METHODS FOR CHARACTERIZING A TARGET POLYNUCLEOTIDE, AND FOR FORMING A SENSOR, USE OF A HELICASE, KIT

专利摘要:
methods for characterizing a target polynucleotide, and for forming a sensor, use of a helicase, kit, analysis apparatus, and, uses of a molecular motor and a helicase. the invention relates to a new method for characterizing a target polynucleotide. the method uses a pore and a hel308 helicase or a molecular motor that is capable of binding the target polynucleotide into an inner nucleotide. the helicase or molecular motor controls the movement of the target polynucleotide through the pore.
公开号:BR112014009579B1
申请号:R112014009579-5
申请日:2012-10-18
公开日:2021-06-22
发明作者:Ruth Moysey；Andrew John Heron
申请人:Oxford Nanopore Technologies Limited；
IPC主号:

专利说明:

Field of Invention
[0001] The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or a molecular motor that is capable of binding to target polynucleotides on an inner nucleotide. The helicase or molecular motor controls the movement of target polynucleotides through the pore. Fundamentals of the Invention
[0002] There is currently a need for fast and inexpensive polynucleotide sequencing and identification technologies (eg DNA or RNA) across a wide range of applications. Existing technologies are slow and expensive, mainly because they rely on amplification techniques to produce large volumes of polynucleotide and require a high amount of specialized fluorescent chemicals for signal detection.
[0003] Transmembrane pores (nanopores) have great potential as direct electrical biosensors for polymers and a variety of small molecules. In particular, recent focus is on nanopores as a DNA sequencing technology.
[0004] When a potential is applied through a nanopore, there is a change in current flow when an analyte, such as a nucleotide, transiently resides in the drum for a certain period of time. The detection of nucleotide nanopores gives a current shift of known signature and duration. In the “Stranded Sequencing” method, a single polynucleotide strand is passed through the pore and the identity of the nucleotides is derived. Strand sequencing may involve the use of a nucleotide manipulation protein to control the movement of the polynucleotide through the pore. Invention Summary
[0005] It has been shown that a Hel308 helicase can control the movement of a polynucleotide through a pore especially when a potential, such as a voltage, is applied. The helicase is capable of moving a target polynucleotide in a controlled and stepwise fashion against or with the field resulting from the applied voltage. Surprisingly, the helicase is able to function with a high salt concentration which is advantageous for characterizing the polynucleotide and, in particular, for determining its sequence using strand sequencing. This is discussed in more detail below.
[0006] Thus, the invention provides a method of characterizing a target polynucleotide, comprising: contacting the target polynucleotides with a transmembrane pore and a Hel308 helicase so that the helicase controls the movement of the target polynucleotides through the pore and nucleotides in the target polynucleotides interact with the pore; and measuring one or more characteristics of the target polynucleotides during one or more interactions thus characterizing the target polynucleotide.
[0007] The invention also provides: a method of forming a sensor to characterize a target polynucleotide, comprising forming a complex between a pore and a Hel308 helicase and thus forming a sensor to characterize the target polynucleotide; use of a Hel308 helicase to control the movement of a target polynucleotide through a pore; a kit for characterizing a target polynucleotide comprising (a) a pore and (b) a Hel308 helicase; and an analysis apparatus for characterizing target polynucleotides in a sample, comprising a plurality of pores and a plurality of a Hel308 helicase.
[0008] It has also been shown that a molecular motor that is capable of binding with a target polynucleotide at an inner nucleotide can control the movement of the polynucleotide through a pore especially when a potential, such as a voltage, is applied. The motor is capable of moving the target polynucleotides in a controlled and stepwise fashion against or with the field resulting from the applied voltage. Surprisingly, when the motor is used in the method of the invention it is possible to control the movement of the entire strand of target polynucleotides through a nanopore. This is advantageous for characterizing the polynucleotide and, in particular, for determining its sequence using strand sequencing.
[0009] Thus, the invention also provides a method of characterizing a target polynucleotide, comprising: contacting the target polynucleotide with a transmembrane pore and a molecular motor that is capable of binding with the target polynucleotide in an internal nucleotide so that the molecular motor controls the movement of target polynucleotides through the pore and nucleotides within the target polynucleotides interact with the pore; and measuring one or more characteristics of the target polynucleotides during one or more interactions and thereby characterizing the target polynucleotide. Description of Figures
[0010] Figure 1. a) Schematic example of the use of a helicase to control the movement of DNA through a nanopore. 1) An ssDNA substrate with a ring primer containing a cholesterol tag is added on the cis side of the bilayer. The cholesterol tag binds the bilayer, enriching the substrate on the bilayer surface. 2) The helicase added to the cis compartment binds to DNA. In the presence of divalent metal ions and NTP substrate, the helicase moves along the DNA. 3) Under the applied voltage, the DNA substrate is captured by the nanopore via the leader section in the DNA. The DNA is pulled through the pore under the force of the applied potential until a DNA-bound helicase contacts the top of the pore, preventing another uncontrolled DNA translocation. During this process the dsDNA sections (like the initiator) are removed. Moving the helicase along the DNA in a 3’ to 5’ direction pulls the DNA in strands out of the pore against the applied field. 4) The helicase pulls the DNA out of the nanopore, feeding it back to the cis compartment. The last section of DNA to pass through the nanopore is the 5’ leader. 5) When the helicase moves DNA out of the nanopore it is lost back to the cis compartment. b) The design of the DNA substrate used in the Example.
[0011] Figure 2. The helicase is able to move DNA through a nanopore in a controlled fashion, producing changes in current as the DNA moves through the nanopore. Example of helicase-DNA events (180 mV, 400 mM KCl, Hepes pH 8.0, 0.15 nM 400-mer DNA, 100 nM Hel308 Mbu, 1 mM DTT, 1 mM ATP, 1 mM MgCl2). Top) Current section versus time of acquisition of DNA Hel308 400 mer events. The pore current is ~180 pA. DNA is captured by the nanopore under the force of the applied potential (+180 mV). Enzyme-fixed DNA results in a long block (at ~60pA in this condition) that shows the step changes in the current as the enzyme moves the DNA through the pore. Middle) The middle section is an enlargement of one of the DNA events, showing the capture of DNA by the enzyme, the current changes in steps as the DNA is pulled through the pore, and ending up at a long poly-T level. characteristic before leaving the nanopore. Background) increasing step changes in current as DNA is moved through the nanopore.
[0012] Figure 3. The helicase controlled the movement of the DNA resulting in a consistent pattern of current transitions as the DNA is passed through the nanopore. Examples of the last ~80 current transitions of four typical DNA events that terminate at the poly-T level. The four examples (two in 3a and two in 3b) illustrate that a consistent pattern of current transitions is observed.
[0013] Figure 4. The increased salt concentration increases the pore current and gives a greater DNA discrimination range (range = minimum current to maximum current across the DNA current transitions). Examples of helicase-DNA events (180 mV, Hepes pH 8.0, 0.15 nM 400 mer DNA SEQ ID NOs: 59 and 60, 100 nM Hel308 Mbu, 1 mM DTT, 1 mM ATP, 1 mM MgCl2) at 400 mM, 1M, and 2M KCl are shown in Figures 4 and 4c. The top traces show a complete event ending at the poly-T level, and the bottom traces show a zoomed-in section of the last 10 seconds of each event with a 150 pA y-axis constant current scale. Increasing the salt concentration to 400mM KCl to 2M KCl leads to a ~350% increase in the open pore current (I-open from ~180pA to ~850pA), and a ~200% increase in the discrimination range (~ 25pA to ~75pA). Figure 4d is a diagram of the DNA discrimination band as a function of salt concentration.
[0014] Figure 5. The helicase can control the movement of DNA in at least two modes of operation. The helicase moves along the DNA in the 3'-5' direction, but the orientation of the DNA in the nanopore (depending on which end the DNA is captured) means that the enzyme can be used to either move the DNA out of the nanopore. against the applied field, or move the DNA inside the nanopore with the applied field. a) When the 5' end of the DNA is captured, the helicase works against the direction of the field applied by the voltage, pulling the DNA in strands out of the nanopore until the DNA is ejected back into the cis chamber. On the right is an example of a Hel308 DNA helicase event running 5' down against the applied field. b) When DNA is captured 3’down in the nanopore, the enzyme moves the DNA inside the nanopore towards the field until it is completely translocated through the pore and lost in the trans side of the bilayer. On the right is an example of Hel308 DNA helicase running 3’down with the applied field. Current traces vary between the 5’down and 3’down orientations of DNA.
[0015] Figure 6. Fluorescence test to test enzyme activity. a) A custom fluorescent substrate was used to test the ability of the helicase to displace hybridized dsDNA. 1) The fluorescent substrate filament (100 nM final) has a 3' ssDNA overhang, and a 40 base section of hybridized dsDNA. The top main filament has a base of carboxyfluorescein at the 5' end, and the hybridized complement has a base with a black hole quencher (BHQ-1) at the 3' end. When hybridized, fluorescein fluorescence is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 µM of a capture filament that is complementary to the shorter filament of the fluorescent substrate is included in the test. 2) In the presence of ATP (1 mM) and MgCl2 (5 mM), helicase (100 nM) added to the substrate binds the 3' tail of the fluorescent substrate, moves along the main filament, and displaces the main filament as shown . 3) Since the complementary filament with BHQ-1 is completely displaced the fluorescein in the main filament fluoresces. 4) Excess capture strand preferably loops with complementary DNA to avoid initial substrate reannealing and loss of fluorescence. b) Graph of the initial activity rate of buffer solutions (10 mM Hepes pH 8.0, 1 mM ATP, 5 mM MgCl2, 100 nM fluorescent substrate DNA, 1 μM capture DNA) containing different concentrations of 400 mM KCl to 2 M.
Figure 7 shows examples of helicase-controlled DNA events using different Hel308s helicases (180 mV, Hepes pH 8.0, 0.15 nM 400 mer DNA SEQ ID NOs: 59 and 60, 100 nM Hel308, 1 mM DTT , 1 mM ATP, 1 mM MgCl2): Hel308 Mhu (a), Hel308 Mok (b) and Hel308 Mma (c). These represent typical examples of controlled movement of DNA through MspA nanopores that terminate at the poly-T level.
