and PCR products. Single-stranded DNA (ssDNA) can be converted to dsDNA and vice versa. The exact sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, expressed sequence tag (EST), or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, primer RNA, or amplified copy of any of the above.

[0068] As used in the present invention, “hybridizes” is intended to mean the non-covalent attachment of a first polynucleotide to a second polynucleotide. The strength of the bond between the first and second polynucleotides increases with the complementarity between these polynucleotides.

[0069] As used in the present invention, the term “protein” is intended to mean a molecule that includes, or consists of, a polypeptide that is folded into a three-dimensional structure. The polypeptide includes portions that, when folded into the three-dimensional structure, give the protein biological activity.

[0070] As used in the present invention, the term “enzyme” is intended to mean a molecule that catalytically modifies another molecule. Enzymes can include proteins as well as other types of molecules such as polynucleotides. Examples of enzymes that are also proteins include polymerases, exonucleases, and helicases.

[0071] As used in the present invention, a “polymerase” is intended to mean an enzyme with an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to a primed single-stranded polynucleotide template and can sequentially add nucleotides to the growing primer to form a polynucleotide with a sequence that is complementary to that of the template.

Exemplary compositions, systems and methods for nucleotide sequencing

[0072] Exemplary compositions, including chains anchored to polymerases adjacent to the nanopores, will now be described with reference to figures 1A to 1D. In one aspect, a composition includes a nanopore including a first side, a second side, and an opening extending through the first and second sides. The composition may also include a plurality of nucleotides, wherein each of the nucleotides includes an elongated tag. The composition may also include a first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide. The composition may also include a polymerase disposed adjacent the first side of the nanopore, with the polymerase configured to add nucleotides from the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide. The composition may also include a permanent chain including a head region, a tail region and an elongated body disposed therebetween, the head region being anchored to the polymerase, wherein the elongated body occurs at the opening of the nanopore. The composition may also include a first portion disposed on the elongated body, wherein the first portion is configured to bind to the elongated marker of a first nucleotide on which the polymerase is acting. The composition may also include one or more reporter regions disposed on the elongate body, wherein the one or more reporter regions are configured to indicate when the first nucleotide is complementary or not complementary to a next nucleotide in the sequence of the second polynucleotide.

[0073] For example, Figure 1A schematically illustrates a cross-section of an exemplary composition that includes the nanopore 100, the permanent chain 110, a plurality of nucleotides 120 (one of which is shown, for reasons of simplicity of illustration), the polymerase 130, the first polynucleotide 140, and the second polynucleotide 150. The nanopore 100 includes the first side 101, the second side 102, the opening 103, and the constriction 104. The permanent chain 110 includes the head region 111, the tail region 112 and the elongated body 113. In the embodiment illustrated in Figure 1A, the polymerase 130 is disposed adjacent to the first side 101 of the nanopore 100, the head region 111 of the permanent chain 110 is anchored to the polymerase 130, the tail region 112 of the chain The permanent current 110 is freely disposed on the second side 102 of the nanopore 100 and the elongated body 113 is movable within the opening 103 of the nanopore 100. The elongated body 113 of the permanent current 110 includes a first portion 115 and one or more reporter regions 114. The nucleotide 120 includes the elongated marker 121, which includes the second portion 122. The polymerase 130 is configured to add nucleotides from the plurality of nucleotides 120 to the first polynucleotide 140 based on a sequence of the second polynucleotide 150.

[0074] As illustrated in Figure 1B, based on the action of polymerase 130 on nucleotide 120, the first portion 115 of the elongated body 113 of permanent chain 110 is configured to bind to the elongated marker 121 of nucleotide 120, e.g. configured to bind to the second portion 122. For example, the polymerase 130 may include an active site (not specifically illustrated) that receives and binds the nucleotide 120, which places the second portion 122 relatively close to the first portion 115 for a period of sufficient time for the first portion 115 and the second portion 122 to interact with each other, for example, to bond with each other (which can also be referred to as forming a duplex with each other). For example, in some embodiments, the first portion 115 includes a first oligonucleotide, and the second portion 122 includes a second oligonucleotide that is complementary to and hybridizes to the first oligonucleotide.

[0075] The first portion 115 may be configured to generate at least one first signal state responsive to polymerase 130 acting on nucleotide 120, and nucleotide 120 may be identifiable based on the first signal state as described. in more detail elsewhere in the present invention. For example, the formation of a duplex between portion 115 and portion 122 may cause one or more reporter regions 114 to be disposed at a location within the opening 103 that results in a single current or flow through the opening 103 based on which nucleotide 120 can be identified. Illustratively, the first signal state may include an electrical signal or an optical signal.

[0076] Additionally, one or more reporter regions 114 of the elongated body 113 of the permanent chain 110 may be configured to indicate when the nucleotide 120 is complementary or not to a next nucleotide in the sequence of the second polynucleotide 150. For example, in some embodiments , the position of one or more reporter regions 114 in opening 103 may be based on the particular conformation of polymerase 130, which may be based on whether or not nucleotide 120 is a match to the next nucleotide in the sequence of second polynucleotide 150. To For more details on the differences in polymerase conformation and kinetics between the corresponding and mismatched nucleotides, see the following references, the entire contents of each of which are incorporated by reference in the present invention: Freudenthal et al., “New structural snapshots provide molecular insights into the mechanism of high fidelity DNA synthesis,” DNA Repair, doi:10.2016/j.dnarep/2015.04.007 (available online April 30, 2015); Freudenthal et al., “Watching a DNA polymerase in action,” Cell Cycle 13: 691–692, doi:10.4161/cc.27789 (2014); and Freudenthal et al., “Observing a DNA polymerase choose right from wrong,” Cell 154: 157–168, doi:10.1016/j.cell.2013.05.048 (2013).

[0077] For example, as illustrated in Figure 1B, polymerase 130 may have a modified conformation 130' based on nucleotide 120 being a match to the next nucleotide in the sequence of second polynucleotide 150, which causes one or more regions reporters 114 are placed at a location within the opening 103 that results in a single current or flow through the opening 103, based on which it can be determined that the nucleotide 120 is a match. In comparison, as illustrated in Figure 1B, polymerase 130 may have a modified conformation 130' based on nucleotide 120 being a match to the next nucleotide in the sequence of second polynucleotide 150, which causes one or more reporter regions 114 to be placed at a location within opening 103 that results in a single current or flow through opening 103 based on which it can be determined that nucleotide 120 is a mismatch.

[0078] It is observed that, in the embodiment illustrated in Figure 1B, the reporter region/regions 114 can provide an indication of correspondence or not, while the portion 115 and the portion 122 are linked to each other. Alternatively, as illustrated in Figure 1D, the polymerase 130 may again have a modified conformation 130' based on the nucleotide 120 being a match to the next nucleotide in the sequence of the second polynucleotide 150, which causes the region(s) reporter(s) 114 are placed at a location within the opening 103 that results in a single current or flow through the opening 103 based on whether it can be determined whether the nucleotide 120 is a match or non-match. However, portion 115 and portion 122 do not necessarily need to be linked to each other for the reporter region(s) 114 to provide such an indication. For example, portion 115 and portion 122 may dissociate from each other, e.g., in response to thermal fluctuations within opening 103, or in response to the application of a sufficient potential difference between the first side 101 and the second side 102 and the position of the reporter region(s) 114 within the opening 103, while the portions 115 and 122 dissociated from each other can indicate whether the nucleotide 120 is a match or not. In comparison, if nucleotide 120 is a mismatch, then polymerase 130 may have a conformation 130" analogous to that illustrated in Figure 1C, which causes the reporter region(s) 114 to be at a location within the opening 103 that results in a single current or flow through opening 103 based on which it can be determined that nucleotide 120 is a mismatch.

[0079] Furthermore, based on nucleotide 120 being a match, polymerase 130 can incorporate nucleotide 120 into the first polynucleotide, and can cleave the elongated tag 121 from nucleotide 120, which can diffuse away on the cis side (e.g. , the first side) of the nanopore 100. Additionally, based on the polymerase 130 successfully incorporating the nucleotide 120 into the first polynucleotide 140, the polymerase 130 may release pyrophosphate or other phosphorus oxyanion that may have two or more phosphate subunits, e.g. , two, three, four, five or six phosphate subunits (not specifically illustrated). Furthermore, based on polymerase 130 not successfully incorporating nucleotide 120 into the first polynucleotide 140, polymerase 130 does not release pyrophosphate.

[0080] Reporter region 114 is configured to generate one or more signals based on one or more of the effects resulting from nucleotide 120 being activated by polymerase 130 or resulting from nucleotide 120 being complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide 150, for example, based on whether or not one or more of the effects of the nucleotide 120 is linked to or resides in an active site of the polymerase 130 or the polymerase 130 successfully incorporates the nucleotide 120 into the first polynucleotide 140. Other details of such exemplary secondary signal states are described in more detail below with reference to Figures 3, 4A to 4F, 5A to 5D and 6. It may be detectable based on one or more signals whether the first nucleotide is being activated by polymerase 130 or whether or not the first complementary nucleotide is complementary to the following nucleotide of the second polynucleotide, or whether both are being activated by polymerase 130 and whether the first nucleotide is complementary or not complementary to the following nucleotide of the second polynucleotide. Illustratively, each one or more signals may independently include an electrical signal or an optical signal.

[0081] Optionally, the elongate body 113 may include more than one reporter region, for example, it may include any suitable number of reporter regions, for example, one, two, three, four, five or more than five reporter regions. Each of these reporter regions can be the same as another of the reporter regions. Alternatively, each of these reporter regions may be different from the other reporter regions. Or some reporter regions may be the same as each other, while other reporter regions may be different from the others. In an exemplary non-limiting illustrative example, the elongated body 113 includes a polynucleotide that includes one or more abasic nucleotides that define the reporter region 114. An abasic nucleotide can be detected within an opening of a nanopore as described, for example, in Wilson , “Electronic Control of DNA Polymerase Binding and Unbinding to Single DNA Molecules Tethered in a Nanopore,” doctoral thesis, University of California at Santa Cruz (2009), the contents of which are incorporated by reference in the present invention. Illustratively, the movement or presence of one or more abasic nucleotides or other suitable reporter regions 114 may cause a measurable change in a current through the opening 103 or constriction 104, a measurable change in the flow of molecules through the opening 103 or the constriction 104, or an optical signal. For example, a change in the flow of molecules through opening 103 or constriction 104 can be detected electrically, chemically, biologically, or optically.

[0082] In the embodiments illustrated in Figures 1A to 1D, the head region 111, the tail region 112 and the elongated body 113 of the chain 110 may include any suitable material or combination of materials. For example, the head region 111 may be configured to be anchored to the polymerase 130 through a chemical bond, e.g., through a covalent bond, hydrogen bond, ionic bond, dipole-dipole bond, dispersion forces. London or any suitable combination thereof. For example, the head region 111 may include a first portion that is linked, for example, covalently, to a second portion of the polymerase 130. Exemplary covalent bonds that may anchor the head region 111 to the polymerase 130 include carbon-carbon bonds, carbon-nitrogen bonds, carbon-oxygen bonds, oxygen-oxygen bonds, sulfur-sulfur bonds, phosphorus-oxygen bonds, phosphorus-sulfur bonds, amide bonds, thioether bonds, hydrazide bonds, carbon-sulfur bonds and bonds that result from the reaction of oxyamine with carbonyls (aldehydes and ketones), from Staudinger reagent pairs such as phosphine and azides, or click chemistry pairs such as azides and alkynes. However, the bond does not need to be covalent. For example, such a bond may be formed by hybridizing a first head region oligonucleotide to a second polymerase 130 oligonucleotide. Alternatively, such a bond may be formed using physical or biological interactions, e.g., an interaction between a first structure of head region protein and a second polymerase protein structure 130 that inhibits separation of the polymerase head region 130. For example, the head region 111 may include a first alpha-helix and the polymerase may include a second alpha-helix. helix that blocks the 111 head region so as to inhibit dissociation of the 111 head region from the polymerase. Interactions between receptors and ligands are also useful, examples of which include avidin-biotin, or analogues thereof; antibody- epitope; lectin-carbohydrate and similar.

[0083] The elongated body 113 can be linked, for example, by covalent bonding, to the head region 111, and the tail region 112 can define an end of the elongated body 113 that is distal from the head region 111. The elongated body 113 may include any suitable material of biological origin or non-biological origin, or a combination thereof. As described in more detail in the present invention, the elongated body 113 may include one or more reporter regions that indicate when a nucleotide, on which the polymerase 130 is acting, is complementary or is not complementary to a following nucleotide in the sequence of the second polynucleotide 150. The elongated body 113 may also include the first portion 115 that may interact, for example, bind, to the elongated marker 121 of the nucleotide 120 on which the polymerase 130 is acting. Exemplary biological materials that may be included in the elongate body 113 include biological polymers, such as polynucleotides, polypeptides, polysaccharides, and analogs thereof. Exemplary synthetic polymers that may suitably be included in an elongate body 113 include PEG (polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinyl alcohol), PE (polyethylene), LDPE (low density polyethylene), HDPE (high density polyethylene) , polypropylene, PVC (polyvinyl chloride), PS (polystyrene), NYLON (aliphatic polyamides), TEFLON® (tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes, polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters) , poly(alkyl methacrylates) and other polymeric chemical and biological binders, such as described in Hermanson et al., further mentioned above.

[0084] The nanopore 100 may have any suitable configuration that allows for the arrangement of the polymerase 130 adjacent to the first side 101 of the nanopore 100, such that the head region 111 of the chain 110 is anchored to the polymerase 130 and the elongated body 113 of the chain 110, for example, may be disposed in the opening 103 of the nanopore 100. In some embodiments, the nanopore 100 may be a biological pore, a solid-state pore, or a hybrid solid-state and biological pore. A biological pore is intended to mean a pore that is made from one or more materials of biological origin. “Biological origin” refers to material derived or isolated from a biological environment such as an organism or cell, or a synthetically manufactured version of a biologically available structure. Biological pores include, for example, polypeptide pores and polynucleotide pores.

[0085] A polypeptide pore is intended to mean a pore that is made from one or more polypeptides. The one or more polypeptides may include a monomer, a homopolymer or a heteropolymer. Pore and polypeptide structures include, for example, an α-helix bundle pore and a β-barrel pore, as well as all others well known in the art. Exemplary polypeptide pores include α-hemolysin, Mycobacterium smegmatis porin A, gramicidin A, maltoporin, OmpF, OmpC, PhoE, Tsx, F-pilus, SP1, mitochondrial porin (VDAC), Tom40, outer membrane phospholipase A, and lipoprotein Neisseria autotransporter (NaIP). “Mycobacterium smegmatis porin A (MspA)” is a membrane porin produced by mycobacteria, which allows hydrophilic molecules to enter the bacteria. MspA forms a tightly interconnected octamer and transmembrane beta barrel that resembles a cup and includes a central constriction. For more details about α-hemolysin, see U.S. Patent No. 6,015,714, the contents of which are incorporated into the present invention by reference. For more details on SP1, see Wang et al., Chem. Commun., 49: 1741-1743, 2013, the content of which is incorporated into the present invention by reference. For more details on MspA, see Butler et al., “Single-molecule DNA detection with an engineered MspA protein nanopore,” Proc. Natl. Academic. Sci. 105: 20647-20652 (2008) and Derrington et al., “Nanopore DNA sequencing with MspA,” Proc. Natl. Academic. Sci. USA, 107:16060-16065 (2010), the total contents of both of which are incorporated into the present invention by reference. Other pores include, for example, the MspA homolog from Norcadia farcinica and lysenin. For more details about lysenin, see PCT publication No. WO 2013/153359, the entire contents of which are incorporated into the present invention by reference.