[0017] Figure 8. Fluorescence test to test the internal binding activity of the helicase. A) Customized fluorescent substrates were used to test the ability of helicases to bind to native 3' ends lacking DNA, allowing them to subsequently displace hybridized dsDNA. The fluorescent substrate filament (50 nM final) has a 3' ssDNA overhang, and a 40 base section of hybridized dsDNA. The top main strands are modified with four consecutive non-DNA-derived triethylene glycol spacers (referred to as "spacer 9" groups), either at the 3' end, or internally, at the junction between the overhang and the dsDNA (as a negative control ). In addition, the top main filament has a carboxyfluorescein base at the 5' end, and the hybridized complement has a base with a black hole quencher (BHQ-1) at the 3' end. When hybridized, fluorescein fluorescence is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. A capture filament (1 µM), which is complementary to the shorter filament of the fluorescent substrate, is included in the test. B) In the presence of ATP (1 mM) and MgCl2 (1 mM), a homologous Hel308 helicase (20 nM), added to the substrate containing 3'-terminal “spacer 9” groups, can bind to the ssDNA overhang of the substrate fluorescent, move along the main filament, and displace the complementary filament. C) Since the complementary filament with BHQ-1 is completely displaced the fluorescein in the main filament fluoresces. D) An excess capture strand preferably anneals with the complementary DNA to avoid initial substrate reannealing and loss of fluorescence.
[0018] Figure 9 shows the relative rates of position change of Hel308-mediated dsDNA compared to unmodified DNA in 3' and -"spacer 9" DNA in 3' in 400 mM NaCl, 10 mM Hepes, pH 8, 0.1 mM ATP, 1 mM MgCl2, 50 nM fluorescent substrate DNA, 1 μM capture DNA.
[0019] Figure 10. Schematic diagram of the use of a helicase to control the movement of DNA through a nanopore that is employed in example 5. A) A DNA substrate (SEQ ID NOs 67 and 68) with a ring primer (SEQ ID NO 69) with an attached cholesterol tag is added to the cis of the bilayer. The cholesterol tag binds the bilayer, enriching the substrate on the bilayer surface. The helicase added to the cis compartment binds to the 4 bp leader of SEQ ID NO 67. B). Under an applied voltage, the DNA substrate is captured by the nanopore via the 5' leader section of the DNA, which strips SEQ ID NO 69. C) Under the applied field strength the DNA is pulled into the pore until the helicase bonded contact the top of the pore and prevent further uncontrolled translocation. In this process the antisense strand of SEQ ID NO 68 is removed from the DNA strand. D) In the presence of divalent metal ions and NTP substrate, the helicase at the top of the pore moves along the DNA and controls the translocation of the DNA through the pore. Moving the helicase along the DNA in a 3’ to 5’ direction pulls the strand DNA out of the pore against the applied field. Single-stranded DNA exposed on the cis side (3' in this case) is available for other helicases to bind or at the terminal nucleotide on an inner nucleotide. E) If the helicase in the pore disengages from the DNA, the DNA is pulled inside the pore into the field until the next helicase in the DNA reaches the pore. The helicase in the pore pulls the DNA out of the nanopore, feeding it back into the cis compartment. The last section of DNA to pass through the nanopore is the 5’ leader. F) When the helicase moves the DNA out of the nanopore it is lost back to the cis compartment. Arrows indicate the direction of DNA movement.
[0020] Figure 11 shows diagrams of the data indicating how the position of the DNA region in the 900-mer nanopore (y-axis) varied as the Mbu homologous Hel308 helicase controlled the translocation of the DNA strand through the MspA pore (x-axis) ) during each helicase event. AC shows examples of typical full-strand DNA translocation events from approximately the start of the strand through the end of the strand (exiting via the poly-T leader), while event D shows an example of incomplete DNA translocation, where the enzyme detachment means that the DNA will never make it to the end of the strand. Slips (for example, such as large slips highlighted by dotted circles) indicate the sequence falling back to a previous point on the filament, and are the result of enzyme detachment. When an enzyme detaches, the DNA will be pulled back under the force of the field within the nanopore until another enzyme further along the filament contacts the pore, then continuing the helicase movement.
Figure 12 shows diagrams of the data indicating how the position of 900 mers varied as the Tga homologous Hel308 helicase controlled the translocation of the DNA strand through the MspA pore. Events A-D show the translocation of the complete DNA strand.
Figure 13 shows a fluorescence test to compare the processability of the enzyme Hel308 Mbu helicase (SEQ ID NO: 10) with that of the Hel 308 Mok helicase (SEQ ID NO: 29). A custom fluorescent substrate was used to test the ability of the helicase to displace hybridized dsDNA. The fluorescent substrate (50 nM final) has a 3' ssDNA overhang, and 80 and 33 base pair sections of the hybridized dsDNA (section A, SEQ ID NO: 70). The main "template" filament from the background is hybridized to an 80 nt "blocker" filament (SEQ ID NO: 71), adjacent to its 3' overhang, and a 33 nt fluorescent probe (SEQ ID NO: 72), labeled at its 5' and 3' ends with carboxyfluorescein (FAM) and bases of the black hole extinguisher (BHQ-1), respectively. When hybridized, FAM is distant from BHQ-1 and the substrate is essentially fluorescent. In the presence of ATP (1 mM) and MgCl2 (10 mM), the helicase (20) binds to the 3' overhang of the substrates (SEQ ID NO: 70), moves along the lower filament, and begins to displace the 80 nt blocking filament (SEQ ID NO: 71), as shown in section B. If processive, helicase displaces the fluorescent probe as well (section C, SEQ ID NO: 72 labeled with carboxyfluorescein (FAM) at its 5' end a “black hole” fire extinguisher (BHQ-1) at its 3' end). The fluorescent probe is designed in such a way that its 5’ and 3’ ends are self-complementary and thus form a kinetically stable clamp once displaced, preventing the probe from re-annealing the template filament (section D). Upon formation of the staple product, FAM is brought into the vicinity of BHQ-1 and its fluorescence is quenched. An enzyme with processability, capable of displacing the 80-mer “blocker” (SEQ ID NO: 71) and fluorescent filaments (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5' end and a “black extinguisher” -hole” (BHQ-1) at its 3' end) will thus lead to a decrease in fluorescence over time. However, if the enzyme has a processability of less than 80 nt it would be unable to displace the fluorescent filament (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5' end and a black hole extinguisher ( BHQ-1) at its 3' end) and therefore the “blocker” filament (SEQ ID NO: 71) would re-anneal with a main filament from the bottom (section E).
[0023] Figure 14 shows additional fluorescent substrates that were used for control purposes. The substrate used as a negative control was identical to that described in Figure 3, but lacking the overhang in (section A, (SEQ ID NOs: 71, 72 (labeled with a carboxyfluorescein (FAM)) at its 5' end and an extinguisher " black hole” (BHQ-1) at its end in 3') and 73)). A substrate similar to that depicted in Figure 3, but lacking the 80 base pair section (SEQ ID NOs: 72 (labeled with a carboxyfluorescein (FAM) at its 5' end and a black hole extinguisher (BHQ-1) in its end in 3') and 74), was used as a positive control for active but not necessarily processive helicases (section B).
[0024] Figure 15 shows a graph of time-dependent fluorescence changes when testing Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase (SEQ ID NO: 29) against a processability substrate shown in Figure 13 in buffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl2, 50 nM fluorescent substrate DNA (SEQ ID NOs: 70, 71 and 72 (labeled with a carboxyfluorescein (FAM)) at its end in 5' and a black hole extinguisher (BHQ-1) at its 3' end.) The decrease in fluorescence exhibited by Hel308 Mok denotes the increased processability of these complexes as compared to Hel308 Mbu (SEQ ID NO: 10).
[0025] Figure 16 shows a graph of time-dependent fluorescence changes when testing Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase (SEQ ID NO: 29) against substrate with positive control processability (shown in Figure 14 section B, SEQ ID NOs: 72 (labeled with a carboxyfluorescein (FAM) at its 5' end and a black hole fire extinguisher (BHQ-1) at its 3' end) and 74) in buffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl2, 50 nM fluorescent substrate DNA (SEQ ID NOs: 72 (labeled with a carboxyfluorescein (FAM)) at its 5' end and a fire extinguisher " black hole” (BHQ-1) at its end in 3') and 74)). This positive control demonstrated that both helicases were indeed active, as denoted by a decrease in fluorescence for both samples. Sequence Listing Description
[0026] SEQ ID NO: 1 shows the codon-optimized polynucleotide sequence encoding the MS-B1 mutant MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.
SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B1 mutant of the MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.
[0028] SEQ ID NO: 3 shows the sequence of polynucleotides encoding an α-haemolysin-E111N/K147N subunit (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19): 7702-7707).
[0029] SEQ ID NO: 4 shows the amino acid sequence of an α-HL-NN subunit.
[0030] SEQ ID NOs: 5 to 7 shows the amino acid sequence of MspB, C and D.
[0031] SEQ ID NO: 8 shows the amino acid sequence of the motif of Hel308.
[0032] SEQ ID NO: 9 shows the amino acid sequence of the extended Hel308 motif.
SEQ ID NOs: 10 to 58 shows the amino acid sequence of Hel308 helicases and motifs in Table 5.
[0034] SEQ ID NOs: 59 to 74 show the sequences used in the Examples.
[0035] SEQ ID NO: 75 shows the sequence of Hel308 Dth in alignment from page 57 onwards.
[0036] SEQ ID NO: 76 shows the sequence of Hel308 Mmar in alignment on page 57 onwards.
[0037] SEQ ID NO: 77 shows the sequence of Hel308 Nth in alignment on page 57 onwards.
[0038] SEQ ID NO: 78 shows the consensus sequence in the alignment on page 57 onwards. Detailed Description of the Invention
[0039] It should be understood that the different applications of the products and methods described can be adapted to the specific needs of the technique. It should also be understood that the terminology used herein is for the purpose of describing the particular embodiments only, and is not intended to be limiting.
[0040] In addition, as used in this report and appended claims, the singular forms “a”, “an”, and “o, a,” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a pore" includes two or more such pores, reference to "a helicase" includes two or more such helicases, reference to "a polynucleotide" includes two or more such polynucleotides, and the like .
[0041] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Hel308 Methods of the Invention
[0042] The invention provides a method of characterizing a target polynucleotide. The method comprises contacting the target polynucleotide with a transmembrane pore and a Hel308 helicase such that the helicase controls movement of the target polynucleotide through the pore and the nucleotides in the target polynucleotide interact with the pore. One or more characteristics of the target polynucleotide are then measured using standard methods known in the art. Steps (a) and (b) are preferably carried out with a potential applied through the pore. As discussed in more detail below, the applied potential typically results in the formation of a complex between the pore and the helicase. The applied potential can be a voltage potential. Alternatively, the applied potential can be a chemical potential. An example of this is the use of a salt gradient across the lipid membrane. A salt gradient is described in Holden et al., J Am Chem Soc. 2007 Jul 11;129(27):8650-5.
[0043] In some cases, current passing through the pore during one or more interactions is used to determine the target polynucleotide sequence. This is filament sequencing.
[0044] Method has several advantages. First, it was surprisingly shown that Hel308s helicase has a surprisingly high salt tolerance, and thus the method of the invention can be carried out with high salt concentrations. In the context of Strand Sequencing, a charge vehicle, such as a salt, is needed to create a conductive solution to apply a voltage shift to capture and translocate the target polynucleotide and to measure the resulting sequence-dependent current changes as the polynucleotide passes through the pore. Since signal measurement is dependent on salt concentration, it is advantageous to use high salt concentrations to increase the amplification of the acquired signal. High salt concentrations provide a high signal-to-noise ratio and allow currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations. For strand sequencing, salt concentrations in excess of 100 mM are ideal and salt concentrations of 1 M and above are preferred. It has surprisingly been shown that Hel308s helicase will work effectively with salt concentrations as high as, for example, 2M.
[0045] Second, when a voltage is applied, the Hel308s helicase can surprisingly move the target polynucleotide in two directions, ie, with or against the field resulting from the applied voltage. Thus, the method of the invention can be carried out in one of the two ways mentioned. Different signals are obtained depending on which direction the target polynucleotide moves through the pore, ie, towards or against the field. This is discussed in more detail below.
[0046] Third, the Hel308s helicase typically moves the target polynucleotide through the pore one nucleotide at a time. The Hel308s helicase can therefore function as a single base coil. This is of course advantageous when sequencing a target polynucleotide because substantially all, if not all, of the nucleotides in the target polynucleotide can be identified using the pore.
[0047] Fourth, Hel308 helicases are capable of controlling the movement of single-stranded polynucleotides and double-stranded polynucleotides. This means that a variety of different polynucleotide targets can be characterized according to the invention.
[0048] Fifth, the Hel308s helicase appears very resistant to the field resulting from the applied voltages. It was found that very little polynucleotide movement under an “unzipped” condition. This is important because it means that there are no complications from unwanted “backward” movement when moving polynucleotides against the field resulting from the applied voltage.
[0049] Sixth, Hel308 helicases are easy to produce and easy to manipulate. Its use has therefore contributed to a straightforward and less expensive method of sequencing.
[0050] The method of the invention is intended to characterize a target polynucleotide. A polynucleotide, like a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid can comprise any combination of any nucleotides. The nucleotides can be naturally or artificially occurring. One or more nucleotides in the target polynucleotide can be oxidized or methylated. One or more nucleotides in the polynucleotide can be damaged. One or more nucleotides in the target polynucleotide can be modified, for example, with a label or tag. The target polynucleotide can comprise one or more spacers.
[0051] A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine, guanine, thymine, uracil and cytosine. Sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates can be attached to the 5' or 3' side of a nucleotide.
Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), adenosine monophosphate cyclic (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) and deoxycatidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
[0053] A nucleotide can be abasic (ie, a nucleobase is missing).
[0054] The polynucleotide can be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded.
The polynucleotide can be a nucleic acid such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target polynucleotide can comprise an RNA strand hybridized to a DNA strand. The polynucleotide can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or other synthetic polymers with side chains of nucleotide.
[0056] All or only part of the target polynucleotide can be characterized using this method. The target polynucleotide can be of any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs in length, or 100,000 or more nucleotide pairs in length.
[0057] Target polynucleotide is present in any appropriate sample. The invention is typically performed on a sample that is known to contain or suspected to contain the target polynucleotide. Alternatively, the invention can be performed on a sample to confirm the identity of one or more target polynucleotides whose presence in the sample is known or expected.
[0058] The sample may be a biological sample. The invention can be carried out in vitro on a sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically Archean, prokaryotic or eukaryotic and typically belongs to one of five kingdoms: plantae, animalia, fungi, monera and protist. The invention can be carried out in vitro on a sample obtained from or extracted from a virus. The sample is preferably a fluid sample. The sample typically comprises a fluid from the patient's body. The sample can be urine, lymph, saliva, mucus or amniotic fluid, but preferably blood, plasma or serum. Typically, the sample is of human origin, but alternatively it could be from another mammalian animal such as commercially bred animals such as horses, calf, sheep or pigs or it could alternatively be from pets such as cats or dogs. Alternatively, a plant-originated sample is typically obtained from a commercial crop, such as a cereal, vegetable, fruit or vegetable, for example, wheat, barley, oats, canola, corn, soybeans, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton.
[0059] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory testing.
[0060] The sample is typically processed before being tested, for example, by centrifugation or by passing through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample can be measured immediately after being taken. The sample can also typically be stored prior to testing, preferably below -70°C.
[0061] A transmembrane pore is a structure that allows hydrated ions triggered by an applied potential to flow from one side of the membrane to the other side of the membrane.
[0062] Any membrane can be used according to the invention. Appropriate membranes are well known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic layer can be a monolayer or bilayer.
[0063] The membrane is preferably a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by recording from a single channel. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Appropriate lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer, or a liposome. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are described in International Patent Application PCT/GB08/000563 (published as WO 2008/102121), International Patent Application PCT/GB08/004127 (published as WO 2009/077734) and International Patent Application PCT/GB2006 /001057 (published as WO 2006/100484).
[0064] Methods to form lipid bilayers are known in the art. Appropriate methods are described in the Example. Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is transported in an aqueous solution/passage. air interface on each side of an opening that is perpendicular to the interface.
[0065] Montal and Mueller's method is popular because it is a relatively straightforward and cost-effective method of forming good quality lipid bilayers that are suitable for pore protein insertion. Other common methods of bilayer formation include binding clips, painted bilayers, and liposome bilayer patch clips.
[0066] In a preferred embodiment, the lipid bilayer is formed as described in International Patent Application PCT/GB08/004127 (published as WO 2009/077734).
[0067] In another preferred embodiment, the membrane is a solid state layer. A solid state layer is not of biological origin. In other words, a solid state layer is not derived from or isolated from a biological environment such as an organism or cell, or a synthetically fabricated version of a biologically available structure. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si3N4, A1203, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as rubber silicone with two-component curing addition, and glazing. The solid-state layer can be formed from monoatomic layers, such as graphene, or layers that are only a few atoms thick. Graphene layers are described in International Patent Application PCT/US2008/010637 (published as WO 2009/035647).
[0068] The method is typically carried out using (i) an artificial bilayer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted thereto. The method is preferably carried out using an artificial lipid bilayer. The bilayer can comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. Appropriate apparatus and conditions are discussed below. The method is typically carried out in vitro.
[0069] The polynucleotide can be coupled with the membrane. This can be done using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer (as discussed in detail below), the polynucleotide is preferably coupled to the membrane via a polypeptide present on the membrane or a hydrophobic anchor present on the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube or amino acid.
[0070] The polynucleotide can be directly coupled to the membrane. The polynucleotide is preferably coupled to the membrane via a linker. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycols (PEGs) and polypeptides. If a polynucleotide is directly coupled to the membrane, then some data will be lost as the characterization cycle cannot continue to the end of the polynucleotide due to the distance between the membrane and the helicase. If a linker is used, then the polynucleotide can be processed to completion. If a linker is used, the linker can be attached to the polynucleotide at any position. The linker is preferably affixed to the polynucleotide in the polymer tail.
[0071] Coupling can be either stable or transient. For certain applications, the transient nature of the coupling is preferred. If a stable coupling molecule has been attached directly to either the 5' or 3' end of a polynucleotide, then some data will be lost as the characterization cycle cannot continue to the end of the polynucleotide due to the distance between the bilayer and the site active helicase. If coupling is transient, when the coupled end becomes randomly freed from the bilayer, then the polynucleotide can be processed to completion. Chemical groups that form stable or transient bonds with the membrane are discussed in more detail below. The polynucleotide can be transiently coupled to an amphiphilic layer or lipid bilayer using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of 6 to 30 carbon atoms, such as hexanoic acid, can be used.
[0072] In preferred embodiments, the polynucleotide is coupled to a lipid bilayer. The coupling of polynucleotides to synthetic lipid bilayers has been previously performed with several different binding strategies. These are summarized in Table 1 below. Table 1


[0073] Polynucleotides can be functionalized using a modified phosphoramidite in the synthesis reaction, which is easily compatible for the addition of reactive groups such as thiol, cholesterol, lipid and biotin groups. These different chemical attachments give a menu of binding options for polynucleotides. Each different modification group ties the polynucleotide in a slightly different way and the coupling is not always permanent thus giving different residence times for the polynucleotide to the bilayer. The advantages of transient coupling are discussed below.
[0074] Coupling of polynucleotides can also be achieved by various other means, provided that the reactive group can be added to the polynucleotide. The addition of reactive groups to both ends of the DNA was previously recorded. A thiol group can be added at the 5' of the ssDNA using polynucleotide kinase and ATPYS (Grant, GP and PZ Qin (2007). "A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids." Nucleic Acids Res 35 (10): e77). A more diverse selection of chemical groups, such as biotin, thiols and fluorophores, can be added using terminal transferase to incorporate oligonucleotides modified to the 3' of ssDNA (Kumar, A., P. Tchen, et al. (1988). labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169(2): 376-82).
[0075] Alternatively, the reactive group can be considered to be the addition of a short piece of DNA complementary to one already coupled with the bilayer, so that binding can be achieved via hybridization. Ligation of short pieces of ssDNA have been described using T4 RNA ligase I (Troutt, AB, MG McHeyzer-Williams, et al. (1992) "Linkage-anchored PCR: a simple amplification technique with single-sided specificity." Acad Sci USA 89(20):9823-5). Alternatively either ssDNA or dsDNA can be linked to native dsDNA and then the two strands are separated by thermal or chemical denaturation. For native dsDNA, you can add either a piece of ssDNA to one or both ends of, or dsDNA to one or both ends. Then, when the duplex is fused, each single strand will have either a 5' or 3' modification if ssDNA was used for binding or a modification at the 5' end, the 3' end or both if dsDNA was used for the call. If the polynucleotide is a synthetic strand, chemical coupling can be incorporated during chemical synthesis of the polynucleotide. For example, the polynucleotide can be synthesized using a primer and a reactive group attached to it.