[0086] A polynucleotide pore is intended to mean a pore that is made from one or more nucleic acid polymers. A polynucleotide pore may include, for example, a polynucleotide origami.

[0087] A solid state pore is intended to mean a pore that is made from one or more materials of non-biological origin. “Solid state” refers to materials that are not of biological origin. A solid-state pore can be made of inorganic or organic materials. Solid-state pores include, for example, silicon nitride pores, silicon dioxide pores and graphene pores.

[0088] A hybrid solid state and biological pore is intended to mean a hybrid pore that is made of materials of biological and non-biological origin. Materials of biological origin are defined above and include, for example, polypeptides and polynucleotides. A solid-state and biological hybrid pore includes, for example, a polypeptide-solid-state hybrid pore and a polynucleotide-solid-state hybrid pore.

[0089] It should be noted that different types of nanopores may have different dimensions from each other in various aspects. For example, as illustrated in Figure 1A, the nanopore 100 can be characterized as having a first dimension H1 defining a nanopore thickness 100, e.g., a thickness between the outer surface of the first side 101 and an outer surface of the adjacent second side 102 to the opening 103. In embodiments wherein the nanopore 100 includes the constriction 104, the nanopore 100 may also be characterized as having a second dimension H2 defining a depth of constriction, e.g., a depth between the outer surface of the first side 101 and the narrowest part of the constriction 104 adjacent to the opening 103. The nanopore 100 can also be characterized as having a first diameter D1 defining an opening diameter 103, for example, an opening diameter 103 at the widest point of the opening. In embodiments where the nanopore 100 includes the constriction 104, the nanopore 100 may also be characterized as having a second diameter D2 defining a constriction diameter, for example, a constriction diameter 104 at the narrowest point of the constriction. It should be noted that such dimensions of the nanopore 100 should not be interpreted as being limiting, and that other dimensions of the nanopore 100 may be suitably defined. For example, the first dimension H1 of the nanopore 100 may vary along the lateral dimension, for example, if the nanopore 100 includes a relatively thin barrier, in which a relatively thick pore is disposed. Or, for example, in embodiments in which the nanopore 100 includes the constriction 104, the second H2 dimension of the nanopore 100 may vary depending on the relative location of the constriction 104 with respect to the outer surface of the first side 101. That is, the constriction 104 may be located disposed at any appropriate location in the nanopore 100 and, in fact, it may even be disposed distal to the outer surface of the first side 101 or the second side 102. The opening 103 and the constriction 104 need not necessarily be perfectly circular, and in addition Furthermore, they may be characterized as having an approximate diameter or using any other suitable dimensions. Furthermore, the nanopore 100 may include multiple constrictions, each of which is characterized using appropriate dimensions.

[0090] In some embodiments, the first H1 dimension of nanopore 100 is about 100 nm or smaller, or about 50 nm or smaller, or about 20 nm or smaller, or about 10 nm or smaller, or about 5 nm or smaller or about 2 nm or smaller. For example, H1 may be between about 2 nm and about 100 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 20 nm. In embodiments including constriction 104, the second H2 dimension of nanopore 100 is about 100 nm or smaller, or about 50 nm or smaller, or about 20 nm or smaller, or about 10 nm or smaller, or about 5 nm or smaller, or about 2 nm or smaller, or about 1 nm or smaller. For example, H2 may be between about 1 nm and about 100 nm, or between about 2 nm and about 50 nm, or between about 5 nm and about 20 nm. By way of illustration, H1 can be between about 5 nm and about 50 nm and H2 can be between about 1 nm and about 5 nm. In an exemplary embodiment, H1 is about 10 nm and H2 is about 5 nm. In another exemplary embodiment, H1 is about 10 nm and H2 is about 6 nm. In another exemplary embodiment, H1 is about 10 nm and H2 is about 7 nm. In another exemplary embodiment, H1 is about 10 nm and H2 is about 8 nm. In another exemplary embodiment, H1 is about 10 nm and H2 is about 9 nm. In another exemplary embodiment, H1 is about 10 nm and H2 is about 10 nm. In another exemplary embodiment, H1 is about 5 nm and H2 is about 2 nm. In another exemplary embodiment, H1 is about 5 nm and H2 is about 2 nm. In another exemplary embodiment, H1 is about 5 nm and H2 is about 2 nm. In another exemplary embodiment, H1 is about 5 nm and H2 is about 2 nm. The terms “approximately” and “about” are intended to mean within 10% above or below the stated value.

[0091] In some embodiments, the first diameter D1 of opening 103 of nanopore 100 is about 100 nm or smaller, or about 50 nm or smaller, or about 20 nm or smaller, or about 10 nm or smaller, or about 5 nm or smaller or about 2 nm or smaller. For example, D1 may be between about 2 nm and about 100 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 20 nm. In embodiments including constriction 104, the second diameter D2 of constriction 104 of nanopore 100 is about 100 nm or smaller, or about 50 nm or smaller, or about 20 nm or smaller, or about 10 nm or smaller, or about 5 nm or smaller, or about 2 nm or smaller, or about 1 nm or smaller. For example, D2 may be between about 1 nm and about 100 nm, or between about 2 nm and about 50 nm, or between about 5 nm and about 20 nm. Illustratively, D1 may be between about 5 nm and about 50 nm and D2 (if applicable) may be between about 1 nm and about 5 nm.

[0092] In an illustrative embodiment, D1 is about 5 to 10 nm and D2 is about 1 to 1.2 nm. In another illustrative embodiment, D1 is about 5 to 10 nm and D2 is about 1.2 to 1.4 nm. In yet another illustrative embodiment, D1 is about 5 to 10 nm and D2 is about 1.4 to 1.6 nm. In yet another illustrative embodiment, D1 is about 5 to 10 nm and D2 is about 1.6 to 1.8 nm. In yet another illustrative embodiment, D1 is about 5 to 10 nm and D2 is about 1.8 to 2.0 nm. In exemplary embodiments where the pore is MspA, D1 may be, for example, about 4.8 nm, D2 may be, for example, about 1.1 to 1.2 nm, H1 may be, for example, about 9.6 nm and H2 may be, for example, about 7.9 to 8.1 nm. In exemplary embodiments where the pore is α-hemolysin, D1 may be, for example, about 2.6 nm, D2 may be, for example, about 1.4 to 1.5 nm, H1 may be, e.g. about 10 nm and H2 can be, for example, about 5 nm. Other suitable combinations of dimensions can be appropriately selected for other pore types.

[0093] The characteristics of the permanent current 110 can be suitably selected based on one or more of the dimensions of the nanopore 100. For example, the elongated body 113 of the current 110 can have a width selected based on D1 or D2, or either D1 as in D2. For example, the width of the elongated body 113 may be selected so that the elongated body 113 is movable within the opening 103, for example, the elongated body 113 has a width that is smaller than the first diameter D1 of the opening 103. In In embodiments that include constriction 104, the width of the elongate body 113 may also be selected so that at least a portion of the elongate body 113 is movable adjacent to the constriction 104, e.g., that it has a width that is equal to or less than the second diameter D2. Optionally, in embodiments that include constriction 104, the width of the elongate body 113 may also be selected so that at least a portion of the elongate body 113 is movable through the constriction 104, e.g., has a width that is sufficiently smaller than the second diameter D2 to allow movement of the elongated body 113 through the constriction 104. If the nanopore 100 includes multiple constrictions (not specifically illustrated), then the width of the elongated body 113 may be selected such that the elongated body 113 is movable through of some or all of these constrictions, as appropriate.

[0094] The length of the elongated body 113 of the chain 110 can be selected based on H1 or H2, or based on H1 and H2. For example, the length of the elongated body 113 may be selected to be less than H1 so that that tail region 112 does not extend beyond the outer surface of the second side 102 of the nanopore 100 even if the elongated body 113 were fully extended through the constriction 104 toward the second side 102. Or, for example, in embodiments including the constriction 104, the length of the elongate body 113 may be selected to be shorter than H2, so that the tail region 112 does not overlap. extend beyond the constriction 104 of the nanopore 100 even if the elongated body 113 were fully extended toward the second side 103. In other embodiments, the length of the elongated body 113 may be selected to be longer than H1, so that tail region 112 could extend beyond the outer surface of the second side 102 of the nanopore 100 if the elongated body 113 were fully extended through the constriction 104 toward the second side 102. Or, for example, in embodiments including the constriction 104 , the length of the elongated body 113 can be selected to be longer than H2, so that the tail region 112 could extend beyond the constriction 104 of the nanopore 100 if the elongated body 113 were fully extended towards the second side 103.

[0095] The length of the elongated body 113 may be selected so as to permit relatively free movement of the elongated body 113 within the opening 103, at least on the first side 101 of the nanopore 100, substantially without steric hindrance or other interference caused by the elongated body in itself. That is, the elongated body 113 may be configured so as to occupy only a portion of the volume of the opening 103 in the first side 101 of the nanopore 100, for example, so as to occupy less than 50% of the volume of the opening 103 in the first side 101 of the nanopore 100, or less than 20% of the volume of the opening 103 in the first side 101 of the nanopore 100, or less than 10% of the volume of the opening 103 in the first side 101 of the nanopore 100, or less than 5% of the volume of the opening 103 on the first side 101 of the nanopore 100, or less than 1% of the volume of the opening 103 on the first side 101 of the nanopore 100. Furthermore, in the embodiment illustrated in Figures 1A to 1D, the tail region 112 of the chain 110 can be released from the nanopore 100 or any other member, thereby allowing relatively free movement of the entire elongated body 113 relative to the head region 111. Alternatively, in embodiments as described below with reference to Figures 4A to 4F, 5A to 5D and 6 , the tail region 112 can be linked to another member in order to inhibit the dissociation of the polymerase 130 from the nanopore 100.

[0096] In a non-limiting example, one or more reporter regions 114 may facilitate the measurement of translational, rotational or conformational movement or the presence (or a combination thereof) of the elongated body 113 responsive to polymerase 130 acting on nucleotide 120 or is responsive to a conformational change of polymerase 130 based on nucleotide 120 being complementary or noncomplementary to a next nucleotide in the sequence of the second polynucleotide 140, or responsive to both polymerase 130 acting on nucleotide 120 and a conformational change of polymerase 130 based on the nucleotide 120 being complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide 140. In an exemplary embodiment, the D1 dimension of the opening 103 is suitably selected in order to facilitate the use of the reporter region(s). es) to measure the movement of the elongated body 113. For example, the aperture 103 may be narrow enough to interact measurably with one or more reporter regions sensitive to the movement of the reporter region(s) 114. As an example, the reporter region 114 has an electrical or flow blocking characteristic, and the aperture 103 has a width selected such that movement of the reporter region(s) 114 causes a detectable change in current or flow through the aperture. opening 103 under a voltage applied across the nanopore 100. For example, nucleotides that are larger (such as A and G) may result in more blocking when they are arranged in an opening, for example, with an elongated tag disposed at the constriction of MspA. , compared to the T, which has a smaller elongated marker. Exemplary ranges of blocking currents or flows in terms of % open pore current or flow include 0 to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, from 60% to 70%, from 70% to 80%, from 80% to 90% and from 90% to 100%. In an exemplary embodiment, the range is between 20% and 70% for MspA in 300 mM KCl with a bias of 180 mV and a current or open pore flux of 110 pA. Other non-limiting examples of reporter regions 114 are provided in greater detail in the present invention.

[0097] Although Figure 1A illustrates an exemplary arrangement of the components of the nanopore 100, the polymerase 130 and the permanent current 110, it should be understood that other arrangements may be suitably used. For example, the polymerase 130 with the head region 111 anchored thereto may instead be disposed adjacent to the second side 102. Or, for example, the tail region 112 of the permanent chain 110 may instead be disposed on the first side 101 of the nanopore 100. Or, for example, the tail region 112 of the permanent current 110 may instead be anchored to the first side 101 or the second side 102 of the nanopore 100, or to another member that is placed on the first side 101 or the second side 102 of the nanopore 100. Some of such combinations of features are described in the present invention and in the provisional patent applications incorporated by reference in the present invention, but it should be noted that all such combinations of features are contemplated and can be readily envisioned based on the teachings in the present invention.

[0098] The lengths of the present elongated bodies may suitably vary, such that the present tail regions may be disposed at any suitable location relative to the nanopore 100. For example, the elongated body 113 does not necessarily need to be sufficiently long, so that the tail region 112 is disposed beyond the second side 102 of the nanopore 100 in the manner illustrated in Figures 1A to 1D. Instead, the elongated body 113 may be long enough so that the tail region 112 is disposed on the first side 101 of the nanopore 100, or it may be long enough so that the tail region 112 is disposed on the second side 102 of the nanopore 100, but not beyond the second side 102. Furthermore, the tail regions present do not necessarily need to extend freely, but instead, they can be attached to any appropriate member.

[0099] Furthermore, it should be noted that any suitable type of nanopore 100, any suitable type of polymerase 130 and any suitable type of permanent current 110 can be used in the embodiments provided herein, for example, as illustrated in Figures 1A to 1D. For example, as noted above, the nanopore may include a biological pore, a solid-state pore, or a hybrid solid-state and biological pore. For example, nanopore 100 may include one or more solid state materials. Exemplary solid-state materials suitable for use in a solid-state nanopore include silicon (Si), silicon nitride (SiN or SiNx), graphene, and silicon oxide (SiO2 or SiOx). For more details on solid-state nanopores, see the following references, the entire contents of which are incorporated by reference in the present invention: Dekker, “Solid-state nanopores,” Nature Nanotechnology 2: 209-215 (2007); Schneider et al., “DNA Translocation through Graphene Nanopores,” Nano Letters 10: 3163–3167 (2010); Merchant et al. Nano Letters 10:2915-2921 (2010); and Garaj et al., “Graphene as a subnanometre trans-electrode membrane,” Nature 467: 190–193 (2010). Biological pores include, for example, polypeptide pores and polynucleotide pores. The biological pores can be arranged within a barrier that includes a membrane of biological origin, or a solid-state membrane. Non-limiting examples of biological nanopores include MspA and alpha-hemolysin. Exemplary membranes of biological origin include lipid bilayers. Exemplary solid-state membranes include silicon and graphene. For further details on exemplary hybrid nanopores and their preparation, see the following references, with the entire contents of each being incorporated by reference into the present invention: Hall et al., “Hybrid pore formation by directed insertion of alpha hemolysin into solid-state nanopores,” Nature Nanotechnology 5: 874–877 (2010), and Cabello-Aguilar et al., “Slow translocation of polynucleotides and their discrimination by α-hemolysin within a single track-etched nanopore designed by atomic layer deposition,” Nanoscale 5: 9582–9586 (2013).

[0100] In some embodiments, as described in greater detail below with reference to figures 7A to 7D and 8, the polymerase 130 illustrated in figures 1A to 1D can be optionally modified in order to delay one or more reaction parameters during use of the composition illustrated in Figures 1A to 1D to sequence a polynucleotide. For example, polymerase 130 may be optionally modified in order to delay the release of pyrophosphate responsive to the incorporation of nucleotide 130 into the first polynucleotide 140.