[0076] A common technique for modifying genomic DNA sections is the use of polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, multiple copies of the same section of DNA can be generated, where for each 5' copy of each strand in the duplex will be a synthetic polynucleotide. By using an antisense primer that has a reactive group, such as a cholesterol, thiol, biotin or lipid, each copy of the amplified target DNA will contain a reactive group for coupling.
The transmembrane pore is preferably a protein transmembrane pore. A protein transmembrane pore is a polypeptide or grouping of polypeptides that allows hydrated ions, such as analyte, to flow from one side of a membrane to the other side of the membrane. In the present invention, the protein transmembrane pore is capable of forming a pore that allows hydrated ions to direct an applied potential to flow from one side of the membrane to the other. The protein transmembrane pore preferably allows analyte like nucleotides to flow from one side of the membrane, as a lipid bilayer, to the other. The protein transmembrane pore lets a polynucleotide, such as DNA or RNA, be removed through the pore.
[0078] Protein transmembrane pore can be a monomer or an oligomer. The pore is preferably composed of several repeating subunits, such as 6, 7 or 8 subunits. The pore is most preferably a heptameric or octameric pore.
[0079] Protein transmembrane pore typically comprises a barrel or channel through which ions can flow. Pore subunits typically encircle a central axis and contribute filaments to a β-drum or transmembrane channel or a helical α-beam or transmembrane channel.
[0080] A protein transmembrane pore barrel or channel typically comprises amino acids that facilitate interaction with the analyte, such as nucleotides, polynucleotides or nucleic acids. These amino acids are preferably located near the drum or channel constriction. The protein transmembrane pore typically comprises one or more positively charged amino acids such as arginine, lysine or histidine, or aromatic amino acids such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides or nucleic acids.
[0081] Protein transmembrane pores for use in accordance with the invention may be derived from β-trum pores or α-beam helix pores. The pores of the β-drum comprise a drum or channel which is formed from β filaments. Suitable β-drum pores include, but are not limited to, β-toxins such as α-haemolysin, anthrax toxin and leucocidins, and outer membrane proteins/bacterial pores such as Mycobacterium smegmatis (Msp), e.g., MspA, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and autotransport lipoprotein (NalP). The pores of the helix α-beam comprise a drum or channel which is formed from α helices. Helix α-beam pores include, but are not limited to, inner membrane proteins and α-outer membrane proteins such as WZA toxin and ClyA. The transmembrane pore can be derived from Msp or from α-hemolysin (α-HL).
The protein transmembrane pore is preferably derived from Msp, preferably from MspA. Such a pore will be oligomeric and will typically comprise 7, 8, 9 or 10 monomers derived from Msp. The pore may be an Msp-derived homo-oligomeric pore comprising identical monomers. Alternatively, the pore may be an Msp-derived hetero-oligomeric pore comprising at least one monomer that differs from the others. Preferably the pore is derived from MspA or a homologue or paralog thereof.
[0083] An Msp-derived monomer comprises the sequence shown in SEQ ID NO: 2 or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. It includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence that varies from that of SEQ ID NO: 2 and that retains its ability to form a pore. The ability of a variant to form a pore can be tested using any method known in the art. For example, the variant can be inserted into a lipid bilayer along with other appropriate subunits and its ability to oligomerize to form a pore can be determined. Methods are known in the art for inserting subunits into membranes such as lipid bilayers. For example, subunits can be suspended in a purified form in a solution containing a lipid bilayer so that it diffuses with the lipid bilayer and is inserted by bonding with the lipid bilayer and assembled into a functional state. Alternatively, the subunits can be inserted directly into the membrane using the "pick and place" method described in M.A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Patent Application No. PCT/GB2006/001057 (published as WO 2006/100484).
Over the full length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, a variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least minus 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 throughout the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a span of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids (“difficult homology”).
[0085] Methods standard in the art can be used to determine homology. For example, the UWGCG Package provides the BESTFIT program that can be used to calculate homology, eg used in its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate sequence homology or alignment (such as identifying equivalent residues or corresponding sequences (typically in their default settings)), for example, as described in Altschul SF (1993) J Mol Evol 36 :290-300; Altschul, S.F et al (1990) J Mol Biol 215:40310. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
[0086] SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer monomer. The variant may comprise any of the mutations in the MspB, C or D monomers compared to MspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7. In particular, a variant may comprise the following substitution present in MspB: A138P. The variant may comprise one or more of the following substitutions present in MspC: A96G, N102E and A138P. The variant may comprise one or more of the following mutations present in MspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant may comprise combinations of one or more of the Msp B, C and D mutations and substitutions. The variant preferably comprises the L88N mutation. The SEQ ID NO: 2 variant has the L88N mutation in addition to all MS-B1 mutations and is called MS-B2. The pore used in the invention is preferably MS-(B2)8.
Amino acid substitutions can be made in the amino acid sequence of SEQ ID NO: 2 in addition to those discussed above, for example, up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or similar charge to the amino acids they replace. Alternatively, conservative substitution introduces another amino acid that is aromatic or aliphatic in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 major amino acids as defined in Table 2 below. Where amino acids have similar polarity, this can be determined also by referring to the hydropathy scale for amino acid side chains in Table 3.

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 can be further deleted from the above described polypeptides. Up to 1, 2, 3, 4, 5, 10, 20 or 30 can be deleted, or more.
[0089] Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore-forming activity. Fragments can be at least 50, 100, 150 or 200 amino acids in length. Such fragments can be used to produce the pores. A fragment preferably comprises the pore-forming domain of SEQ ID NO: 2. Fragments should include one of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2. Typically, fragments include all 88 residues. , 90, 91, 105, 118 and 134 of SEQ ID NO:2.
[0090] One or more amino acids may alternatively be or added to the polypeptides described above. An extension may be provided at the amino terminus or carboxy terminus of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The span can be quite short, for example 1 to 10 amino acids in length. Alternatively, the extension can be longer, for example up to 50 or 100 amino acids. A carrier protein can be fused to the amino acid sequence in accordance with the invention. Other fusion proteins are discussed in more detail below.
[0091] As discussed above, a variant is a polypeptide that has an amino acid sequence that varies from that of SEQ ID NO: 2 and that retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 2 that are responsible for pore formation. The pore-forming capacity of Msp, which contains a β-drum, is provided by β-sheets in each subunit. A variant of SEQ ID NO:2 typically comprises the regions in SEQ ID NO:2 that form β-sheets. One or more modifications can be made to the regions of SEQ ID NO: 2 that form β-sheets so long as the resulting variant retains its ability to form a pore. A variant of SEQ ID NO: 2 preferably includes one or more modifications, such as substitutions, additions or deletions, within its a-helix and/or loop regions.
Msp-derived monomers can be modified to aid identification or purification, for example, by the addition of histidine residues (an his tag), aspartic acid residues (an asp tag), a streptavidin tag, or a Flag tag , or by adding a signal sequence to promote its secretion from a cell where the polypeptides do not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag over a native or engineered position in the pore. An example of this would be to react a gel exchange reagent with an engineered cysteine on the outside of the pore. This has been demonstrated as a method to separate hemolysin heterooligomers (Chem Biol. 1997 Jul; 4(7):497-505).
[0093] Msp-derived monomer can be labeled with a revealing label. The developer label can be any suitable label that allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, for example 125 I, 35 S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.
[0094] Msp-derived monomer can be produced also using D-amino acids. For example, the Msp-derived monomer can comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins and peptides.
[0095] Msp-derived monomer contains one or more specific modifications to facilitate nucleotide discrimination. The Msp-derived monomer may also contain other non-specific modifications as long as they do not interfere with pore formation. A number of non-specific side chain modifications are known in the art and can be made to the side chains of the Msp-derived monomer. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methylacetimidate or acylation with acetic anhydride.
[0096] The Msp-derived monomer can be produced by standard methods known in the art. The Msp-derived monomer can be produced synthetically or by recombinant means. For example, the pore can be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing pores are discussed in International Patent Applications PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133 (published as WO 2010/086603). Methods for inserting pores into membranes are discussed.
[0097] Protein transmembrane pore is also preferably derived from α-hemolysin (α-HL). The wild-type α-HL pore is made up of seven identical monomers or subunits (ie, it is heptameric). The sequence of an α-haemolysin-NN monomer or subunit is known in SEQ ID NO: 4. The protein transmembrane pore preferably comprises seven monomers each comprising the sequence shown in SEQ ID NO: 4 or a variant thereof. Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290 and 294 of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4 form part of an α-HL drum or channel constriction.
In such embodiments, a pore comprising seven proteins or monomers each comprising the sequence shown in SEQ ID NO: 4 or a variant thereof is preferably used in the method of the invention. The seven proteins can be the same (homoheptamers) or different (heteroheptamers).
A variant of SEQ ID NO: 4 is a protein that has an amino acid sequence that varies from that of SEQ ID NO: 4 and that retains its pore-forming ability. The ability of a variant to form a pore can be tested using a method known in the art. For example, a variant can be inserted into a lipid bilayer along with other appropriate subunits and its ability to oligomerize to form a pore can be determined. Methods are known in the art for inserting subunits into membranes such as lipid bilayers. Appropriate methods are discussed above.
The variant may include modifications that facilitate covalent binding to or interaction with the helicase. The variant preferably comprises one or more reactive cysteine residues that facilitate binding with the helicase. For example, one may include a cysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45, 50, 51, 237, 239 and 287 and/or at the amino or carboxy terminus of SEQ ID NO: 4. Preferred variants comprise a substitution of the residue at position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 with cysteine (A8C, T9C, N17C, K237C, S239C or E287C). The variant is preferably any of the variants described in International Patent Application PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).
[00101] The variant may also include modifications that facilitate any interaction with nucleotides.
[00102] The variant can be a naturally occurring variant that is naturally expressed by an organism, for example, by a Staphylococcus bacterium. Alternatively, the variant can be expressed in vitro or recombinantly by bacteria such as Escherichia coli. Variants also include non-naturally occurring variants produced by recombinant technology. Over the entire length of the amino acid sequence of SEQ ID NO: 4, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, a variant polypeptide can be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 4 throughout the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a span of 200 or more, for example 230, 250, 270 or 280 or more, of amino acids contiguous (“hard homology”). Homology can be determined as discussed above.