[0101] Furthermore, it should be noted that a head group of a chain can be linked to a polymerase in different ways. For example, a well-known bioconjugate chemistry, such as that described by Hermanson, mentioned above, can be used. In illustrative embodiments, the polymerase includes a chemical moiety for forming a bond, such as a cysteine, or a linker peptide, such as a SpyTag. Further information regarding spytags and their use to form links can be found, for example, in the following references, the entire contents of each of which are incorporated by reference in the present invention: Zakeri et al., “Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin,” Proc. Nat. Acad. Sci. USA 109: E690-E697 (2012), and Fierer et al., “SpyLigase peptide-peptide ligation polymerases affibodies to enhance magnetic cancer cell capture,” Proc. Nat. Acad. Sci. USA 111: E1176-E1181 (2014). Other exemplary ligands include: NHS-esters, isocyanates and isothiocyanate ligand conjugation to amines, maleimides to cysteines, click chemistry with azides to alkynes, use of fusion tags such as Halotag, Spycatcher-Spytag and other protein-bioconjugation methods. similar proteins. For additional information on exemplary ligands that may be used, see the following references, the entire contents of each of which are incorporated by reference into the present invention: Hermanson, Bioconjugate Techniques, 2nd Ed., Elsevier, 2008; and Liu et al., “Specific Enzyme Immobilization Approaches and Their Application with Nanomaterials,” Topics in Catalysis 55(16-18): 1146-1156 (2012).

[0102] Exemplary systems for sequencing polynucleotides using polymerase-anchored chains adjacent to nanopores will now be described with reference to Figures 2A and 2B. Figure 2A schematically illustrates the system 270 including a composition as illustrated in Figures 1A to 1D and the measurement circuit (e.g., electrical or optical measurement circuit) configured to measure a state of current or flow based on one or more in both the binding of the first portion 115 of an elongated body 113 to the elongated marker 121 of the nucleotide 120 upon which the polymerase 130 is acting, and an indication of one or more reporter regions 114 when that nucleotide is complementary or non-complementary to a next nucleotide in the sequence of a second polynucleotide (second polynucleotide not specifically illustrated in Figure 2A, but may be configured in an analogous manner as shown in Figures 1A to 1D). System 270 includes nanopore 100, permanent current 110, nucleotide 120, polymerase 130, and measurement circuit 230. Nanopore 100 includes first side 101, second side 102, opening 103, and constriction 104. permanent chain 110 includes the head region 111, the tail region 112, and the elongated body 113. The nucleotide 120 includes the elongated marker 121 and the second portion 122. The polymerase 130 may be configured in a manner analogous to as described in another section in the present invention. Furthermore, the elongated body 113 may include the first portion 115 that is configured to bind the elongated marker 121 of the nucleotide 120 on which the polymerase 130 is acting, as well as reporter region(s) 114 configured to indicate when nucleotide 120 is complementary or non-complementary to a following nucleotide in the sequence of a second polynucleotide (not specifically illustrated in Figure 2A, but may be configured in a manner analogous to as described above with reference to Figures 1A to 1D).

[0103] In an illustrative example, the nanopore 100, the chain 110, the nucleotide 120, the polymerase 130 and the first and second polynucleotides, as illustrated in Figures 1A to 1D, can be immersed in a conductive fluid, e.g. an aqueous saline solution. The measurement circuit 230 may be in communication with the first electrode 231 and the second electrode 232, and may be configured to apply a voltage between the first electrode 231 and the second electrode 232 in order to impose a voltage across the nanopore 100. A direct current (DC) or alternating current (AC) voltage can be appropriately used. In some embodiments, the measuring circuit 230 may be further configured to use the first electrode 231 and the second electrode 232 to measure the magnitude of a current or flow through the opening 103. In some embodiments, the measuring circuit 230 may additionally include an optical, biological or chemical sensor respectively configured to optically, biologically or chemically sense the magnitude of a molecular flow through aperture 203. Exemplary optical sensors include CCDs and photodiodes. In some embodiments, the measurement circuit includes one or more agents that react, chemically or biologically, with molecular flow through aperture 103 to generate an optically detectable signal. Exemplary signals (e.g., optical or electrical signals) that can be generated using the system 270 illustrated in Figure 2A are described below with reference to Figures 4F and 6.

[0104] The first portion 115 of the elongated body 113 of the chain 110 may be configured to generate at least one polymerase-responsive signal 130 acting on the nucleotide 120, the nucleotide 120 being identifiable based on such signal. For example, as described above with reference to Figure 1B and as illustrated in Figure 2A, the first portion 115 may interact, for example by binding, for example, hybridizing with the second portion 122 of the elongated label 121. Binding of the first portion 115 to second portion 122 may define a duplex that is large enough so that it cannot pass through the constriction 104 of the nanopore 100 and, instead, which may lie within or adjacent to the constriction 104 under the voltage that the first electrode 231 and the second electrode 232 apply through the nanopore 100. The portion 122 of the elongated marker 121 may be selected so that the nucleotide 120 is identifiable based on the state of current or flow through the opening 103 when the duplex is housed within or adjacent to constriction 104. For example, each different type of nucleotide 120 (e.g., A, C, T, or G) may include a respective different elongated marker 121 than the other, e.g., each includes a different second portion 122 that binds to a different portion of the first portion 115 of each other. In a non-limiting example, the elongated marker 121 includes a first nucleotide sequence, and the first portion 115 includes a second nucleotide sequence that is complementary to the first nucleotide sequence. Measuring circuitry 230 may be configured to measure a corresponding current or flow state through opening 103. Such current or flow state may be based on the elongated marker, the nucleotide 120 being identifiable based on such current or flow state. .

[0105] For example, the portion 122 of the elongated marker 121 of a first nucleotide 120 (e.g., one of A, C, T or G) may include a different nucleotide sequence relative to the portion 122 of the elongated marker 121 of a second nucleotide 120 (e.g., another from A, C, T, or G) and a nucleotide sequence other than the portion 122 of the elongated marker 122 of a third nucleotide 120 (e.g., yet another from A, C, T, or G), and a nucleotide sequence other than the portion 122 of the elongated marker 122 of a fourth nucleotide 120 (e.g., the remainder of A, C, T or G). Illustratively, each of the different portions may bind (e.g., hybridize) with a different portion of each other's first portion 115 and, when the resulting duplex becomes housed within or adjacent to the constriction 104 (or otherwise, when interacting with constriction 104), results in a current or flow state different from each other. Additionally or alternatively, housing the resulting duplex in or adjacent to the constriction 104 (or otherwise interacting with the constriction 104) may cause the reporter region(s) 114 to be disposed within of opening 103 result in a current or flow state different from the other duplexes and that correspond to that nucleotide. Therefore, based on the current or state of flow through the opening corresponding to the elongated marker 122 of the particular nucleotide 120 under the action of the polymerase 130, such a nucleotide is identifiable.

[0106] The reporter region(s) 114 may have a suitable physical, chemical, optical, electrical, biological or other flow-blocking property different from that of one or more other regions of the elongate body 113 , and may be configured to identify nucleotide 120 or to indicate when nucleotide 120 is under the action of polymerase 130 or is complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide (not specifically shown in Figure 2A, but may be configured as illustrated in Figures 1A to 1D), or both, to identify nucleotide 120 and to indicate when nucleotide 120 under the action of polymerase 130 is complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide. For example, the reporter region(s) may provide or generate one or more unique current or flow signatures (e.g., signal states) that individually identify the nucleotide under action, or individually indicate whether that nucleotide is complementary or not complementary to the next nucleotide of the second polynucleotide, or both individually identify the nucleotide under the action and individually indicate whether that nucleotide is complementary or not complementary to the next nucleotide of the second polynucleotide. For example, the reporter region(s) may generate a signal state (e.g., current or flow state) responsive to some or all of the nucleotides 120 being bound by, or residing in, m) in an active site of the polymerase 130, the nucleotide 120 being complementary or non-complementary to a following nucleotide in a polynucleotide being sequenced, or the polymerase 130 successfully incorporating the nucleotide 120 into the second polynucleotide at a position that complements the following nucleotide ( not specifically shown in Figure 2A, but can be configured as illustrated in Figures 1A to 1D).

[0107] For example, a magnitude or duration of the conformational change of polymerase 130 may be responsive to nucleotide 120 being complementary or non-complementary to the next nucleotide of the second polynucleotide, and a magnitude or duration of time, or both, of corresponding signal may be based on the conformational change of the polymerase 130. Illustratively, in some embodiments, the measurement circuit 130 may be configured to detect the position of the reporter region(s) 114 relative to the constriction 104, the position of which may indicate when the nucleotide on which polymerase 130 is acting is complementary or not complementary to the next nucleotide in the sequence of the second polynucleotide. For example, the position of the reporter region(s) 114 may be based on a conformational change of polymerase 130 that may occur when polymerase 130 successfully adds nucleotide 120 to the first polynucleotide in a manner as described. in other sections of the present invention. Furthermore, as noted in other sections of the present invention, in some embodiments, the position of the reporter region(s) 114 relative to the constriction 104 may be based on the identity of the nucleotide 120. Therefore, a A particular signal state (e.g., a current or flow state) that can be measured (e.g., optically or electrically) by system 270 may indicate one or both of the identity of nucleotide 120 and whether or not nucleotide 120 is complementary to a following nucleotide in a polynucleotide being sequenced.

[0108] As another example, the reporter region 114 may have a different physical property than some or all of the other regions of the elongate body 113. For example, the reporter region 114 may cause a differential blocking current or flow through the opening 103 in comparison with other regions of the elongate body 113. Additionally or alternatively, the reporter region 114 may have a different electrical or flow blocking property than some or all of the other regions of the elongate body 113. For example, the reporter region 114 may include an electrostatic charge, while some or all other regions of the elongated body 113 may include a different electrostatic charge, or may be discharged (e.g., may be electrically neutral). Or, for example, the reporter region 114 may be discharged, while some or all of the other regions of the elongate body 113 may include an electrostatic charge. Or, for example, reporter region 114 may have a physical property. Physical properties include the volume and shape of the reporter region 114. In an illustrative example, movement of the reporter region 114 within the opening 103 causes a measurable change in current or flow through the opening, or an optional constriction 104 thereof. by modulating a flow or blocking current through the opening or constriction. Or, for example, reporter region 114 may have a chemical or biological property that facilitates chemical or biological detection. Chemical or biological properties include the presence of a chemical or biological group, for example, a radioactive group or a group with enzymatic activity.

[0109] One or more electrical, physical, chemical, optical, biological or other flow-blocking properties of the reporter region may cause a measurable change in current through the opening 103 or constriction 104, a measurable change in the flow of molecules through the opening 103 or constriction 104, or an optical signal. In an illustrative example, the movement or presence of the reporter region 114 within the opening 103 causes a measurable change in a current through the opening 103 or constriction 104, or causes a measurable change in the flow of molecules through the opening 103 or constriction 104. , wherein the change in flow may be electrically, chemically, biologically, or optically detectable. For example, the presence or movement of the reporter region 114 within the opening 103 or the constriction 104 can cause an ionic current block or a molecular flow block, which can be detected optically, electrically, chemically, or biologically. Illustratively, a gradient of a molecule on the trans side can create a natural molecule flow that can be partially blocked by the reporter region 114. The measurement circuit 130 can be configured to measure such molecular flow non-electrically (e.g., optically). using streams of luminescent (e.g., fluorescent or chemiluminescent) molecules, or streams of reactants that become chemiluminescent in the presence of other reactants. For example, Ca2+ can flow from one side of the nanopore to the other side, where it encounters a sensitive dye to calcium, such as Fluo-2, Fluo-4, Fluo-8 or similar, to induce fluorescence. Other pairs of reagents that can be used include, but are not limited to, luminol and oxidants, calcium and aequorin, or ATP and oxidants. luciferase, to name a few. For more details regarding the optical detection of molecular flows through an opening or constriction, see Ivankin et al., “Label-Free Optical Detection of Biomolecular Translocation through Nanopore Arrays,” ACSNano 8(10) : 10774-10781 (2014), the entire contents of which are incorporated by reference in the present invention.

[0110] In non-limiting embodiments, as described in greater detail below with reference to Figures 4A to 4F, 5A to 5D and 6, application of a sufficiently high voltage across the nanopore 100 can cause the first portion 115 and the second portion 122 that form a duplex dissociate from each other, after which one or more reporter regions 114 may be arranged in a position within the opening 103 that is based on the conformational state of the polymerase 130. In this sense, based on a first state signal state, nucleotide 120 may be identifiable, and a second signal state may indicate the conformational state of polymerase 130, which may indicate when nucleotide 120 is complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide. The voltage across the nanopore 100 can be repeatedly reversed in order to facilitate repeated binding of the first portion and the second portion to each other, followed by measurement of the first signal state, followed by dissociation of the first portion and the second portion from each other. , followed by measuring the second signal state, for a desired number of times, in order to identify the nucleotide 120 on which the polymerase 130 is acting and to determine whether the nucleotide 120 is complementary or not complementary to a next nucleotide in the sequence of the second polynucleotide with the desired precision.

[0111] In this regard, note that previously known real-time single-molecule sequencing technologies are relatively error-prone and have relatively poor accuracy as these technologies cannot sufficiently distinguish whether a nucleotide being triggered is complementary or non-complementary. to a next nucleotide in the sequence of a second polynucleotide, or if such a nucleotide is instead a member of an “incompatibility” complex with the polymerase and the primed nucleic acid. In comparison, the present compositions, systems and methods can identify a nucleotide that is being triggered, for example, based on a first state of the signal, and can also determine whether the nucleotide is complementary or non-complementary to a following nucleotide in the sequence. of a second polynucleotide (i.e., a match), based on a second signal state, and can therefore greatly increase the accuracy of polynucleotide sequencing. It should be noted that other schemes for generating signal states indicating whether or not the complementary nucleotide is complementary to a following nucleotide in the sequence of a second polynucleotide, such as is presented herein, can analogously increase the accuracy of polynucleotide sequencing. .

[0112] For example, each of the one or more reporter regions 114 may have a physical property different from some or all of the other regions of the elongate body 113. In some embodiments, the reporter region(s) 114 may cause a differential blocking current or flow through the opening 103 compared to other regions of the elongate body 113. Additionally, or alternatively, the reporter region/regions 114 may have a blocking property. different electrical or flux than some or all other regions of the elongated body 113. For example, the reporter region(s) 114 may include an electrostatic charge, while some or all of the other regions of the elongated body 113 may include a different electrostatic charge, or may be discharged (e.g., may be electrically neutral). Or, for example, the reporter region(s) 114 may be discharged, while some or all of the other regions of the elongate body 113 may include an electrostatic charge. The state (e.g., magnitude) of the current or flow through the opening 103 may be based on the position of the reporter region(s) 114 within the opening 103, which may be based on the conformational state of the polymerase. 130, and the time period for the current or flow state may be based on the duration of the change in position of one or more reporter regions. In a non-limiting illustrative example, the elongated body 113 includes a polynucleotide that includes one or more abasic nucleotides that define one or more reporter regions 114.