[00103] Amino acid substitutions can be made to the amino acid sequence of SEQ ID NO: 4 in addition to those discussed above, for example, up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions can be made as discussed above.
[00104] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 4 can be further deleted from the above described polypeptides. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues can be deleted, or more.
[00105] Variants can be fragments of SEQ ID NO: 4. Such fragments retain the pore-forming activity. Fragments can be at least 50, 100, 200 or 250 amino acids in length. A fragment preferably comprises the pore forming domain of SEQ ID NO: 4. Fragments typically include residues 119, 121, 135, 113 and 139 of SEQ ID NO: 4.
[00106] One or more amino acids may alternatively or additionally be added to the polypeptides described above. An extension may be provided at the amino terminus or carboxy terminus of the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. The extension can be very short, for example 1 to 10 amino acids in length. Alternatively, the extension can be longer, for example up to 50 or 100 amino acids. A carrier protein can be fused to a pore or variant.
[00107] As discussed above, a variant of SEQ ID NO:4 is a subunit that has an amino acid sequence that varies from that of SEQ ID NO:4 and that retains its ability to form a pore. The variant typically contains the regions of SEQ ID NO: 4 that are responsible for pore formation. The pore-forming capacity of α-HL, which contains a β-drum, is provided by β-filaments in each subunit. A variant of SEQ ID NO:4 typically comprises the regions in SEQ ID NO:4 that form β-strands. The amino acids of SEQ ID NO: 4 that form β-strands are discussed above. One or more modifications can be made to the regions of SEQ ID NO: 4 that form β-strands as long as the resulting variant retains its ability to form a pore. Specific modifications that can be made to the β-strand regions of SEQ ID NO: 4 are discussed below.
[00108] A variant of SEQ ID NO: 4 preferably includes one or more modifications, such as substitutions, additions or deletions, within its a-helix and/or loop regions. Amino acids that form a-helices and loops are discussed above.
[00109] A variant can be modified to aid its identification or purification as discussed above.
[00110] Pores derived from α-HL can be produced as discussed above with reference to pores derived from Msp.
[00111] In some embodiments, the protein transmembrane pore is chemically modified. The pore can be chemically modified in any way and at any location. The protein transmembrane pore is preferably chemically modified by binding a molecule to one or more cysteines (cysteine binding), binding a molecule to one or more lysines, binding a molecule to one or more unnatural amino acids, enzyme modification of an epitope or modification of a terminal. Appropriate methods for carrying out such modifications are well known in the art. The protein transmembrane pore can be chemically modified by the binding of any molecule. For example, the pore can be chemically modified by the attachment of a dye or a fluorophore.
[00112] Any number of monomers in the pore can be chemically modified. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers is preferably chemically modified as discussed above.
[00113] The reactivity of cysteine residues can be increased by modifying adjacent residues. For example, the basic groups of the flanking residues of arginine, histidine or lysine will shift the pKa of cysteines from the thiol group to that more reactive S-group. The reactivity of cysteine residues can be protected by thiol protecting groups like dTNB. These can be reacted with one or more cysteine residues from the pore before the binder is fixed.
[00114] The molecule (with which the pore is chemically modified) can be attached directly to the pore or attached via a linker as described in International Patent Applications PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09 /001679 (published as WO 2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).
[00115] Any Hel308 helicase can be used according to the invention. Hel308 helicases are also known as ski2 type helicases and the two terms can be used interchangeably.
The Hel308 helicase typically comprises the amino acid motif Q-X1-X2-G-R-A-G-R (in the following parts called the Hel308 motif; SEQ ID NO: 8). The Hel308 motif is typically part of the helicase VI motif (Tuteja and Tuteja, Eur. J. Biochem. 271, 1849-1863 (2004)). X1 can be C, M or L. X1 is preferably C. X2 can be any amino acid residue. X2 is typically a hydrophobic or neutral residue. X2 can be A, F, M, C, V, L, I, S, T, P or R. X2 is preferably A, F, M, C, V, L, I, S, T or P. X2 is more preferably A, M or L. X2 is most preferably A or M.
The Hel308 helicase preferably comprises the motif Q-X1-X2-G-R-A-G-R-P (in the following parts called the extended Hel308 motif; SEQ ID NO: 9) wherein X1 and X2 are as described above.
[00118] The most preferred Hel308 motifs and extended Hel308 motifs are shown in Table 5 below. Hel308 helicase can comprise any of these preferred motifs.
[00119] Hel308 helicase is preferably one of the helicases shown in Table 4 below or a variant thereof.











The Hel308 helicase is most preferably one of the helicases shown in Table 5 below, or a variant thereof. The Hel308 helicase most preferably comprises the sequence of one of the helicases shown in Table 5, i.e., one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58, or a variant thereof.


The Hel308 helicase more preferably comprises (a) the sequence of Hel308 Mbu (i.e., SEQ ID NO: 10) or a variant thereof, (b) the sequence of Hel308 Pfu (i.e., SEQ ID NO: 13 ) or a variant thereof, (c) the Hel308 Mok sequence (i.e., SEQ ID NO: 29) or a variant thereof, (d) the Hel308 Mma sequence (i.e., SEQ ID NO: 45) or a variant thereof, (e) the Hel308 Fac sequence (i.e., SEQ ID NO:48) or a variant thereof, or (f) the Hel308 Mhu sequence (i.e., SEQ ID NO:52) or a variant of the same. The Hel308 helicase most preferably comprises the sequence shown in SEQ ID NO: 10 or a variant thereof.
The Hel308 helicase most preferably comprises (a) the Hel308 Tga sequence (i.e., SEQ ID NO: 33) or a variant thereof, (b) the Hel308 Csy sequence (i.e., SEQ ID NO: 22 ) or a variant thereof, or (c) the Hel308 Mhu sequence (i.e., SEQ ID NO:52) or a variant thereof. The Hel308 helicase most preferably comprises the sequence shown in SEQ ID NO: 33 or a variant thereof.
[00123] A variant of a Hel308 helicase is an enzyme that has an amino acid sequence that varies from the wild-type helicase and that retains polynucleotide binding activity. In particular, a variant of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 is an enzyme that has an amino acid sequence that varies from any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 and which retains polynucleotide binding activity. A variant of SEQ ID NO: 10 or 33 is an enzyme that has an amino acid sequence that varies from that of SEQ ID NO: 10 or 33 and that retains polynucleotide binding activity. The variant retains helicase activity. The variant must work in at least one of the two ways discussed below. Preferably, the variant works in two modes. The variant may include modifications that facilitate manipulation of the polynucleotide encoding the helicase and/or facilitate its activity at high salt concentrations and/or room temperature. Variants typically differ from wild-type helicase in regions outside of the Hel308 motif or extended Hel308 motif discussed above. However, variants may include modifications within this reason(s).
[00124] About the full length of an amino acid sequence of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58, as SEQ ID NO: 10 or 33, the variant will preferably be at least 30% homologous to sequence based on amino acid identity. More preferably, a polypeptide variant may be at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to an amino acid sequence of any one of SEQ ID NOs: 10, 13 , 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 , 53, 54, 55 and 58, as SEQ ID NO: 10 or 33, over the complete sequence. One can have at least 70%, for example at least 80%, at least 85%, at least 90% or at least 95%, amino acid identity over a piece of 150 or more, for example 200, 300, 400 , 500, 600, 700, 800, 900 or 1000 or more, contiguous amino acids ("difficult homology"). Homology is determined as described above. The variant may differ from the wild-type sequence in any of the ways discussed above with reference to SEQ ID NOs: 2 and 4.
[00125] A variant of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 preferably comprises the Hel308 motif, the extended Hel308 motif of the relevant wild-type sequence. For example, a variant of SEQ ID NO: 10 preferably comprises the Hel308 motif of SEQ ID NO: 10 (QMAGRAGR; SEQ ID NO: 11) the extended Hel308 motif of SEQ ID NO: 10 (QMAGRAGRP; SEQ ID NO: 12). The Hel308 motif and extended Hel308 motif of each of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43 , 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 are shown in Table 5. However, a variant of any one of SEQ ID NOs: 10, 13, 16 , 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55 and 58 may comprise the Hel308 motif or Hel308 motif extended from a different wild-type sequence. For example, a variant of SEQ ID NO: 10 can comprise the Hel308 motif of SEQ ID NO: 13 (QMLGRAGR; SEQ ID NO: 14) the extended Hel308 motif of SEQ ID NO: 13 (QMLGRAGRP; SEQ ID NO: 15). A variant of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 may comprise any of the preferred motifs shown in Table 5. Variants of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 may also include modifications within the Hel308 motif, from the extended Hel308 motif of the relevant wild-type sequence. Appropriate modifications to X1 and X2 are discussed above when defining the two reasons.
[00126] A variant of SEQ ID NO: 10 may lack the first 19 amino acids of SEQ ID NO: 10 and/or lack the last 33 amino acids of SEQ ID NO: 10. A variant of SEQ ID NO: 10 preferably comprises a sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more preferably at least 95%, at least 97% or at least 99% homologous based on amino acid identity with amino acids 20 to 211 or 20 to 727 of SEQ ID NO: 10.
[00127] The helicase can be covalently attached to the pore. The helicase is preferably non-covalently attached to the pore. Applying a voltage to the pore and helicase typically results in the formation of a sensor that is capable of targeting polynucleotide sequencing. This is discussed in more detail below.
[00128] Any of the proteins described herein, i.e. the transmembrane protein pores or Hel308 helicases, can be modified to aid in their identification or purification, for example, by the addition of histidine residues (an his-tag), residues of aspartic acid (an asp tag), a streptavidin tag, a FLAG tag, a SUMO tag, a GST tag or an MBP tag, or by adding a signal sequence to promote its secretion from a cell in which the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag to a native or engineered position on the pore or helicase. An example of this would be the reaction of a gel displacement reagent to an engineered cysteine outside the pore. This has been demonstrated as a method for the separation of hetero-oligomers from hemolysins (Chem Biol 1997 Jul; 4(7):497-505).
[00129] Pore and/or helicase can be marked with a revealing label. The developer label can be any suitable label that allows the pore to be detected. Appropriate labels include, but are not limited to, fluorescent molecules, radioactive isotopes, for example 125 I, 35 S, enzymes, antibodies, antigens, polynucleotides and linkers such as biotin.
[00130] Proteins can be made synthetically or by recombinant means. For example, the pore and/or helicase can be synthesized by translation and in vitro transcription (IVTT). The amino acid sequence of the pore and/or helicase can be modified to include non-naturally occurring amino acids or to increase protein stability. When a protein is produced by synthetic means, such amino acids can be introduced during production. The pore and/or helicase can also be altered after synthetic or recombinant production.
[00131] Poro and/or helicase can also be produced using D-amino acids. For example, the helicase pore or can comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.
[00132] Pore and/or helicase may also contain other non-specific modifications, as long as they do not interfere with pore formation or helicase function. A number of non-specific side chain modifications are known in the art and can be made to the side chains of the protein(s). Such modifications include, for example, the reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methylacetimidate or acylation with acetic anhydride.
[00133] Poro and helicase can be produced using conventional methods known in the art. Polynucleotide sequences encoding a pore or helicase can be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a pore or helicase can be expressed in a bacterial host cell using techniques standard in the art. The pore and/or helicase can be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
The pore and/or helicase can be produced on a large scale after purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system, and Gilson's HPLC system.
[00135] The method of the invention involves measuring one or more characteristics of the target polynucleotide. The method may involve measuring two, three, four or five or more characteristics of the target polynucleotide. The one or more characteristics are preferably selected from (i) the length of the polynucleotide in question, (ii) the identity of the target polynucleotide, (iii) the target polynucleotide sequence, (iv) the secondary structure of the target polynucleotide and ( v) whether the target polynucleotide is modified. Any combination of (i) to (v) can be measured according to the invention.
[00136] For (i), the length of the polynucleotide can be measured using the number of interactions between the target polynucleotide and the pore.
[00137] For (ii), the identity of the polynucleotide can be measured in several ways. Polynucleotide identity can be measured in conjunction with measurement of target polynucleotide sequence or without measurement of target polynucleotide sequence. The first is straightforward; the polynucleotide is sequenced and thus identified. The latter can be done in a number of ways. For example, the presence of a particular pattern in the polynucleotide can be measured (without measuring the remaining sequence of the polynucleotide). Alternatively, measuring a particular electrical and/or optical signal in the method can identify the target polynucleotide as coming from a particular source.
[00138] For (iii), the polynucleotide sequence can be determined as described above. Appropriate sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010;132( 50):17961-72, and international application WO 2000/28312.
[00139] For (iv), secondary structure can be measured in a variety of ways. For example, if the method involves an electrical measurement, the secondary structure can be measured using a change in residence time or a change in current flow through the pore. This allows single-stranded and double-stranded polynucleotide regions to be distinguished.
[00140] For (v), the presence or absence of any modification can be measured. The method preferably comprises determining whether or not the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. Specific modifications will result in specific interactions with the pore that can be measured using the methods described below. For example, methylcytosine can be distinguished from cytosine in terms of the current flowing through the pores during its interaction with each nucleotide.
[00141] Several different types of measurements can be made. This includes without limitation: electrical measurements and optical measurements. Possible electrical measurements include: current measurements, impedance measurements, tunnel formation measurement (Ivanov AP et al., Nano Lett. 2011 Jan 12;11(1):279-85), and FET measurements (WO international application 2005/124888). Optical measurements can be combined 10 with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan; 81(1):014301). The measurement can be a transmembrane current measurement such as a measurement of ionic current flowing through the pore.
[00142] Electrical measurements can be made using standard single-channel recording equipment as described in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010;132(50):17961-72, and international application
[00143] WO-2000/28312. Alternatively, electrical measurements can be made using a multi-channel system, for example as described in international application WO-2009/077734 and international application WO-2011/067559.
[00144] In a preferred embodiment, the method comprises: contacting the target polynucleotide with a transmembrane pore and a Hel308 helicase such that the helicase controls movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and measuring current passing through the pore during one or more interactions to measure one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide.
[00145] The methods can be performed using any apparatus that is suitable for investigating a membrane/pore system in which a pore is inserted into a membrane. The method can be performed using any apparatus that is suitable for transmembrane pore detection. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier has an opening in which the membrane containing the pore is formed.
[00146] The methods can be carried out using the apparatus described in international application No. PCT/GB08/000562 (WO 2008/102120).
[00147] The methods may involve measuring the current passing through the pore during one or more interactions with the nucleotide(s). Therefore, the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane, and pore. Methods can be performed using a link clamp or a voltage clamp. The methods preferably involve the use of a voltage clamp.
[00148] The methods of the invention may involve measuring a current passing through the pore during one or more interactions with the nucleotide. Appropriate conditions for measuring ionic currents through transmembrane protein pores are well known in the art and described in the Example. The method is typically performed with a voltage applied across the membrane and pore. The voltage used is typically +2V to -2V, typically -400mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is most preferably in the range 100 mV to 240 mV and most preferably in the range 120 mV to 220 mV. It is possible to increase the discrimination between different nucleotides by a pore using an increased applied potential.
[00149] Methods are typically carried out presence of all charge carriers such as metal salts, eg alkali metal salt, halide salts eg chloride salts like alkali metal chloride salt. Cargo vehicles can include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl-imidazolium chloride. In the exemplary apparatus discussed above, salt is present in the aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl) or cesium chloride (CsCl) is typically used. KCl is preferred. The salt concentration can be in saturation. The salt concentration can be 3M or less and is typically 0.1 to 2.5 M, 0.3 to 1.9 M, 0.5 to 1.8 M, 0.7 to 1.7 M, from 0.9 to 1.6M or from 1M to 1.4M. The salt concentration is preferably from 150mM to 1M. As discussed above, Hel308s helicase surprisingly works with high concentrations of salts. The method is preferably carried out using a salt concentration of at least 0.3M, such as at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1 .0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High concentrations of salts provide a high signal-to-noise ratio and allow currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations.
[00150] Methods are typically performed in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer can be used in the methods of the invention. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The methods are typically carried out at a pH of 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8 ,7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.
[00151] The methods can be performed from 0oC to 100oC, from 15oC aoooooo oo oo 95 C, from 16 C to 90 C, from 17 C to 85 C, from 18 C to 80 C, 19 C to 70 C, or from 20oC to 60oC. Methods are typically carried out at room temperature. Methods are optionally performed at a temperature that supports enzyme function, such as about 37oC.
[00152] The method is typically performed in the presence of free nucleotides or free nucleotide analogues and an enzyme cofactor that facilitates the action of the helicase. Free nucleotides can be one or more of any of the individual nucleotides discussed above. Free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate ( GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP ), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate ( dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP ), deoxyuridine diphosphate (dUDP ), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). Enzyme cofactor is a factor that allows the helicase to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg2+, Mn2+, Ca2+ or Co2+. The enzyme cofactor is most preferably Mg2+.
[00153] The target polynucleotide can be contacted with the Hel308 helicase and the pore in any order. It is preferred that when the target polynucleotide is contacted with the Hel308 helicase and the pore, the target polynucleotide first forms a complex with the helicase. When voltage is applied across the pore, the target polynucleotide/helicase complex then forms a complex with the pore and controls movement of the polynucleotide through the pore.
[00154] As discussed above, Hel308 helicases can work in two ways with respect to nanopores. First, the method is preferably carried out using the Hel308 helicase such that it moves from the target sequence through the pores with the field resulting from the applied voltage. In this mode, the 3' end of the DNA is first captured in the nanopore, and the enzyme moves to the DNA in the nanopore in such a way that the target sequence is passed through the nanopore with the field, until it finally translocates to the trans side of the bilayer. Alternatively, the method is preferably carried out such that the enzyme moves the target sequence through the pore against the field resulting from the applied voltage. In this mode, the 5' end of the DNA is first captured in the nanopore, and the enzyme moves the DNA through the nanopore so that the target sequence is pulled out of the nanopore against the applied field until it is finally ejected back to the cis side. of the bilayer.
[00155] The method of the invention most preferably involves a pore derived from MspA and a helicase comprising the sequence shown in SEQ ID NO: 8 or 10 or a variant thereof. Any of the embodiments as discussed above with reference to MspA and SEQ ID NO: 8 and 10 can be used in combination. Other Methods
[00156] The invention also provides a method of forming a sensor to characterize a target polynucleotide. The method comprises forming a complex between a pore and a Hel308 helicase. The complex can be formed by contacting the pore and helicase in the presence of the target polynucleotide and then applying a potential through the pore. The applied potential can be a chemical potential or a voltage potential as described above. Alternatively, the complex can be formed by covalently bonding the pore to the helicase. Methods for covalent attachment are known in the art and described, for example, in international application Nos. PCT/GB09/001679 (published as WO 2010/004265) and PCT/GB10/000133 (published as WO 2010/086603). The complex is a sensor to characterize the target polynucleotide. The method preferably comprises forming a complex between an Msp-derived pore and a Hel308 helicase. Any of the embodiments as discussed above with reference to the method of the invention apply equally to this method. Kits
[00157] The present invention also provides kits for characterizing a target polynucleotide. The kits comprise (a) a pore and (b) a Hel308 helicase. Any of the embodiments as discussed above with reference to the method of the invention will apply equally to kits.
[00158] The kit can further comprise components of a membrane, such as the phospholipids needed to form a lipid bilayer.
[00159] The kits of the invention may additionally comprise one or more other reagents or instruments that enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: appropriate buffer(s) (aqueous solutions), means for obtaining a sample from an individual (such as a container or an instrument comprising a needle), means for amplifying and/ or expressing polynucleotides, a membrane as defined above, or a clamp-connector or voltage apparatus. Reagents may be present in the kit in a dry state, so a fluid sample resuspends the reagents. The kit may also optionally include instructions to enable the kit to be used in the method of the invention or details regarding patients with whom the method may be used. The kit can optionally comprise nucleotides. Device
[00160] The invention also provides an apparatus for characterizing a target polynucleotide. The apparatus comprises a plurality of pores and a plurality of a Hel308 helicase. The apparatus preferably further comprises instructions for carrying out the method of the invention. The apparatus can be any conventional apparatus for analyzing polynucleotides, such as an array or a chip. Any of the embodiments as discussed above with reference to the methods of the invention are equally applicable to the apparatus of the invention.