[0113] In an illustrative embodiment, nanopore 100 is a biological nanopore. Non-limiting examples of biological nanopores include MspA and alpha-hemolysin. The reporter region 114 of the chain 111 may include one or more basic residues configured to be positioned within or adjacent to one or more constrictions 104 of the biological nanopore. Movement of one or more suitably positioned abasic residues through a constriction of any pore can result in a readily detectable signal, for example, in a detectable change in current or flow through the constriction/constrictions 104. Biological nanopores, such as MspA and alpha-hemolysin may usefully include constrictions that may serve to concentrate the effect of the reporter region(s) 114. For example, MspA includes a single constriction with a diameter of approximately 1.2 nm and a length of approximately 0.5 nm, can provide adequate spatial resolution because the magnitude of flow blockage or ionic current through the constriction is mainly based on the elongated body segment threaded into the narrow region (constriction) of the nanopore.

[0114] In an illustrative embodiment, one or more reporter regions 114 include an electrostatic charge, and each of the first and second signal states generated by system 270, which are respectively responsive to the polymerase acting on nucleotide 120 (for the first signal state) and indicate when nucleotide 120 is complementary or non-complementary to the next nucleotide of the second polynucleotide (for the second signal state) includes a state of current or flow through constriction 104. However, it should be understood that the measurement circuit 230 may include, or be in communication with, any element or combination of elements that facilitates measurement of any suitable reporter region(s) and need not necessarily be based on measurement of current or flow through constriction 104 or even in the movement of the reporter region(s). Furthermore, the reporter region(s) 114 need not necessarily be linked to chain 110 and, instead, they may bind to a nucleotide 120 or other driven molecule, or may be linked to polymerase 130. For example, U.S. Patent Application Publication No. 2011/0312529, to He et al., the contents of which are incorporated by reference in the present invention, discloses exemplary modified polymerases that can indicate a conformational state of the polymerase, for example, being conformationally marked.

[0115] The particular properties of the one or more reporter regions, e.g., of the reporter region(s) 114 or of a reporter region located elsewhere in the composition and system, may be selected based on the configuration particular system 270 in order to facilitate the measurement of that reporter region(s). For example, one or more of the reporter regions may have an optical property, and the system 270 may include, or be in communication with, an optical sensor configured to measure the optical property and generate one or more signal states based on the identity of the nucleotide 120 or when the nucleotide 120 on which the polymerase 130 is acting is complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide (not specifically illustrated in Figure 2A, but may be configured as described with reference to Figures 1A to 1D) or based on both the identity of the nucleotide 120 and whether the nucleotide 120 on which the polymerase 130 is acting is complementary or non-complementary to a following nucleotide in the sequence of the second polynucleotide. In an illustrative embodiment, the reporter region(s) 114 or other reporter region disposed elsewhere in the composition and system may include a first FRET pair partner, e.g., a FRET donor or acceptor. , which interacts with a corresponding second FRET pair partner, e.g., with a FRET acceptor or donor, so as to emit light of a particular wavelength that measurement circuit 230 is configured to detect. Or, for example, the reporter region(s) 114 or other reporter region disposed elsewhere in the composition and system may have a chemical or biological property, and the system 270 may include, or be in communication with a chemical or biological sensor configured to measure the chemical or biological property, and this generates a second signal state when the nucleotide 120 on which the polymerase 130 is acting, is complementary or is not complementary to a following nucleotide in the sequence of second polynucleotide (not specifically illustrated in Figure 2A, but may be configured as described with reference to Figures 1A to 1D). As another example, the reporter region can provide a molecular flow block that modulates the flow of molecules through the opening or constriction, which flow can be detected optically, electrically, chemically or biologically.

[0116] In a non-limiting example, measurement circuit 230 is configured to measure a state of the signal generated by reporter region(s) 114 responsive to the release of pyrophosphate or other phosphorus oxyanion responsive to polymerase 130 successfully incorporating nucleotide 120 into the first polynucleotide (not specifically illustrated in Figure 2A, but can be configured as described with reference to Figures 1A to 1D). For example, the reporter region(s) 114 may be configured to bind pyrophosphate that is released in response to polymerase 130 successfully incorporating nucleotide 120 into the first polynucleotide in a manner analogous to described below, with reference to figures 11A to 11C.

[0117] In this regard, note that polymerase 130 can be optionally modified in order to delay the release of the pyrophosphate responsive to the incorporation of nucleotide 120 into the first polynucleotide. For example, in some embodiments, polymerase 130 may include a polymerase Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp- 7, PR4, PR5, PR722 or modified recombinant L17. In some embodiments, the polymerase 130 may include a modified recombinant DNA polymerase Φ29 having at least one amino acid substitution or combination of substitutions selected from the group consisting of: an amino acid substitution at position 484; an amino acid substitution at position 198 and an amino acid substitution at position 381. In some embodiments, the polymerase 130 may include a modified recombinant DNA polymerase Φ29 having at least one amino acid substitution or combination of substitutions selected from the group consisting of E375Y, K512Y , T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W and L381A. For more details on exemplary modified polymerases that can delay the release of pyrophosphate responsive to the incorporation of a nucleotide into a polynucleotide, see U.S. Patent No. 8,133,672 to Bjornson et al., the contents of which are incorporated into the present invention by reference.

[0118] In another non-limiting example, the measurement circuit 230 is configured to measure a state of the signal generated by the reporter region(s) 114 responsive of the elongated marker 121 responsive to the polymerase 130 successfully incorporating nucleotide 120 in the first polynucleotide (not specifically illustrated in Figure 2A, but may be configured as described with reference to Figures 1A to 1D). For example, the reporter region(s) 114 may be configured to bind to a portion of the elongated tag 121 that is released in response to polymerase 130 successfully incorporating nucleotide 120 into the first polynucleotide. Illustratively, if an allosteric aptamer that can recognize newly cleaved pyrophosphate and undergo a conformational change to expose an oligonucleotide is positioned at the linker portion of the tag, this aptamer can expose an oligonucleotide that binds to the chain upon successful incorporation. . This binding can create a new state of the removal signal. For example, in a non-limiting embodiment, the elongated marker linked to the nucleotide may include two portions capable of hybridizing to the chain in the manner shown in Figures 11A to 11C. A portion like this can be close to the phosphates and, therefore, can be kept away from the current and close to the active site of the polymerase. This second portion may be distal to the nucleotide and may be capable of binding to the chain while the nucleotide is in the active site of the polymerase. A first removal signal can be created by the distal portion, as long as the nucleotide is maintained in the active site and the voltage cycle (CA) is occurring as described in another section in the present invention. This signal from the distal portion may be indicative of the type of nucleotide in the active site. Based on the nucleotide leaving the active site without phosphate cleavage and incorporation, the proximal portion can hybridize with the chain and can create a removal signal that is affected by virtue of its remaining attachment to the nucleotide. Such a state of the signal may be indicative of the non-incorporation of the nucleotide. Based on phosphate cleavage and incorporation occurring, the proximal portion may hybridize to the chain and may create a different removal signal as long as the nucleotide is no longer present. Such a signal state may be indicative of nucleotide incorporation and phosphate cleavage.

[0119] It should be noted that a nanopore array can be provided to sequence a plurality of polynucleotides in parallel with each other. For example, Figure 2B schematically illustrates a plan view of a system 260 including a measurement circuit 240 configured to measure the movement of one or more respective reporter regions within respective openings of a nanopore array. A plurality of systems 250, each of which may be configured analogously to the system 270 described above with reference to Figure 2A, may be integrally disposed on a common substrate as each other, or may be separately prepared and disposed adjacent to each other. other. Each system 250 may include the nanopore 100, a current (current not specifically illustrated), and an addressable electrode 241. The measurement circuit 240 may be configured in a manner analogous to the measurement circuit 230 and may be in electrical communication with each addressable electrode 241. of each system 250 through a suitable communication path, e.g., a conductor (communication illustrated for only a single system 250) and with a common electrode 242. The measurement circuit 240 can be configured to selectively apply a voltage across each nanopore 100 by applying a voltage across the addressable electrode 241 of that nanopore and across the common electrode 242, and to selectively measure a current or flow through that nanopore at the applied voltage. Analogous arrays can be readily envisioned for other types of detection systems, for example, light, chemical or biological detection systems.

[0120] Some exemplary methods for sequencing polynucleotides, and exemplary compositions that can be used during such methods, will now be described. In one aspect, a method includes providing a nanopore including a first side, a second side, and an opening extending through the first and second sides; providing a plurality of nucleotides, each of the nucleotides comprising an elongated tag; and providing the first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide. The method may also include providing a polymerase disposed adjacent the first side of the nanopore, with the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide, wherein the polymerase is anchored to a permanent chain including a head region, a tail region, and an elongated body arranged between them, with the elongated body occurring at the opening of the nanopore. The method may also include determining that a first nucleotide is being activated by the polymerase based on binding of the elongated tag to a first portion disposed in the elongated body; and with one or more reporter regions disposed in the elongated body, indicating when the first nucleotide is complementary or not complementary to a following nucleotide in the sequence of the second polynucleotide.

[0121] For example, Figure 3 shows an illustrative method 300 for sequencing a polynucleotide using a composition including a chain anchored to a polymerase adjacent to a nanopore. Method 300 includes providing a nanopore, including a first side, a second side, and an opening extending through the first and second sides (step 301). The nanopore may have any suitable configuration, for example, as described above with reference to Figures 1A to 1D. For example, the nanopore 100 illustrated in Figure 1A includes the first side 101, the second side 102, and the opening 103 extending through the first and second sides.

[0122] Step 301 may also, but does not necessarily include, preparing the nanopore. For example, step 301 may include defining a barrier and disposing a nanopore on or in the barrier. Methods for preparing nanopores are known in the art. For example, illustrative methods of preparing an MspA nanopore can be found in Butler et al, “Single-molecule DNA detection with an engineered MspA protein nanopore,” Proc. Natl. Academic. Sci. 105: 20647-20652 (2008), the entire contents of which are incorporated by reference in the present invention. Or, for example, illustrative methods of preparing an alpha-hemolysin nanopore can be found in Howorka et al, “Sequence-specific detection of individual DNA strands using engineered nanopores,” Nature Biotechnology 19: 636-639 (2001), and in Clarke et al., “Continuous base identification for single-molecule nanopore DNA sequencing,” Nature Nanotechnology 4: 265-270 (2009), the entire contents of both documents being incorporated by reference in the present invention.

[0123] The method 300 illustrated in Figure 3 also includes providing a plurality of nucleotides, wherein each of the nucleotides includes an elongated label (step 302). For example, Figure 9A schematically illustrates an exemplary nucleotide 920 including an elongated tag 921 including a second portion 922 that interacts with the first portion 115 of chain 110 described in Figures 1A to 1D during use in detecting a nucleotide at which the polymerase 130 is acting. In the non-limiting example illustrated in Figure 9A, the elongated marker 921 of exemplary nucleotide 920, e.g., T, may include an oligonucleotide moiety 922 linked to the gamma-phosphate of nucleotide 920, e.g., via a delta-phosphate bond. The oligonucleotide portion 922 may include any suitable nucleotide sequence selected to hybridize to a corresponding sequence of nucleotides within portion 115 of chain 110. For example, the oligonucleotide portion 922 illustrated in Figure 9A may include the exemplary sequence 5' GCAT 3 ', and portion 115 may include the complementary sequence 5' ATGC 3'.

[0124] Each different type of nucleotide may include a corresponding elongated marker that is linked to its gamma phosphate in a manner analogous to that illustrated in Figure 9A, or linked in another suitable way. For example, Figures 9B and 9C schematically illustrate exemplary nucleotides, including elongated markers including respective portions that interact with an exemplary chain during use in sequencing a polynucleotide. As illustrated in Figure 9B, nucleotides A, T, C and G may respectively include elongated markers that include different portions of each other, for example, as represented by triangle, diamond, square and circle respectively. Particular parts can be suitably selected in order to interact with, for example, hybridize with a corresponding portion of the permanent current, and to induce different conformational changes respective to the current. Figure 9C illustrates non-limiting examples of the portions that may be included in the elongated markers illustrated in Figure 9B. Each such moiety may interact, for example, hybridize with a different respective portion of a corresponding portion of the permanent current, in order to induce a different respective conformational change of the current.

[0125] For example, Figure 9D schematically illustrates an exemplary chain that includes the head region 911, the tail region 912 and the elongated body 913 that includes one or more reporter region 914 and the portion 915. The head region 911 may include a chemical linker such as a 3' maleimide group (“Mal”) for conjugation with a cysteine (Cys) residue in the pore. The tail region 912 may include a 5' phosphate group (“phos”) that is charged and thus assists with the delivery of current through the opening of the pore responsive to an applied voltage. The elongate body 913 may include a polymer, for example, a polynucleotide, as illustrated in Figure 9C, or any other suitable polymer, such as a biological polymer or a synthetic polymer. Reporter region 914 includes one or more abasic nucleotides denoted as “X” and, in an exemplary embodiment, may be located about 14 to 15 bases from the maleimide, which may be approximately the H2 distance from the mouth of the pore to the constriction of the pore for certain types of nanopores. Portion 915 may include a nucleotide sequence, for example, GGGTATAT, with which each of the nucleotide-linked portions A, T, C, and G to be activated may interact, for example, hybridize differently than to each other.

[0126] As defined herein, different types of interactions between a portion of a permanent chain bound to a polymerase and portions of the nucleotides being triggered can generate signal states based on which the nucleotide can be identified or based on which it can be determined whether that nucleotide is complementary to a following nucleotide in a polynucleotide being sequenced. Figures 10A and 10B schematically illustrate the movement of an exemplary chain responsive to hybridization with a portion of an elongated marker of an exemplary nucleotide during use in sequencing a polynucleotide, in accordance with some embodiments of the present invention. The head region 1011 of the chain is anchored in the polymerase 1030 that is disposed adjacent to the nanopore that optionally includes the constriction 1004, and the elongated body 1013 extends through the opening 1003, for example, so that the reporter region 1014 10A and 10B, the elongated body 1013 includes a polynucleotide, such as an ssDNA, the nucleotides of which are represented by horizontal bars 1016. The reporter region 1014 includes one or more abasic sites of the polynucleotide, e.g. ssDNA. Portion 1015 includes a nucleotide sequence that is selected to hybridize with a corresponding portion, e.g., a complementary nucleotide sequence of an elongated tag of a nucleotide being driven by polymerase 1030 (the nucleotide being driven and its elongated tag are not specifically illustrated in figures 10A and 10B).

[0127] As illustrated in Figure 10A, prior to binding of portion 1015 to the corresponding portion of the nucleotide being triggered, nucleotides 1016 are spaced from each other by approximately 5 angstroms. Furthermore, the 1030 polymerase may have a conformational state that corresponds to a nucleotide not bound by or not resident in an active state of the polymerase. As illustrated in Figure 10B, responsive to the portion 1015 that interacts with, for example, hybridizes with the corresponding portion of the nucleotide being driven by, for example, responsive to the portions that form a double-stranded DNA duplex, the spacing between the nucleotides 1016 at portion 1015 it decreases to about 3.3 angstroms. The change in chain length can be induced by the creation of double-stranded DNA (dsDNA) from the ssDNA that is respectively included in the 1015 portion and the tag portion of the nucleotide being activated. For example, ssDNA is longer than dsDNA by about 1.5 angstroms per nucleotide, which is within the resolution limits of the present systems, e.g., the 270 system illustrated in Figure 2A or the 260 system illustrated in Figure 2C. . Each nucleotide may include a corresponding oligonucleotide portion that is selected to create a different length of dsDNA upon hybridization of that portion to the 1015 portion, thereby shortening the chain by a distance that corresponds to the nucleotide being driven by the 1050 polymerase. The formula for the amount of shortening of a fully stretched chain, as a chain stretched across the pore responsive to an applied voltage, can be expressed as: where N is the number of bases that are hybridized, Ds is the distance by which the chain is shortened, Lss is the length between nucleotides in ssDNA (approximately 5 Angstroms), and Lds is the length between nucleotides in dsDNA (approximately 3, 3 Angstroms). The short black vertical line in Figure 10B indicates a 5-base hybridization event, which shortens the chain and moves the 1014 reporter region to a new location. Duplexes of 6, 7 or 8 bases can be even shorter than what is shown in Figure 10B, for example, as shown in Figures 9C and 9D.

[0128] Furthermore, based on whether or not the nucleotide being triggered is complementary to the following nucleotide in a polynucleotide being sequenced (polynucleotide not specifically illustrated), the conformation of the polymerase 1030 may change. For example, as illustrated in Figure 10B, the polymerase 1030 may have a modified conformation 1030' based on the nucleotide being a match to the next nucleotide in the sequence of the polynucleotide being sequenced, which causes the reporter region(s) (s) 1014 are arranged at a location within the opening 1003 that results in a single current or flow through the opening 1003, based on which it can be determined whether the nucleotide is a match. In comparison, the 1030 polymerase may have a different modified conformation (e.g., the 1030” conformation, as illustrated in Figure 10C), based on the nucleotide not being a match to the next nucleotide in the sequence of the polynucleotide being sequenced, which causes the reporter region(s) 1014 to be arranged at a location within the opening 1003 that results in a unique current or flow through the opening 1003, based on which it can be determined whether that nucleotide is a non correspondence. In this regard, note that the state of the particular signal (e.g., the current or flow state) can indicate both the identity of the nucleotide and whether that nucleotide is a match or not. Therefore, it must be understood that different signal states, which may represent the identities of the nucleotides or whether those nucleotides are a match or a mismatch, need not necessarily occur at different times relative to one another and, in fact, they may occur at the same time as one another. For example, a measured signal (e.g., optical or electrical) may include a composite of more than one signal state (e.g., two signal states), wherein each of the signal states provides information about one or more of a nucleotide identity or whether such nucleotide is a match or not.

[0129] Note that the portions illustrated in Figures 9A to 9D are intended to be purely exemplary and not limiting of the invention. However, the nucleotide-linked portions to be activated may be selected to satisfy one or more of the following parameters and, optionally, all of the following parameters: 1. Portions linked to different types of nucleotides than one another may interact with the portion of the current so as to be distinguishable from each other, for example, by measuring the current or flow through the pore constriction. 2. The stability of a duplex between the nucleotide portion and the corresponding portion of the chain is sufficiently low that such portions bound to “free” nucleotides (nucleotides that are not being acted upon by the polymerase and thus interact transiently with current) interact only briefly with the current portion, e.g., for less than 1 msec. For example, the stability of the duplex between the nucleotide portion and the chain portion can be expressed as the Tm, or melting temperature of the duplex. The operating temperature of the system is expected to be about 20°C, or room temperature. Moieties that are about 5 to 8 nucleotides in length are expected to have Tm < 12°C, which may provide sufficiently low stability at room temperature that the moieties bound to the “free” nucleotides will interact only briefly with the current portion. 3. The stability of a duplex between the nucleotide portion and the corresponding portion of the chain is sufficiently high that when the nucleotide is triggered and held in place by the polymerase for the several milliseconds (1 to 30 ms, for example) during incorporation, such action increases the effective concentration of the nucleotide portion relative to the chain portion, which drives the reaction between the portions forward and increases stability, so that the effective Tm of the duplex is greater than 20 °C (or the expected operating temperature of the system), for example, is greater than 30°C, greater than 40°C, or greater than 50°C. 4. The length of the elongated tag of the nucleotide being driven, for example, the length between the moiety and the gamma-phosphate, may be sufficiently long so that when the moiety is stably hybridized to the corresponding moiety of the chain, there is substantially no force in the nucleotide. If this length is too short, the current may impose a force on the elongated nucleotide marker, which should result in reduced polymerase effectiveness. 5. With the chain bound to the polymerase, the position of the reporter is expected to vary based on whether the nucleotide in the active site of the polymerase is a match or a mismatch, because the conformation of the polymerase can be affected by the complementarity of the nucleotide to the nucleotide next in the polynucleotide being sequenced and therefore the relative position of the reporter region within the opening, for example, relative to the constriction. In this way, nucleotide identity as well as nucleotide complementarity can be discernible based on the position of the reporter in the pore constriction.

[0130] For more information on the hybridization of oligonucleotides to each other, see North American patent No. 8,652,779 to Turner et al., the entire contents of which are incorporated by reference in the present invention. According to Turner et al., on such a size scale, an oligonucleotide must sample its configuration space about 100 times faster than a polymerase can incorporate a nucleotide. Applying this principle to the present compositions, it is believed likely that the portion of the nucleotide being triggered will readily “encounter” and interact with the corresponding portion of the chain, and will also dissociate from the chain after the portion is cleaved from the nucleotide being triggered.

[0131] It should be noted that, in the exemplary portions illustrated in Figure 9D, each nucleotide-linked portion being activated has only a single corresponding hybridization with the portion of the corresponding chain 915 that includes between 5 to 8 bases. Although other hybridization options exist that do not cause complete hybridization, these options can be anticipated to be significantly less stable than the complete options.

[0132] Referring again to Figures 1A to 1C, the action of polymerase 130 on nucleotide 120 may keep portion 122 relatively close to portion 115 of chain 110, resulting in a transient increase in the local concentration of oligonucleotide portion 122 which may induce temporary hybridization between portions 122 and 115 preferentially relative to portions that are linked to nucleotides not currently engaged by polymerase 130. Furthermore, as noted above, polymerase 130 can cleave the elongated tag 121 by incorporating nucleotide 120 into a polynucleotide, responsive to which portion 122 can dissociate from portion 115. Suitable methods for providing nucleotides, including elongated labels, can be readily envisioned.

[0133] The method 300 illustrated in figure 3 also includes providing the first and second nucleotides, the first polynucleotide being complementary to the second polynucleotide (step 303). For example, step 303 may include providing a second polynucleotide, which may also be referred to as a “target” or “template,” that is desired to sequence. Step 303 also includes providing a first polynucleotide that is complementary to the second polynucleotide, but which may be shorter than the second polynucleotide using known methods. Illustratively, the first polynucleotide may include a primer that is complementary to, and which hybridizes to, a portion of the second polynucleotide.

[0134] The method 300 illustrated in figure 3 also includes providing a polymerase arranged adjacent to the first side of the nanopore (step 304). The polymerase can be configured to add the nucleotides of the plurality of nucleotides (provided in step 302) to the first polynucleotide (provided in step 303) based on a sequence of the second polynucleotide (also provided in step 303). Exemplary suitable polymerases are described in other sections of the present invention or are known in the art. Optionally, the polymerase can be modified in order to generate a signal based on the conformation of the polymerase, e.g., as described in U.S. Patent Publication No. 2011/0312529 to He et al., or in order to delay release. of pyrophosphate, for example, as described in US Patent No. 8,133,672, the total contents of both of which are incorporated into the present invention by reference.

[0135] Furthermore, the polymerase provided in step 304 can be anchored to a permanent chain including a head region, a tail region and an elongated body therebetween, with the elongated body including one or more reporter regions, and being the head region anchored to or adjacent to the first side or second side of the nanopore. The chain may have any suitable configuration, for example, as described above with reference to Figures 1A to 1D, or as otherwise defined in the present invention. For example, the elongated body of the chain may have a length that is longer than a first dimension H1 defining a thickness of the nanopore, for example, as illustrated in Figures 1A to 1D. Or, for example, the elongated body may have a length that is equal to a first dimension H1 defining a thickness of the nanopore, or that is shorter than the first dimension H1. Or, for example, in embodiments that include a constriction, the elongated body may have a length that is longer than a second dimension H2 defining a depth of the constriction, for example, as illustrated in Figures 1A to 1D. Or, for example, in embodiments that include a constriction, the elongate body may be of a length that is shorter than the second dimension H2. Or, for example, the one or more reporter regions may be disposed at a location along the selected elongate body such that, based on the elongate body being fully or partially extended when the head region is anchored in polymerase, the polymerase is adjacent to the nanopore, and the first portion 115 of the elongated body being dissociated from the second portion 122 of the elongated label, the reporter region(s) is/are positionable within the opening of the nanopore, e.g. example, adjacent to or within a constriction, as illustrated in Figures 1A to 1D. Any suitable combination of such characteristics may be used.

[0136] Step 304 may also, but does not necessarily need to, include preparing the chain. For example, step 304 may include defining an elongate body that includes portions thereof defining a head region, a tail region, and one or more reporter regions. For example, as described in another section of the present invention, a stream may include DNA. A DNA oligonucleotide of sufficient length can be prepared using procedures known in the art. For example, oligonucleotides with a 5' or 3' primary amine can be purchased commercially from suppliers such as Integrated DNA Technologies, Inc. (Coralville, Iowa). The oligonucleotide can be ordered to include one or more abasic portions, which can be used as one or more reporter regions, as described in the present invention. A bifunctional ligand such as sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) that includes a reactive amine group (NHS) and a thiol (maleimide) reaction group can be readily obtained from sources commercials, for example, from Thermo Fisher Scientific, Inc. (Rockford, Illinois). Such a linker can react with the oligonucleotide under appropriate reaction conditions well known in the art to form a stable amide bond. After purification of the oligonucleotide from the unreacted sulfo-SMCC, the modified oligonucleotide (which is now thiol reactive by virtue of its maleimide group) can react with the polymerase. The polymerase can be prepared in advance to include at least one solvent-accessible cysteine residue that has its thiol group (SH) in reduced form. The reduced form can be obtained by incubation with 5 mM tris(2-carboxyethyl)phosphine (TCEP), for example, which is a readily available commercial compound. The modified oligonucleotide can be combined, for example, in molar excess, with the reduced polymerase and under reaction conditions well known in the art, so that the maleimide forms a stable thioether bond. The polymerase-oligonucleotide conjugate can be removed by purification from excess unreacted oligonucleotide. In another example, compounds suitable for inclusion in a polyethylene glycol (PEG)-based stream, e.g., maleimide-PEG, are readily available from commercial sources such as Laysan Bio, Inc. (Arab, Alabama). For example, the maleimide can be conjugated to a reduced cysteine thiol in a manner analogous to that described above. One or more suitable reporter regions may be defined within the PEG. In another example, a disulfide bond between an oligonucleotide and an alpha-hemolysin nanopore can be prepared as described in Howorka et al, “Kinetics of duplex formation for individual DNA strands within a single protein nanopore,” PNAS 98: 12996- 13301 (2001), the contents of which are incorporated, in full, by reference in the present invention.

[0137] Step 304 may further include arranging the elongated body of the chain within an opening of the nanopore. Based on the length of the elongated body, it may, although not necessarily, extend across the entire nanopore opening. For example, in embodiments as described above with reference to Figures 1A to 1D, the elongate body of the chain may optionally be long enough so that the tail region of the chain is disposed on the other side of a constriction (if any) of the nanopore than is the current head region, and may optionally be arranged beyond the other side of the nanopore such as the current head region. Illustratively, the elongated chain body can be disposed within the nanopore opening by applying a suitable directional force to the elongated chain body. For example, a voltage may be applied across the nanopore in a manner as described in the present invention with reference to Figures 2A to 2C, and the elongated body of the chain may include at least one charged portion which, based on the voltage, attracts the tail region of the chain toward the side of the nanopore opposite to which the chain region is bound or to which the head region of the chain is bound to a first member, and causes translocation of the tail region so that arranging all or a portion of the elongated body of the chain within the nanopore opening.

[0138] Additionally, step 304 may optionally include arranging the polymerase adjacent to the first side of the nanopore. For example, the polymerase can be linked to be adjacent to the first side of the nanopore using a chemical bond, e.g., a covalent bond, hydrogen bond, ionic bond, dipole-dipole bond, London dispersion forces, or any suitable combination of the same. Or, for example, the polymerase can be linked to the first side of the nanopore using an interaction between a first protein structure in the polymerase and a second protein structure that is linked to or adjacent to the first nanopore. For example, the first and second structures may include alpha helices that connect to each other. The binding of the polymerase adjacent to the first side of the nanopore can be permanent, such that the polymerase is maintained in a generally fixed position relative to the first side of the nanopore.

[0139] In an illustrative embodiment, the reduced thiol group (-SH) (also called sulfhydryl group) of a cysteine residue of the polymerase or nanopore can react with a reactive thiol group of the other polymerase or nanopore. Examples of such groups include maleimide and iodoacetamide. As described in more detail at www.lifetechnologies.com/us/en/home/references/molecular- probes-the-handbook/thiol-reactive-probes/introduction-to- thiol-modification-and-detection.html#head2, reagents primary reactive thiols, including iodoacetamides, maleimides, benzylic halides and bromomethyl ketones, can react by S-alkylation of thiols to generate stable thioether products; arylation reagents such as 7-nitrobenz-2,1,3-oxadiazole halides (NBD) can react with thiols or amines by a similar substitution of the aromatic halide for the nucleophile; and because the thiolate anion is a better nucleophile than the neutral thiol, cysteine is more reactive above its pKa. Additionally, as described in more detail at www.piercenet.com/method/sulfhydryl-reactive-crosslinker-chemistry, sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinylsulfones, pyridyl disulfides, TNB -thiols (2-nitro-5-thiobenzoic acid) and disulfide reducing agents; such groups can conjugate with the sulfhydryls through alkylation (e.g., through the formation of a thioether bond) or disulfide exchange (e.g., the formation of a disulfide bond). Sulfhydryl exchange reactions can also be appropriately used. Alternatively, amines (-NH2) can be targeted. For example, the primary amine of lysine residues and the N-terminal polypeptide are relatively reactive. Amine residues can be targeted with N-hydroxysuccinimide esters (NHS esters), which can form a stable amide bond, or imidoester crosslinkers, which can react with primary amines to form amidine bonds. There are many other reactive amine compounds. For example, as described at www.piercenet.com/method/amine-reactive- crosslinker-chemistry, synthetic chemical groups that can form chemical bonds with primary amines include isothiocyanates, isocyanates, acylazides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides and fluorophenyl esters; such groups may be conjugated to amines, for example, through acylation or alkylation. In still other embodiments, a modified amino acid residue can be used to introduce a new functionality, such as an azide or alkyne, for use with click chemistry. For example, thiol or amine reactivities, as described above, can be used with ligands that allow the addition of azide or alkyne functionalities to be further used in a click chemical reaction.

[0140] Step 304 illustrated in Figure 3 may additionally and optionally include connecting the chain tail region to the other on the first side of the nanopore, or to a second member. Any suitable connection as provided in the present invention, or otherwise known in the art, may suitably be used. For example, step 304 may optionally include attaching the chain tail region to the second side of the nanopore. Or, for example, step 304 may optionally include linking the tail region of the chain to a second member (such as an oligonucleotide) disposed on the side of the nanopore opposite that of the head region. In this regard, note that polymerase 130 does not necessarily need to be anchored to the first side of the nanopore for polymerase 130 to remain attached to the nanopore. For example, under some conditions, applying a directional force to the elongated body of a chain can cause translocation of the tail region in order to dispose of the entire chain within the nanopore opening. A sufficiently large force can cause the polymerase that is bound to the current to become temporarily lodged in or on the nanopore. Although not intended to be a limitation regarding the physical configuration, the result can be called “corking” of the nanopore by the polymerase. Corking can be inhibited or avoided by limiting the force on the current (e.g., applying less than 180 mV across the nanopore), limiting the duration of time that the force is applied to the system, or using a sufficiently large protein so that the interaction by corking is avoided. Alternatively or in addition, a reverse voltage can be applied to the system to reverse the interaction between the protein and the nanopore (referred to as “uncorking”). Another option to inhibit or prevent corking, or to facilitate uncorking, is to remove charged amino acids from the nanopore opening or complementary charges on the protein surface in order to reduce the charge affinity between the two components. It may be additionally beneficial to add cross-links to the protein structure (e.g., engineered cysteine pairs for disulfide cross-links or chemical cross-linkers) in order to stabilize the globular structure of the protein.