[00161] The apparatus is preferably configured to carry out the method of the invention.
[00162] The apparatus preferably comprises: a sensor device that is capable of supporting the membrane and plurality of pores and being operable to carry out polynucleotide characterization using the pores and helicases; at least one reservoir to hold the material to carry out a characterization; a fluidic system configured to controllably deliver material from at least one reservoir to the sensor device; and a plurality of containers for receiving the respective samples, the fluidic system being configured to selectively deliver the samples from the containers to the sensor device. The apparatus may be any of those described in international application No. PCT/GB08/004127 (published as WO 2009/077734), PCT/GB10/000789 (published as WO 2010/122293), international application No. PCT/GB10 /002206 (not yet published) or International Application No. PCT/US99/25679 (published as WO 00/28312). Internally linked molecular motors
[00163] Molecular motors are generally used as a means to control the translocation of a polymer, particularly a polynucleotide, through a nanopore. Surprisingly, it has been found that molecular motors that are capable of binding to a target polynucleotide by an internal nucleotide, i.e., at a position other than a 5' or 3' terminal nucleotide, can provide increased reading lengths of polynucleotide as the motor Molecular molecular controls the translocation of the polynucleotide through a nanopore. The ability to translocate a complete polynucleotide through a nanopore, under the control of a molecular engine, allows the characteristics of the polynucleotide, such as its sequence, to be estimated with greater precision and speed compared to known methods. This becomes more important as filament lengths increase and molecular motors are required with improved processability. The molecular engine used in the invention is particularly effective in controlling the translocation of target polynucleotides of 500 nucleotides or more, for example 1000 nucleotides, 5000, 10000 or 20000 or more.
[00164] The invention thus provides a method of characterizing a target polynucleotide, comprising: contacting the target polynucleotide with a transmembrane pore and a molecular motor that is capable of binding to the target polynucleotide at an internal nucleotide so that the molecular motor controls movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby characterizing the target polynucleotide.
[00165] Any of the embodiments discussed above with respect to the Hel308 methods of the invention equally apply to this method of the invention.
[00166] One problem that occurs in sequencing polynucleotides, particularly those with 500 nucleotides or more, is that the molecular motor that is controlling the polynucleotide translocation can break loose from the polynucleotide. This allows the polynucleotide to be pulled through the pore quickly and uncontrollably towards the applied field. Multiple instances of the molecular motor used in the invention bind to the polynucleotide over relatively short distances and therefore the length of polynucleotide that can be pulled through pores before another molecular motor docks with the pore is relatively short.
[00167] An inner nucleotide is a nucleotide that is not a terminal nucleotide in the target polynucleotide. For example, it is not a 3' terminal nucleotide or a 5' terminal nucleotide. All nucleotides in a circular polynucleotide are internal nucleotides.
[00168] Generally, a molecular motor that is capable of binding to an inner nucleotide is also capable of binding to a terminal nucleotide, but the tendency of some molecular motors to bind to an inner nucleotide will be greater than in others . For an appropriate molecular motor for use in the invention, typically at least 10% of its binding to a polynucleotide will be in an internal nucleotide. Typically at least 20%, at least 30%, at least 40% or at least 50% of your binding will be on an internal nucleotide. Binding to a terminal nucleotide can involve binding to both a terminal nucleotide and adjacent internal nucleotides at the same time. For the purposes of the present invention, it is not binding to the target polynucleotide on an internal nucleotide. In other words, the molecular engine used in the present invention is not only capable of binding a terminal nucleotide in combination with one or more adjacent internal nucleotides. The molecular motor must be able to bind to an internal nucleotide, without concomitant binding to a terminal nucleotide.
[00169] A molecular motor that is capable of binding at one inner nucleotide can bind to more than one inner nucleotide. Typically, the molecular motor binds to at least 2 internal nucleotides, for example at least 3, at least 4, at least 5, at least 10 or at least 15 internal nucleotides. Typically the molecular motor binds to at least 2 adjacent internal nucleotides, for example at least 3, at least 4, at least 5, at least 10 or at least 15 adjacent internal nucleotides. The at least 2 inner nucleotides can be adjacent or non-adjacent.
[00170] The ability of a molecular motor to bind to a polynucleotide in an internal nucleotide can be determined by performing a comparative test. The ability of a motor to bind to a control polynucleotide A is compared to the ability to bind to the same polynucleotide, but with a blocking group attached to the terminal nucleotide (polynucleotide B). The blocking group prevents any binding at the B-strand terminal nucleotide, and thus allows only the internal binding of a molecular motor. An example of this type of test is described in Example 4.
Appropriate molecular motors are well known in the art and typically include, but are not limited to, single or double stranded translocases such as polymerases, helicases, topoisomerases, ligases and nucleases such as exonucleases. Preferably the molecular engine is a helicase, for example a Hel308 helicase. Examples of Hel308 helicases that are capable of binding to an internal nucleotide include, but are not limited to, Hel308 Tga, Hel308 Mhu and Hel308 Csy. Thus, the molecular motor preferably comprises (a) the sequence of Hel308 Tga (i.e., SEQ ID NO: 33) or a variant thereof, or (b) the sequence of Hel308 Csy (i.e., SEQ ID NO: 22) or a variant thereof or (c) the sequence of Hel308 Mhu (i.e., SEQ ID NO:52) or a variant thereof. The variant typically has at least 40% homology to SEQ ID NO: 33, 22 or 52 based on amino acid identity over the complete sequence and retains helicase activity. Other possible variants are discussed above.
[00172] The molecular engine used in the invention can be made by any of the methods discussed above and can be modified or labeled as discussed above. The molecular motor can be used in the methods described above or as a part of the apparatus described above. The invention further provides a method of forming a sensor to characterize a target polynucleotide, comprising forming a complex between a pore and a molecular motor that is capable of binding to the target polynucleotide in an internal nucleotide and thus forming a sensor to characterize the target polynucleotide . The invention also provides the use of a molecular motor that is capable of binding the target polynucleotide at an internal nucleotide to control the movement of a target polynucleotide through a pore. The invention also provides a kit for characterizing a target polynucleotide comprising (a) a pore and (b) a molecular motor that is capable of binding the target polynucleotide at an internal nucleotide. The invention also provides an analysis apparatus for characterizing target polynucleotides in a sample, comprising a plurality of pores and a plurality of a molecular motor that is capable of binding the target polynucleotide at an internal nucleotide.
[00173] The following examples illustrate the invention. Example 1
[00174] This example illustrates the use of a Hel308 helicase (Hel308 MBu) to control the movement of intact DNA strands through a nanopore. The general method and substrate used in the example are shown in Figure 1 and described in the figure legend. Materials and methods
The primers were designed to amplify a ~400 bp fragment of PhiX174. Each of the 5' ends of these primers included a non-complementary region of 50 nucleotides, or a homopolymeric stretch or repeating units of homopolymeric sections of 10 nucleotides. These serve as filament controlled translocation identifiers through a nanopore, as well as to determine translocation directionality. In addition, the 5' end of the forward primer was "capped" to include four 2'-O-methyl-uracil (mU) nucleotides and the 5' end of the reverse primer was chemically phosphorylated. These primer modifications then allowed controlled digestion of predominantly only the antisense strand, using lambda exonuclease. The mU termination protects the sense strand from nuclease digestion while the 5' PO4 of the antisense strand promotes it. Right after incubation with lambda exonuclease only the sense strand of the duplex remains intact, now as single-stranded DNA (ssDNA). The ssDNA generated was then purified by PAGE as described above.
[00176] Design of the DNA substrate used in all experiments described here is shown in Figure 6. The DNA substrate consists of a 400 base section of ssDNA from Phix, with a 5'-50T leader to aid capture by nanopore (SEQ ID NO:59). Ringed to this strand just after the 50T leader is a primer (SEQ ID NO: 60) containing a 3'cholesterol tag to enrich the DNA on the surface of the bilayer, and thus improve the capture efficiency.
Buffered solution: 400 mM-2 M KCl, 10 mM Hepes pH 8.0, 1 mM ATP, 1 mM MgCl 2 , 1 mM DTT
[00178] Nanopore: E.coli MS(B2)8 MspA ONLP3271 MS- (L88N/D90N/D91N/D93N/D118R/D134R/E139K)8
[00179] Enzyme: Hel308 Mbu (ONLP3302, ~7.7 µM) 12.5 µl -> 100 nM final.
[00180] Electrical measurements were acquired from individual MspA nanopores inserted into lipid bilayers 1,2-diftanoyl-glycero-3-phosphocholine (Avanti Polar Lipids). Bilayers were formed through ~100 mm diameter openings in 20 mM thick PTFE films (in custom Delrin chambers) using the Montal-Mueller technique, which separates two 1 mL buffered solutions. All experiments were performed in the indicated buffered solution. Single-channel currents were measured on Axopatch 200B amplifiers (Molecular Devices) equipped with 1440A digitizers. Ag/AgCl electrodes were connected to the buffered solutions so that the cis compartment (to which both nanopores and enzyme/DNA are added) is connected to the ground of the 'headstage' Axopatch, and the transport compartment is connected to the active electrode of ' headstage'.
[00181] After reaching a single pore in the bilayer, polynucleotide DNA and helicase were added to 100 μL of buffer and pre-incubated for 5 minutes (DNA = 1.5 nM, Enzyme = 1 μM). This preincubation mix was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate the capture of DNA-helicase complexes in the MspA nanopore (to give final DNA concentrations = 0.15 nM, Enzyme = 0, 1 µM ). ATPase helicase activity was initiated as needed by the addition of bivalent metal (1 mM MgCl2) and NTP (1 mM ATP) to the cis compartment. The experiments were carried out at a constant potential of +180 mV. Results and discussion
[00182] The addition of the DNA-helicase substrate to the nanopores of MspA as shown in Figure 1 produces characteristic current blocks, as shown in Figure 2. DNA without attached helicase transiently interacts with the nanopore producing short-lived blocks in current (< < 1 second). DNA with active linked helicase (ie, moving along the DNA strand under the action of ATPase) produces characteristic long block levels with gradual changes in current as shown in the figure. 2. Different DNA motifs in the nanopore give rise to levels of unique current blocks.
[00183] For a given substrate, there is a characteristic pattern of current transitions that reflects the DNA sequence (examples in Figure 3.).