[0141] Corking can be observed based on a characteristic current or flow pattern that is distinct from patterns resulting from other configurations of the nanopore system. The distinct pattern can be observed, for example, by applying a positive or negative bias to the nanopore system. Therefore, current or flow patterns can be detected during the assembly or use of a system that includes a protein that is localized to a nanopore through the current that is bound to the protein and placed in the lumen of the nanopore. Pattern detection can be used to monitor assembly (e.g., to prevent corking), guide uncorking, or otherwise optimize the desired assembly.

[0142] Note that, because the head region of the chain is linked to the polymerase, such directional force can also place the polymerase adjacent to, or fully or partially disposed within, the nanopore opening as described in the present invention, with reference to figures 1A to 1D. For example, attraction of the tail region to the side of the nanopore opposite to that on which the chain head region is attached to a first member can also cause translocation of the first member to a position adjacent to, or fully or partially disposed within, the nanopore. nanopore opening. Although the polymerase is in such a position, the 112 tail region of the 110 chain may be anchored to another member, for example, it may be hybridized to an oligonucleotide that is complementary to an oligonucleotide in the 112 tail region of the chain, and such anchoring of the tail region 112 can inhibit the dissociation of the polymerase from the nanopore 100.

[0143] The method 300 illustrated in Figure 3 also includes determining that a first nucleotide is being activated by the polymerase based on binding of the elongated marker to a first portion disposed on the elongated body (step 305). For example, as described above with reference to Figures 1A to 1D and 2A and 2B, the connection between the first portion 115 of the elongated body and the second portion 122 of the elongated marker may define a duplex that generates a first signal state, e.g. , a first electrical or optical signal state, which is detectable by a suitable measurement circuit, for example, by the measurement circuit 230 illustrated in Figure 2A, or by the measurement circuit 240 illustrated in Figure 2B. Different nucleotides may include different portions 122 of each other, which may generate different signals from each other, thus facilitating the identification of the particular nucleotide being triggered by the polymerase 130 based on the resulting signal.

[0144] The method 300 illustrated in Figure 3 also includes, with one or more reporter regions arranged along the elongated body, indicating when the first nucleotide is complementary or not complementary to the next nucleotide in the sequence of the second polynucleotide (step 306) . For example, as described above with reference to Figures 1A to 1D and 2A to 2C, the reporter region(s) may be configured to generate a signal state responsive to the first nucleotide being complementary. to a following nucleotide in the second polynucleotide, for example, based on a conformational change of the polymerase, release of pyrophosphate or other suitable phosphorus oxyanion, release of the tag from the elongated nucleotide or the like.

[0145] For example, the polymerase can transition between what is referred to as an “open state,” in which the polymerase does not bind to a nucleotide, to a “closed state,” in which the polymerase binds to a nucleotide. See, for example, Xia et al, “Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics,” Nucl. Acids Res. (2013), nar.gkt674, the entire contents of which are incorporated by reference in the present invention. See also Santoso et al, “Conformational transitions in DNA polymerase I revealed by single-molecule FRET,” Proc. Natl. Academic. Sci. USA, 107(2): 715-720 (2010), the entire contents of which are incorporated into the present invention by reference.

[0146] Conformational changes on the order of several nanometers are known to occur during the catalytic cycle of nucleotide incorporation as the polymerase transitions from the open to the closed state, or as the polymerase changes in editing mode. For example, Figures 7A to 7B schematically illustrate relatively large (>1 nm) polymerase conformational changes in two different polymerases. Figure 7A illustrates the RB69 polymerase, which presents a relatively large conformational change that results in relative movement between the thumb domain and the finger domain, undergoing 3 nm of movement between the open and closed conformations, as described by Xia et al. Figure 7B illustrates Pol I (Klenow fragment, or KF), which undergoes conformational changes during nucleotide incorporation, as disclosed by Santoso et al. The α-carbon structure of the polymerase is shown in beige. The DNA template strand is dark gray, and the primer strand is light gray. The terminal base pair in the active site is magenta. According to Santoso, the β-carbons of the two side chains were used as fluorophore binding sites, shown as green and red spheres, to measure the conformational changes of the polymerase. Arrows indicate the distance in Angstroms between the green and red Cβ positions in the open and closed conformations. There is also evidence that the conformational changes of a polymerase may depend on the identity of the nucleotide being incorporated, for example, as described in Olsen et al., “Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment),” JACS 135: 7855-7860 (2013), its entire contents being incorporated by reference in the present invention. In the nanopore embodiments of the present disclosure, the finger domain can be anchored to a nanopore, while a current is attached to the thumb domain. Alternatively, the thumb domain can be anchored to a nanopore while a current is attached to the finger domain. In either construct, the relative motion that occurs between the finger and thumb domains during polymerase activity can be detected as relative motion between the chain and the nanopore. The binding chemistries used to link the optical probes (e.g., FRET pairs) in the references cited in the present invention can be used in the nanopore embodiments defined in the present invention. Other attachment points can be used in a polymerase-nanopore construct, as long as conformational changes in the polymerase are reliably transmitted as relative motion between the chain and the nanopore.

[0147] Figures 7C and 7D schematically illustrate an exemplary composition including a permanent chain anchored to a polymerase disposed adjacent to a nanopore and configured for use in detecting a conformational change of the polymerase responsive to the action of the polymerase on a nucleotide. The nanopore includes the biological pore 605, which may be disposed in a barrier (not specifically illustrated), for example, a membrane of biological origin, such as a lipid bilayer, or a solid-state membrane. The biological pore 605 includes the opening 603 and the constriction 604. The permanent chain includes the head region 611, the elongated body 613, and one or more reporter region(s) 614. The polymerase 650 is disposed adjacent the biological pore 605 and may be optionally linked to the biological pore 605. The polymerase 650 is configured to receive a template polynucleotide, e.g., circular or linear ssDNA to be sequenced, to synthesize a polynucleotide with a sequence of complementarity to that of the ssDNA by sequentially receiving, ligating and add nucleotides to the polynucleotide according to the ssDNA sequence. The head region 611 of the permanent chain may be anchored at a site of the polymerase 650 that undergoes a conformational change, e.g., responsive to receiving a nucleotide, binding a nucleotide, or adding a nucleotide to the polynucleotide 660, and which moves the reporter region/regions 614 to a location sufficiently different from the constriction 604 in order to produce a signal that indicates when the triggered nucleotide is complementary or non-complementary to a following nucleotide in the ssDNA sequence. For example, the 611 head region may be linked to a polymerase finger region, or to a polymerase thumb. Exemplary attachment points in the finger and thumb regions of the polymerases and chemistries for attaching the moieties to these points are defined in U.S. Patent Application Publication No. 2011/0312529 A1, which is incorporated into the present invention by reference.

[0148] For more details on the structure and function of the family of A and B polymerases, see Patel et al., “Getting a grip on how DNA polymerases function,” Nature Structural Biology 8: 656-659 (2001), the content of which is incorporated by reference in the present invention. For more details on the structure and function of polymerases such as Pol, see the following references, the entire contents of each of which are incorporated by reference in the present invention: Olsen et al., “Electronic measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment,” JACS 135: 7855-7860 (2013); Torella et al., “Identifying molecular dynamics in single-molecule FRET experiments with burst variance analysis,” Biophysics J. 100: 1568-1577 (2011); Santoso et al., “Conformational transitions in DNA polymerase I revealed by single-molecule FRET,” Proc. Sci. molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I,” Nucleic Acids Res. 40: 7975-7984 (2012); 39: 595-599 (2011) and Johnson et al., “Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations,” Proc. Natl. Academic. Sci. USA, 100, 3895-3900, 2003). Any two residues or domains that are known from the above references (or other references cited in the present invention) to undergo a change in relative position during polymerase activity can serve as attachment points to a nanopore and chain, respectively, in a modality of this disclosure.

[0149] In one example, a voltage can be applied across the nanopore 605, for example, using the measuring circuit 230 and electrodes 231, 232, as described above with reference to Figure 2A, or the measuring circuit 240 and electrodes 241, 242, as described above with reference to Figure 2B. The reporter region 614 or the elongated body 613 includes an electrostatic charge that, responsive to applied voltage, causes the elongated body 613 to extend through the constriction 604, such that the reporter region(s) 614 are disposed within or adjacent to constriction 604 when the first portion 615 of the elongated body 613 is not linked to a second portion of an elongated marker of the nucleotide being driven. Optionally, the applied tension may cause the elongated body 613 to be tensioned when the first portion 615 of the elongated body 613 is not linked to a second portion of an elongated marker of the nucleotide being driven. As noted in other sections of the present invention, when the first portion 615 of the elongated body 613 is linked to a second portion of an elongated tag of the nucleotide being driven, the resulting duplex may become lodged within or adjacent to the constriction of the nanopore under a voltage applied across of the nanopore, resulting in a first signal state based on which the nucleotide can be identifiable. As illustrated in Figure 7D, as polymerase protein domains 605 move, for example, changing conformation relative to that shown in Figure 7D, such movements can impose a force on the head region 611 that imposes a force on the elongated body 613 , which imposes a force on the reporter region(s) 614, resulting in translational movement of the reporter region(s) 614 within the opening 603, e.g., movement relative to the constriction 604. As As a result, a conformational change of the polymerase 650 can be translated or transduced into a measurable change in the current or flow through the opening 603, which can also be referred to as a blocking current or flow. It may be detectable, based on such a second signal state, whether the nucleotide being triggered is complementary or non-complementary to the following nucleotide of the ssDNA.

[0150] In an illustrative embodiment, the reporter region(s) 614 are constructed using modified nucleotides. For example, abasic nucleotides typically generate a blocking current of 70 pA compared to residues that include bases, such as dT residues, which generate only a blocking current of 20 pA under conditions that include 4-(2-) acid buffer. 10 mM hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.0, 300 mM KCl, 1 mM MgCl2, 1 mM DL-dithiothreitol (DTT), MspA M2 mutant pore (D90N, D91N, D93N, D118R, D134R and E139K), 180 mV through a 1,2-diphtanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer.

[0151] Movements of abasic residues on the order of, for example, just a few angstroms, can cause easily detectable changes in current or flow, for example, from one to tens of pAs. Since some polymerases move on the order of nanometers, and a single base in the chain corresponds to about 0.5 nanometers, it is predicted that chain movements resulting from conformational changes in the polymerase to which the chain is anchored can be generated and be readily transduced into currents or flows. How the special conformation of the polymerase can be based on whether the nucleotide that the polymerase is acting on is complementary or not complementary to a following nucleotide in the ssDNA can be determined, based on a signal state (current or flow state) when that nucleotide is complementary or not complementary to a following nucleotide in the ssDNA. Furthermore, because nucleotide identity influences both the magnitude of the conformational change and the time spent in the closed state, the signal state (current or flow state) can also potentially and individually identify nucleotides as they are incorporated in order to confirm the identification of the nucleotide of step 305 based on the first state of the signal. For example, in some embodiments, as illustrated in Figure 5B, the identity of the nucleotide can be revealed by the location of the 514 reporter that hybridizes to the 522 portion. A corresponding signal is shown in Figure 6, part “B”. If there is a perfect match as in Figure 5C, the polymerase can adopt a specific conformation. A corresponding signal is shown in figure 6, part “D”. The signs in figure 6, parts “C” and “D”, are distinct and can exceptionally mean a match or incompatibility, respectively.

[0152] The magnitude or duration of time, or both, of the conformational/conformational change(s) of the polymerase may be based on the specific nucleotide upon which the polymerase is acting. Table 1 lists the exemplary single-molecule kinetic parameters that were measured for processing the Klenow fragment templates using the current changes in a SWNT linked to a single polymerase and reported by Olsen et al. In Table 1, Tlo corresponds to the duration of time spent in the closed conformation of the polymerase, rlo corresponds to the mean-normalized variance for Tlo, Thi corresponds to the duration of time spent in the open conformation of the polymerase, rhi corresponds to the mean-normalized variance for Thi, and the rate corresponds to the processing rate, for example, how quickly the polymerase adds the nucleotide to the template. Olson et al. report that the average magnitude H is an indicator for measuring mechanical closure by the enzyme. For the present systems, methods and compositions, the H value can be considered to be the extent of the conformational change between two reference points on the polymerase, as measured in units of distance. For example, H may correspond to the magnitude of mechanical closure to be affected by nucleotide match or mismatch. Table 1 a Mean values ± standard deviation 7858 dx.doi.org/10.1021/ja311603r|J. Am. Chem. Soc. 2013, 135, 7855-7860

[0153] Using the systems, methods and compositions presented here, a signal that correlates with the time duration of the open state Thi, the magnitude of the conformational change H and the processing rate, together, can be used to indicate when the first nucleotide is complementary or non-complementary to the next nucleotide of the ssDNA, and potentially the signals can be differentiated according to a unique signature for each base. Incorporation rates can also be greatly altered by selective use of modified nucleotides, such as alpha or gamma thiol nucleotides. See, for example, U.S. Patent Publication No. 2011/0312529 to He et al., the entire contents of which are incorporated by reference in the present invention.

[0154] In an illustrative embodiment, the template DNA is circularized and polymerase 650 is a strand-shifting polymerase (such as Phi29). In this way, the mold can be sequenced multiple times in a rolling circle mode as is known in the art. Such an embodiment may also inadvertently inhibit the drag of template DNA into or through constriction 604, because only ssDNA can translocate. Any stray ssDNA that finds its way through the 604 constriction (or if a linear template is used) is expected to transit quickly, and is expected to generate noise in the signal. Alternatively, one or more positively charged reporter region(s) 614 may be used under reverse polarity, such that only the reporter region(s) are pulled into the constriction 604, while that negatively charged DNA will be repelled.

[0155] In some embodiments, the polymerase 650 can be linked, for example, anchored to the mouth of the biological pore 605. This could be accomplished using, for example, cysteine/thiol conjugation chemistry. Such conjugation can cause this polymerase 650 to be anchored in a reproducible and stable orientation that can improve the transfer of the conformational movement of the polymerase 650 to the translational movement of the reporter region(s) 614. However, conjugation polymerase to the pore need not be required. For example, the force exerted by the applied voltage-responsive current may be sufficient to hold the polymerase in place, or another member (e.g., an oligonucleotide) may bind to the tail end of the tether to inhibit dissociation of the polymerase from the nanopore.

[0156] Additionally, note that a fast AC current can be used instead of a DC current to produce the necessary electric field. This has the advantage of inhibiting depletion of the AgCl electrode and prolonging the time the device can operate.

[0157] Note that the duplex formed between the elongated marker portion and the chain portion may be in thermodynamic equilibrium. It should be noted that a duplex in thermodynamic equilibrium may have association and dissociation rates that are based on the length and character of the nucleic acid sequence, and that the duplex may dissociate from time to time. It may be useful if the average time in the duplex state (the inverse of the dissociation rate) is shorter than the average lifetime of the polymerase-nucleotide complex during the incorporation event, so that after incorporation and release of the nucleotide marker, the marker will diffuse away and will not block incoming nucleotide markers. The effective association rate may be high enough to result in relatively rapid rebinding compared to the lifetime of the polymerase-nucleotide complex, so that incorporation events are detected and so that the duplex forms again after any such events. of dissociation that occur during nucleotide incorporation. The association rate will be pseudo first order at the nucleotide elongated marker concentration, which can be considered to make such an arrangement a stochastic sensor of the elongated marker concentration. Note that freely diffusing elongated labels can have a relatively low concentration (e.g., from 10 nM to 100 nM, or from 100 nM to 250 nM, or from 250 nM to 500 nM, or from 500 nM to 1 μM) , whereas the elongated marker of the nucleotide being driven will effectively have a relatively high concentration because it is bound to the nucleotide that is held in place by the polymerase during incorporation and is therefore not free to be removed by diffusion.

[0158] In certain exemplary embodiments described herein, the elongated body of the chain and the elongated nucleotide marker being driven by the polymerase, may respectively include, or may even consist solely of, single-stranded DNA (ssDNA). However, it should be noted that other types of molecules can be appropriately used. For example, any chain may be suitably used which includes an elongate body having one or more of the following features, and optionally includes all of the following features: 1. The elongate body may include a region that interacts with portions respectively attached to different types of nucleotides so that the parts are distinguishable from each other, for example, by measuring the current or flow through the pore constriction. 2. The elongated body may include a charged region that causes it to be pulled through the constriction of the pore responsive to an applied voltage. The elongated body can be kept taut in this type of configuration. This charged region may be adjacent to the pore constriction to result in a net force. 3. The elongated body may include one or more reporter regions that generate a clearly distinguishable signal state when the nucleotide on which the polymerase is acting is complementary or not complementary to a following nucleotide in the sequence of the polynucleotide being sequenced. The reporter region(s) and the charged region may be identical to each other, that is, a single region may be both a reporter region and a charged region. Furthermore, it must be understood that different signal states, which may represent the identities of nucleotides or whether those nucleotides are a match or a mismatch, need not necessarily occur at different times relative to one another and, in fact, they may occur at the same time as one another. For example, a measured signal (e.g., optical or electrical) may include a composite of more than one signal state (e.g., two signal states), wherein each of the signal states provides information about one or more of a nucleotide identity or whether such nucleotide is a match or not.

[0159] An exemplary material that can be included in the elongated body of the chain, or in the elongated marker of the nucleotide being driven, or both, is a polymer. Polymers include biological polymers and synthetic polymers. Exemplary biological polymers that are suitable for use in the elongated body of the chain, or the elongated marker of the nucleotide being driven, or both, include polynucleotides, polypeptides, polysaccharides, polynucleotide analogs and polypeptide analogs. Exemplary polynucleotide and polynucleotide analogs suitable for use on the elongated body of the chain, or the elongated tag of the nucleotide being driven, or both, include DNA, enantiomeric DNA, RNA, PNA (peptide-nucleic acid), morpholinos, and LNA (nucleic acid). blocked). Exemplary synthetic polypeptides can include charged amino acids as well as hydrophilic and neutral residues. In some embodiments, the chain is not a nucleic acid or does not include nucleotides. For example, a chain may exclude naturally occurring nucleotides, non-naturally occurring nucleotide analogues, or both. One or more of the nucleotides defined in the present invention or otherwise known in the art may be excluded from a chain.

[0160] Other exemplary polymers that may be suitable for use in the elongated body of the chain, or in the elongated marker of the nucleotide being driven, or both, include synthetic polymers, such as PEG (polyethylene glycol), PPG (polypropylene glycol), PVA (alcohol polyvinyl), PE (polyethylene), LDPE (low density polyethylene), HDPE (high density polyethylene), polypropylene, PVC (polyvinyl chloride), PS (polystyrene), NYLON (aliphatic polyamides), TEFLON® (tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes, polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters), poly(alkyl methacrylates) and other polymeric chemical and biological binders, such as described in Hermanson et al., mentioned further above . Furthermore, as mentioned above, the elongated body portions of the chain, or the elongated nucleotide tag being driven, or both, may be individual short nucleotide sequences that interact with each other. These portions may not interact with the polymerase, such as RNA, PNA or LNA tags, morpholinos or enantiomeric DNA, for example. The portion need not be formed from the same polymer as the other portions of the elongated body of the chain or the elongated marker of the nucleotide being driven. Elongated tags can be readily attached to the gamma-phosphate of nucleotides, as is known in the art. Furthermore, in an illustrative embodiment, the bases isoG and isoC can be used in the elongated nucleotide markers, or in the chain, or in both, so as to inhibit hybridization of the elongated markers with the DNA being sequenced. Additionally, other schemes can be used to induce the secondary structure in the chain to shorten it, like a hairpin.

[0161] Furthermore, it is noted that the chain may include several portions, each of which interacts, respectively, with a portion linked to a certain type of nucleotide. The interaction between the portion of the nucleotide with the corresponding portion of the chain can move the reporter region(s) of the chain by a corresponding amount that facilitates identification of the corresponding nucleotide through a signal, e.g., through a current or flow through the pore opening.

[0162] In a non-limiting illustrative embodiment, the pore includes MspA, which can provide satisfactory separation of nucleotide-specific streams or streams, e.g., a 3.5-fold greater separation of nucleotide-specific streams or streams compared to the alpha-hemolysin. However, it should be noted that alpha-hemolysin or other types of pores can be suitably used with the present compositions, systems and methods.

[0163] Furthermore, it is noted that, in embodiments in which a voltage is applied across the pore and movements of the reporter region(s) is/are measured via the current or flow through the pore, the voltage It can be appropriately applied using direct current (DC) or alternating current (AC). AC current can help increase electrode life and help eject the cleaved elongated tracers from the pore if the tracers become stuck. Furthermore, it is noted that a positively charged current can be used with a reverse bias on the pore in order to prevent the DNA being sequenced from being drawn into the pore. Furthermore, it is noted that a positively charged current and elongated positively charged markers can be used with a reverse bias on the pore in order to prevent the DNA being sequenced from being pulled into the pore.

[0164] Additionally, a stochastic sensing method can be employed. In this arrangement, an AC current can be used to move the hybridized duplex adjacent to the constriction. For example, Figures 4A to 4E schematically illustrate a composition including a chain anchored to or adjacent to a nanopore and configured for use in detecting the action of a polymerase on a nucleotide using a chain anchored to or adjacent to a nanopore responsive to a change. in the electrical potential in the nanopore, and Figure 4F illustrates an exemplary signal that can be generated during the use of such a composition.

[0165] The composition illustrated in Figure 4A includes the nanopore 400, including a first side, a second side, an opening extending through the first and second sides, and a constriction disposed between the first and second sides (components of the nanopore 400 not specifically marked, but may be analogous to those illustrated in figures 7C and 7D); permanent chain 410 including a head region anchored to the first side of the nanopore 400, a tail region that is optionally coupled to another member 460 (e.g., an oligonucleotide) that inhibits the dissociation of polymerase 430 from the nanopore 400, and an elongated body that includes one or more reporter regions 414 and the first portion 415 (other components of chain 410 not specifically labeled, but may be analogous to those illustrated in Figures 7C and 7D); and polymerase 430. As illustrated in Figure 4A, when polymerase 430 is not acting on a nucleotide, the polymerase may be in an open state. The signal illustrated in Figure 4F shown in Figure 4A includes a current or flow state corresponding to the reporter region(s) 414 being arranged in a location that is characteristic of the open state of the polymerase 430, under an applied positive voltage. through nanopore 400, for example, +180 mV, where +180 mV indicates 0 mV on the first side of the nanopore and +180 mV on the second side of the nanopore.

[0166] The composition illustrated in Figure 4B additionally includes nucleotide 420, including the elongated marker 421 that includes the second portion 422. Polymerase 430 acts on nucleotide 420, causing polymerase 430 to change conformation to a closed state, as illustrated in figure 4B. The signal illustrated in Figure 4F shown in Figure 4B includes a current or flow state 414 being arranged in a location that is characteristic of the closed state of the polymerase 430, under a positive voltage applied across the nanopore 400, e.g., +180 mV. Note that under a positive voltage, the first portion 415 can be disposed on the opposite side of the nanopore 400 from the portion 422 of the nucleotide 420, thereby inhibiting the formation of a duplex between the portion 415 and the portion 422 under the positive voltage. Note that portion 415 may be located at any suitable position along the elongated chain marker 410, for example, it may be located between the tail region and the reporter region(s) 414, as illustrated. in Figure 4A, or may be adjacent to the head region, adjacent to the tail region, adjacent to the reporter region, or be between the head region and the reporter region/regions 414.

[0167] As illustrated in Figure 4C, an interaction between portion 422 of nucleotide 420 and portion 415 of chain 410 can set duplex 423 under a reverse voltage, for example, under a negative voltage, based on which portions 415 and 422 are arranged on the same side of the nanopore 400 as another. The signal illustrated in Figure 4F shown in Figure 4C includes a current or flow state corresponding to the positions of the reporter region(s) 414 and the duplex 423 under the negative voltage applied across the nanopore 400, e.g. 180 mV, where -180 mV indicates 0 mV on the first side of the nanopore and -180 mV on the second side of the nanopore. The position of the 414 reporter region can be sensitized, depending on where the chain is bound to the polymerase, to distinguish between matched and mismatched nucleotides in a manner analogous to that described in the Freudenthal references mentioned above.

[0168] As illustrated in Figure 4D, by inverting the voltage to a positive voltage, for example, + 180 mV, the duplex 423 can become housed in or adjacent to the nanopore constriction. The signal illustrated in Figure 4F shown in Figure 4D includes a current or flow state corresponding to the position of the duplex 423, as well as the particular portions defining the duplex 423, under the positive voltage applied across the nanopore 400, e.g., +180 mV. Based on the 422 portion being selected to correspond to a single nucleotide type, the signal can be used to determine which nucleotide is under the action of polymerase 430. Note that the 423 duplex formed between the 415 portion and the 422 portion can be large enough to inhibit movement of the duplex through the constriction.

[0169] As illustrated in Figure 4E, in response to continuous application of positive voltage, for example, + 180 mV, duplex 423 may dissociate. Such dissociation may be considered to “disrupt” the duplex 423 formed between portion 415 and portion 422. In some embodiments, portion 415 may move through the constriction to be disposed on the second side of nanopore 400. The portion of Figure 4F immediately below Figure 4E illustrates an exemplary current or flow through the opening under positive voltage after dissociation of portion 415 from portion 422. The signal illustrated in Figure 4F shown in Figure 4E includes a current or flow state that corresponds to the ) reporter region/regions 414 being arranged in a location that is characteristic of the closed state of the polymerase 430, under a positive voltage applied across the nanopore 400, for example, + 180 mV. The portion 422 can be configured to remain disposed on the first side of the nanopore 400 even if the portion 415 is disposed on the second side of the nanopore 400, so as to temporarily inhibit the interaction between the portions 415 and 422. As illustrated in Figure 4C , after such dissociation, the voltage applied across the opening 403 can be changed again, for example, it can be changed to the first voltage, responsive to which the portions 415 and 422 can interact with each other so as to, again, define the duplex 423 .

[0170] It should be noted that signals corresponding to the relative positions of the reporter region(s) 414 and the duplex 423, and the particular portions defining the duplex 423 may be detectable in any suitable manner. For example, the composition may be in operable communication with a measurement circuit such as is described above with reference to Figure 2A or Figure 2B. The measurement circuit may be configured to detect the current or flow through the opening of the nanopore 400, which may vary over time based on the conformational state of the polymerase 430 and based on the portion 422 of the particular nucleotide 420 being driven by the polymerase. 430. In an illustrative embodiment, the nanopore 400, the chain 410, the polymerase 430, and the nucleotide 420 may be immersed in a conductive fluid, for example, in an aqueous saline solution. A measuring circuit configured analogously to the measuring circuit 230 illustrated in FIG. 2A or the measuring circuit 240 illustrated in FIG. 2B may be in communication with the first and second electrodes, and may be configured to apply a first voltage between those electrodes. , so as to apply a voltage across the nanopore 400, as represented by the “+” and “-” signs illustrated in Figure 4A, and to use the electrodes to measure the magnitude of a current or flow through opening 403 at the first voltage . For example, the portion of Figure 4F immediately below Figure 4A illustrates an exemplary current or flow through the opening of nanopore 400 at a first voltage. Reporter region 414 may have a flow or electrical blocking property different from some or all other regions of the elongate current body (not specifically labeled). For example, the reporter region(s) 414 may include an electrostatic charge, while some or all of the other regions of the elongated body may include a different electrostatic charge, or may be discharged (e.g., may be electrically neutral). Or, for example, the reporter region(s) 414 may be discharged, while some or all of the other regions of the elongate body may include an electrostatic charge. In a non-limiting illustrative example, the elongated body of the chain includes a polynucleotide that includes one or more abasic nucleotides that define the reporter region(s) 414. The magnitude of the current or flow through the opening of the nanopore 400 may change, measurably, in response to the relative location of the reporter region(s) 414 within the opening 403, the relative location of the duplex 423, and the portions defining the duplex 423, based on what the conformational state of polymerase 430 and the action of polymerase 430 can be determined.

[0171] Note that, in some embodiments, the respective lengths of the elongated body of the chain and linker binding the nucleotide and the elongated tag, the respective locations of portions 415 and 422, and the respective location of the reporter region/regions (es) 414 are coselected so as to inhibit or prevent the application of force to the nucleotide 420 while the nucleotide is being driven by the polymerase 430 and thereby to prevent such force from modifying the performance of the polymerase. In an illustrative embodiment, the interaction between portion 415 and portion 422 forms duplex 423. The length of the elongate body of the chain, and the location of portion 415 along the elongate body, may be coselected such that portion 415 may be extended through the constriction of the nanopore 400 in response to an appropriate applied voltage, for example, in order to cause dissociation between the portion 415 and the portion 422. The size of the duplex 423 can inhibit movement of the duplex through the constriction of the nanopore 400, and can protect nucleotides 420 from forces that could otherwise have been applied to nucleotide 420 through the elongated marker 421. In an exemplary embodiment, the reporter region(s) 414 are arranged in a suitable location along the elongated body of the chain 410 in order to be disposed within, or adjacent to, the constriction of the nanopore 400 when the portions 415 and 422 dissociate from each other under positive voltage.

[0172] In another example, Figures 5A to 5D schematically illustrate a composition including a chain anchored to or adjacent to a nanopore and configured for use in detecting the action of a polymerase on a nucleotide using a chain anchored to or adjacent to a nanopore responsive to a change in electrical potential in the nanopore, and Figure 6 illustrates exemplary signals that can be generated during the use of such a composition.

[0173] The composition illustrated in Figure 5A includes the nanopore 500, including a first side, a second side, an opening extending through the first and second sides, and a constriction disposed between the first and second sides (components of the nanopore 400 not specifically marked, but may be analogous to those illustrated in figures 7C and 7D); permanent chain 510 including a head region anchored to the first side of the nanopore 500, a tail region that is optionally coupled to another member 560 (e.g., an oligonucleotide) that inhibits the dissociation of polymerase 530 from the nanopore 500, and an elongated body that includes one or more reporter regions 514 and the first portion 515 (other components of chain 510 not specifically labeled, but may be analogous to those illustrated in Figures 7C and 7D); nucleotide 520 including an elongated (unlabeled) tag that includes the second portion 522; and polymerase 530. As illustrated in Figure 5A, when polymerase 530 is not acting on nucleotide 520, the polymerase can be in an open state or a closed state, each of which has a unique signal relative to its unbound states. The signal illustrated in Figure 6 denoted “A” (long dashed line) includes a current or flow state corresponding to the reporter region(s) 514 being arranged in a location that is characteristic of the unbound states of the polymerase. 530, under a positive voltage applied across the nanopore 500, e.g. +180 mV.

[0174] The composition illustrated in Figure 5B includes polymerase 530 that acts on nucleotide 530, causing polymerase 530 to change conformation to a closed state. Furthermore, an interaction between portion 522 of nucleotide 520 and portion 515 of chain 510 may define duplex 523. Under a positive voltage, e.g., +180 mV, duplex 523 may become lodged in or adjacent to the constriction of the nanopore 500. The signal illustrated in Figure 6, denoted “B” (solid line) includes a current or flow state corresponding to the position of duplex 523, as well as the particular portions that define duplex 523 under the positive voltage applied across nanopore 500. , for example, + 180 mV. Based on portion 522 being selected to correspond to a single type of nucleotide, the signal can be used to determine which nucleotide is under the action of polymerase 530. Note that the duplex 523 formed between portion 515 and portion 522 can be large enough to inhibit movement of the duplex through the constriction. Illustratively, the “reporter” for each different nucleotide may include the bases immediately adjacent to the duplex (e.g., just below the last base of the duplex in Figure 5B). Thus, in a non-limiting example, there may be four reporters to identify the nucleotides. There may also be a fifth reporter, which is reporter 514 who is shown as a circle. Reporter 514 can be used to report the conformational state of polymerase 530 as in Figures 5C and 5D. This conformational state may depend on whether the polymerase is retaining a matching or mismatching nucleotide.

[0175] For example, as illustrated in Figure 5C, responsive to continuous application of positive voltage, e.g., + 180 mV, duplex 523 may dissociate. Such dissociation may be considered to “disrupt” the duplex 523 formed between portion 515 and portion 522. In some embodiments, portion 515 may move through the constriction to be disposed on the second side of nanopore 500. The portion of Figure 6 , denoted “C” (line of short dashes), illustrates an exemplary current or flow through the opening under positive voltage after dissociation of portion 515 from portion 522. The current or flow state denoted “C” corresponds to the reporter region/regions 514 being arranged in a location that is characteristic of the closed state of the polymerase 530 in which the correct (corresponding) nucleotide 520 is bound by the polymerase, under a positive voltage applied across the nanopore 500, e.g. 180mV.

[0176] In this example, polymerase 530 incorporates nucleotide 520 into the polynucleotide being sequenced, cleaves the marker for that nucleotide and subsequently binds to another nucleotide that may or may not be complementary to the next nucleotide in the polynucleotide being sequenced. For example, the “T” nucleotide, illustrated in Figure 5C, becomes complementary to the next nucleotide in the polynucleotide being sequenced, while the “T” nucleotide, illustrated in Figure 5D, becomes non-complementary to the next nucleotide in the polynucleotide being sequenced. sequenced. In the example illustrated in Figure 5D, upon application of positive voltage, e.g., +180 mV, polymerase 530 can change a unique conformation that occurs only when the nucleotide being triggered is a mismatch with the following nucleotide in the polynucleotide being sequenced. This slightly repositions the reporter region/regions 514 in the constriction of the nanopore 500 relative to the position of the reporter region/regions 514 for the case where the nucleotide was a match, e.g. illustrated in figure 5C. The portion of Figure 6, denoted “D” (dotted line) illustrates an exemplary current or flow through the opening under the positive voltage of the polymerase 530 changing to the conformation corresponding to an incompatible nucleotide, under a positive voltage applied across the nanopore 500, for example, + 180 mV. Note that the signal level of the dotted line in “D” is different from the signal level of the dashed line in “C”, thus making it easier to distinguish the corresponding nucleotides from the mismatched nucleotides. Furthermore, each mismatched base can also have a unique polymerase confirmation, resulting in distinguishable signal states.

[0177] Note that the above states are not intended to be limiting, but are provided by way of example. Other detectable states than those indicated may occur. For example, there may be multiple detectable states for the polymerase when there is no nucleotide corresponding to such open and closed configurations, or even multiple states when there is an incompatible nucleotide associated with the polymerase in either the open or closed state.

[0178] It should be noted that signals corresponding to the relative positions of the reporter region(s) 514 and the duplex 523 and the particular portions defining the duplex 523 may be detectable in any suitable manner, such as defined in other sections in the present invention. Furthermore, it should be noted that the signal that indicates when the nucleotide being triggered is complementary or is not complementary to a following nucleotide in the sequence of the polynucleotide being the sequence, need not be electrical or optical and, in fact, can be generated in any suitable way.

[0179] Furthermore, it should be noted that the dissociation of a duplex as may be formed based on an interaction between a first portion of an elongated body and a second portion of an elongated marker responsive to an applied voltage may be characterized as defining a first pathway that is characterized by two or more kinetic constants. Additionally, the indication of when the first nucleotide is complementary or is not complementary to the next nucleotide in the sequence of a polynucleotide being sequenced, e.g., a conformational change of the polymerase, release of pyrophosphate, or release of the elongated nucleotide tag, can be characterized. as defining a second pathway that is characterized by two or more kinetic constants. The statistical distribution of the measured signals (e.g., optically or electrically measured) in the course of obtaining measurements of the first pathway or the second pathway may be based on the relative values of the kinetic constants corresponding to that pathway. For example, based on whether a kinetic constant for the first pathway or the second pathway is significantly greater than the other kinetic constants for that pathway, the kinetics of that pathway may be dominated by that given kinetic constant, and the resulting statistical distribution of signals can be described by an exponential function. Comparatively, two or more of the kinetic constants for the first path or the second path may be selected to be of the same order of magnitude, or even to be substantially equal to each other (for example, differing from each other by a factor of five or less, or four or less, or three or less, or two or less), such that the kinetics of that pathway are not dominated by any kinetic constant, and the resulting statistical distribution of signals can be described by a gamma function, in which there is substantially no probability of zero or very short time events that are substantially unobservable. In comparison, with an exponential distribution, there is a high probability of short or zero-time events that are substantially unobservable.

[0180] One or more of the kinetic constants of the first or second pathway may be suitably modified so as to be of the same order as one or more of the kinetic constants of that pathway, or even so as to be substantially equal to one or more of the kinetic constants of that pathway. For example, as mentioned above, the polymerase can be modified in order to delay the release of pyrophosphate responsive to the incorporation of a nucleotide into the first nucleotide, thereby modifying at least one kinetic constant of that pathway. As another example, the chain may further include a second portion that hybridizes with the first portion to form a hairpin structure. The first and second portions of the current can be configured to dehybridize from each other in a two-step process responsive to a voltage applied across the nanopore, thereby modifying at least one kinetic constant of that pathway. An exemplary tetraphosphate-modified nucleotide with a tag configured to form a hairpin structure is shown in Figure 12A). After hybridizing with the current, a hairpin is formed as shown in Figure 12B. This clamp is expected to have two removal rate constants, k1 and k2, which are shown in Figure 12B. These rate constants can be designed to be of similar magnitude to each other, so that when added together they can form a gamma distribution.

[0181] As noted in other sections of the present invention, a variety of compositions can be suitably included in the elongated nucleotide marker or the elongated chain body, or both. Such compositions may include DNA, PNA, LNA, RNA, morpholinos, PEG (polyethylene glycol) and the like, and may be of any suitable length. An oligonucleotide tag containing a suitably modified nucleotide can be appropriately linked to such different compositions, for example, using precursors compatible with ideally click chemistry. In one example, the nucleotide is labeled with azide, which could facilitate the use of alkyne-labeled oligonucleotides, which are easily synthesized. Exemplary molecules include tetraphosphate-labeled nucleotides as shown below, where (A) corresponds to azide-P4O13-dTTP and (B) corresponds to alkyne-P4O13-dTTP. These nucleotides can be modified with any desired tag using standard click chemistry:

[0182] References for making and labeling tetraphosphate nucleotides include the following, the entire contents of each of which are incorporated by reference in the present invention: Kumar, S., A. Sood, J. Wegener, P. J. Finn, S. Nampalli, J. R. Nelson, A. Sekher, P. Mitsis, J. Macklin, and C. W. Fuller, “Terminal phosphate labeled nucleotides: synthesis, applications, and linker effect on incorporation by DNA polymerases,” Nucleosides Nucleotides Nucleic Acids 24(5-7): 401- 408 (2005); Sood, A., S. Kumar, S. Nampalli, J. R. Nelson, J. Macklin, and C. W. Fuller, “Terminal phosphate-labeled nucleotides with improved substrate properties for homogeneous nucleic acid assays,” J Am Chem Soc 127(8): 2394 - 2395 (2005); Kumar, S., C. Tao, M. Chien, B. Hellner, A. Balijepalli, J. W. Robertson, Z. Li, J. J. Russo, J. E. Reiner, J. J. Kasianowicz, and J. Ju, “PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis,” Sci Rep 2: 684 (8 pages) (2012); Bonnac, L., S. E. Lee, G. T. Giuffredi, L. M. Elphick, A. A. Anderson, E. S. Child, D. J. Mann, and V. Gouverneur, “Synthesis and O-phosphorylation of 3,3,4,4-tetrafluoroaryl- C-nucleoside analogues,” Org Biomol Chem 8(6): 1445-1454 (2010); and Lee, S. E., L. M. Elphick, A. A. Anderson, L. Bonnac, E. S. Child, D. J. Mann, and V. Gouverneur, “Synthesis and reactivity of novel gamma-phosphate modified ATP analogues,” Bioorg Med Chem Lett 19(14): 3804- 3807 (2009).

Other alternative modalities

[0183] Although several illustrative embodiments of the invention are described above, it will be apparent to a person skilled in the art that various changes and modifications can be made thereto without departing from the invention. For example, although certain compositions, systems and methods are discussed above with reference to the sequencing of polynucleotides, such as DNA or RNA, it should be understood that the present compositions, systems and methods may be appropriately adapted for use in detecting any type of event. , for example, the movement of a molecule, or a portion thereof, which may be related to the presence or movement of a reporter region adjacent to a constriction of a nanopore. The appended claims are intended to encompass all such changes and modifications as fall within the true spirit and scope of the invention.

Claims (15)

1. A composition characterized by the fact that it includes: a nanopore including a first side, a second side and an opening extending through the first and second sides; a plurality of nucleotides, wherein each of the nucleotides comprises an elongated marker; first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide; a polymerase disposed adjacent the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide; a permanent chain including a head region, a tail region and an elongated body disposed therebetween, with the head region being anchored to the polymerase, wherein the elongated body occurs at the opening of the nanopore; a first portion disposed on the elongated body, wherein the first portion is configured to bind to the elongated marker of a first nucleotide on which the polymerase is acting; and a reporter region disposed in the elongate body, wherein the reporter region is configured to indicate when the first nucleotide is complementary or is non-complementary to a following nucleotide in the sequence of the second polynucleotide. 2. The composition of claim 1, wherein the first portion is configured to generate a first polymerase-responsive signal state acting on the first nucleotide, the first nucleotide being identifiable based on the first signal state. . 3. Composition, according to claim 2, characterized by the fact that the polymerase acting on the first nucleotide comprises the polymerase that binds to the first nucleotide; or the first signal state that includes an electrical or optical signal; or the reporter region is configured to generate a second signal state, which is detectable based on the second signal state, whether the first nucleotide is complementary or non-complementary to the following nucleotide of the second polynucleotide. 4. Composition according to claim 3, characterized by the fact that the reporter region is configured to generate the second state of the polymerase-responsive signal by successfully incorporating the first nucleotide into the first polynucleotide, preferably, the reporter region is configured to generate the second state of the polymerase-responsive pyrophosphate-responsive signal, successfully incorporating the first nucleotide into the first polynucleotide. 5. Composition according to claim 4, characterized by the fact that the polymerase is modified so as to delay the release of the pyrophosphate responsive to the incorporation of the first nucleotide into the first polynucleotide. 6. Composition according to claim 5, characterized in that the polymerase comprises a polymerase Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5 , Cp-5, Cp-7, PR4, PR5, PR722 or modified recombinant L17; preferably, wherein the polymerase comprises a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of: a amino acid substitution at position 484; an amino acid substitution at position 198 and an amino acid substitution at position 381; more preferably, wherein the polymerase comprises a modified recombinant Φ29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W and L381A. 7. Composition according to claim 3, characterized by the fact that the reporter region is configured to generate the second state of the signal responsive to a conformational change of the polymerase. 8. Composition according to claim 7, characterized by the fact that a magnitude or a time duration of the conformational change of the polymerase being responsive to the first nucleotide being complementary or non-complementary to the next nucleotide of the second polynucleotide, a magnitude or a time duration, or both, of the second state of the signal being based on the conformational change of the polymerase. 9. The composition of claim 3, wherein the second state of the signal includes an electrical signal or an optical signal. 10. Composition according to claim 1, characterized in that the elongated marker comprises a first nucleotide sequence and the first portion comprises a second nucleotide sequence that is complementary to the first nucleotide sequence. 11. System characterized by the fact that it includes the composition as defined in claim 10, and further including a measurement circuit configured to measure a first current or flow state through the opening, preferably, wherein the first current or flow state is based on the elongated marker, the first nucleotide being identifiable based on the first current or flow state. 12. The system of claim 11, wherein the measuring circuit is further configured to measure a second current or flow state through the opening, preferably on which the second current or flow state is based. at a position of the reporter region within the opening, being determinable based on the second current or flow state, whether the first nucleotide is complementary or non-complementary to the following nucleotide in the second polynucleotide. 13. Process characterized by the fact that it includes: (a) providing a nanopore including a first side, a second side and an opening extending through the first and second sides; (b) providing a plurality of nucleotides, wherein each of the nucleotides comprises an elongated marker; (c) providing a first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide; (d) providing a polymerase disposed adjacent the first side of the nanopore, with the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide, wherein the polymerase is anchored to a permanent chain including a region head, a tail region and an elongated body arranged between them, with the elongated body occurring at the opening of the nanopore; (e) determining that a first nucleotide is acting by the polymerase based on binding of the elongated tag to a first portion disposed on the elongated body; and (f) indicating, with a reporter region disposed on the elongated body, when the first nucleotide is complementary or is non-complementary to a following nucleotide in the sequence of the second polynucleotide. 14. Process, according to claim 13, characterized by the fact that it further comprises (g) determining that a subsequent nucleotide is being acted upon by the polymerase based on the binding of the elongated marker to the first portion disposed on the elongated body; and (h) indicating, with a reporter region disposed in the elongated body, when the subsequent nucleotide is complementary or non-complementary to a nucleotide that is a polynucleotide. 15. Process according to claim 14, characterized by the fact that it further comprises repeating steps (g) and (h) for a plurality of subsequent nucleotides that are complementary or non-complementary to a plurality of nucleotides that are subsequent to the next nucleotide .