[00184] In the implementation depicted in Figure 1, the DNA strand is sequenced from a random starting point as the DNA is captured with a helicase at a random position along the strand. However, as long as the enzyme does not dissociate, the filaments will all end in the same way in the 50T leader (Figure 1). As shown in Figure 2 shows, the same characteristic termination is observed for most filaments, with the current transitions ending at a long dwell time polyT level (Figure 3). salt tolerance
[00185] Nanopore filament sequencing experiments of this type require ionic salts. The ionic salts are needed to create a conductive solution for applying a voltage shift to capture and translocate DNA, and to measure the resulting sequence-dependent current changes as the DNA passes through the nanopore. Since the measurement signal is dependent on the ion concentration, it is advantageous to use high concentration ionic salts to increase the magnitude of the acquired signal. For nanopore sequencing, salt concentrations in excess of 100 mM KCl are ideal, and salt concentrations of 1 M KCl and above are preferred.
[00186] However, many enzymes (including some helicases and DNA motor proteins) do not tolerate high salt conditions. Under conditions of high salt content, enzymes do not duplicate or lose structural integrity, or fail to function properly. The current literature for known and studied helicases shows that almost all helicases fail to function above salt concentrations of about 100 mM KCl/NaCl, and there are no reported helicases that show correct activity under conditions of 400 mM KCl and above. While potentially halophilic variants of similar enzymes exist from halotolerant species, they are extremely difficult to express and purify in conventional expression systems (eg, E. coli).
Applicants have surprisingly shown in this example that Hel308 from Mbu exhibits salt tolerance up to very high levels of KCl. Applicants found that the enzyme retains functionality at salt concentrations of 400 mM KCl up to 2 M KCl, either in fluorescence experiments or in nanopore experiments (Figure 4). Figure 4 shows Hel308 Mbu DNA events at 400 mM KCl, 1 M KCl, and 2 M KCl salt conditions performed using the same system described in Figure 1. Applicants observed similar movement across the entire range of concentrations of salt. As the salt concentration is increased, an increase in current through the nanopore (I-open) is observed at a fixed voltage. This reflects the increase in the conductivity of the solution and the increase in the number of ions flowing through the nanopore under the applied field. In addition, an increase in the minimum to maximum range of discrimination in current levels of DNA events was also observed (see Figure 4, magnifications and graph on the lower right). A ~200% increase in the DNA discrimination range is observed as the salt concentration is increased from 400 mM KCl to 2 M KCl (Table 6 below).
Forward and reverse operating modes
[00188] Most helicases move along single-stranded polynucleotide substrates in a unidirectional fashion, moving a specific number of bases for each bound NTPase. While Figure 1 illustrates the use of this motion to pull filamentous DNA out of the nanopore, helicase motion could be exploited in other ways to feed DNA through the nanopore in a controlled manner. Figure 5 illustrates the basic “forward” and “reverse” operating modes. In forward mode, DNA is fed into the pore by the helicase in the same direction that DNA would move under the force of the applied field. For Hel308 Mbu, which is a 3' - 5' helicase, this requires capturing the 3' end of the DNA at the nanopore until a helicase contacts the top of the nanopore, and the DNA is then fed into the nanopore under the control of the helicase with the field of applied potential, finally, exiting on the trans side of the bilayer. The reverse mode requires capturing the 5' end of the DNA, after which the helicase proceeds to pull the stranded DNA back out of the nanopore against the field from the applied potential, finally expelling it from this cis side of the bilayer. Figure 5 shows the two modes of operation, using Hel308 Mbu, and examples of typical DNA events. Example 2
[00189] This example illustrates the salt tolerance of a Hel308 helicase (Hel308 MBu ) using a fluorescence assay for test enzyme activity.
[00190] A custom fluorescent substrate was used to test the ability of the helicase to displace hybridized dsDNA (Figure 6A). As shown in 1) of Figure 6A, the fluorescent substrate filament (100 nM final) has a 3' ssDNA overhang, and a 40 base section of hybridized dsDNA. The top main filament has a carboxyfluorescein base at the 5' end, and the hybridized complement has a base 'Black Hole' BHQ-1) quencher at the 3' end. When hybridized, fluorescein fluorescence is quenched via local BHQ-1, and the substrate is essentially non-fluorescent. 1 µM of a capture filament that is complementary to the shorter filament of the fluorescent substrate is included in the test. As shown in 2), in the presence of ATP (1 mM) and MgCl2 (5 mM), helicase (100 nM) was added to the substrate binds to the 3' tail of the fluorescent substrate, moves along the main filament, and displaces the complementary filament as shown. As shown in 3), once the complementary filament with BHQ-1 is fully displaced the fluorescein in the main filament blooms. As shown in 4), an excess capture strand preferentially anneals with complementary DNA to avoid initial substrate reannealing and loss of fluorescence.
[00191] Substrate DNA: 5'FAM-SEQ ID NO: 61 and SEQ ID NO: 62-BHQ1-3'. FAM = carboxyfluorescein and BHQ1 = 'Black Hole' fire extinguisher -1
[00192] Capture DNA: SEQ ID NO: 62.
[00193] The graph in Figure 6 shows the initial rate of activity in buffer solutions (10 mM Hepes pH 8.0, 1 mM ATP, 5 mM MgCl2, 100 nM fluorescent substrate DNA, 1 μM DNA capture) containing different concentrations of KCl 400 mM at 2 M. The helicase works at 2 M. Example 3
In this Example, three different Hel308s helicases were used, namely Hel308 Mhu (SEQ ID NO: 52), Hel308 Mok (SEQ ID NO: 29) and Hel308 Mma (SEQ ID NO: 45). All experiments were performed as previously described in Example 1 under the same experimental conditions (pore = MspA B2, DNA = 400mers SEQ ID NO: 59 and 60, buffer = 400mM KCl, 10mM Hepes pH 8.0, 1mM dtt, 1mM ATP , 0.1mM MgCl2). The results are shown in Figure 7. Example 4
[00195] This example measures the internal binding capabilities of a series of Hel308 helicases using a fluorescence test.
Customized fluorescent substrates were used to test the ability of helicases to prime native 3' ends lacking DNA, allowing them to subsequently displace hybridized dsDNA (Figure 8). As shown in section A of the Figure. 8, the fluorescent substrate strand (50 nM final) has a 3' overhang of ssDNA, and a 40 base section of hybridized dsDNA. The top main filaments are modified with four consecutive "spacer 9" groups, either at the 3' end or internally at the junction between the overhang and the dsDNA (as a negative control). In addition, the top main filament has a carboxyfluorescein base at the 5' end, and the hybridized complement has a base black-hole quencher (BHQ-1) at the 3' end. When hybridized, fluorescein fluorescence is quenched via local BHQ-1, and the substrate is essentially non-fluorescent. A capture filament (1 µM), which is complementary to the shorter filament of the fluorescent substrate, is included in the test. In the presence of ATP (1 mM) and MgCl2 (1 mM), a helicase homolog Hel308 (20 nM), added to the substrate containing 3'-terminal "9" spacer groups, can bind to the ssDNA hook of the fluorescent substrate , move along the main filament, and displace the complementary filament as shown in section B. once the complementary strand with BHQ -1 is fully displaced (section C) the fluorescein on the main filament fluoresces. An excess capture strand preferentially anneals with complementary DNA to avoid initial substrate reannealing and loss of fluorescence (section D).
[00197] DNA Substrate: SEQ ID NO: 63 with a 5' FAM; SEQ ID NO: 63 with a 5' FAM and 3' spacer ((9 spacer)4); SEQ ID NOs: 64 (with a 5' FAM) and 65 separated with a spacer ((space 9)4); and SEQ ID NO: 62 with a 3' BHQ1.
[00198] Capture DNA: SEQ ID NO: 66.
[00199] Several different homologues of Hel308 helicase have been investigated for their half-bonding capabilities, these including Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mma, Hel308 Mhu, Hel308 Min, Hel308 Mig, Hel308 Mmaz, Hel308 Mac, Hel308 Mok , Hel308 Mth, Hel308 Mba and Hel308 Mzh. The graph in Figure 9 shows the relative turnover rates of Hel308-mediated dsDNA, comparing 3'-unmodified DNA and 3'-"spacer 9" DNA in 400 mM NaCl, 10 mM Hepes, pH 8.0, 1 mM ATP , 1 mM MgCl2, 50 nM fluorescent substrate DNA, 1 μM capture DNA. Several Hel308 homologues were observed to have greater than 20% relative rates of Hel308-mediated dsDNA turnover including Hel308 Csy, Hel308 Tga, Hel308 Mma, Hel308 Mhu, and Hel308 Min.
[00200] This Example compares the use of two Hel308s helicases, Hel308 MBu and Hel 308 Tga, and their ability to control the movement of intact long DNA strands (900 mers) through a nanopore. The general method and substrate employed throughout this Example are shown in Figure 10 and described in the Figure description above. Materials and methods
DNA was formed by ligating a 50-polyT 5' leader to a ~900base PhiX dsDNA fragment. The leader also contains a complementary section to which SEQ ID NO:69 with a Chol tag has been hybridized to allow the DNA to be bilayered. Finally, the 3' end of the PhiX dsDNA was digested with an AatII digestion enzyme to give a 4nt 3'-overhang of ACGT.
[00202] Sequence used: SEQ ID NO: 67 - 900 mer filament including 5' leader and follower; SEQ ID NO: 68 -antisense minus 4 base pairs 5' leader; and SEQ ID NO: 69 with various spacers and a Chol tag at the 3' end.
Buffered solution: 400 mM-2 NaCl, 10 mM potassium ferrocyanide, 10 mM potassium ferrocyanide, 100 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl2,
[00204] Nanopore: MS-(B1-G75S-G77S-L88N-Q126R)8 (ONT Ref B2C)
Enzyme: Hel308 Mbu 1000 nM or Hel308 Tga 400 nM final.
[00206] The electrical experiments were set up as described in Example 1 in order to obtain a single pore inserted into a lipid bilayer. After reaching a single pore in the bilayer, ATP (1 mM) and MgCl2 (1 mM) were added to the chamber. A control recording at +140 mV was cycled for 2 minutes. Polynucleotide DNA SEQ ID NOs: 67, 68 and 69 (DNA = 0.15 nM) were then added and the DNA events observed. Finally, Hel308 helicase (Mbu 1000 nM or Tga, 400 nM) was added to the cis behavior of the chamber and electrophysiology to initiate the capture of helicase-DNA complexes in the MspA nanopore. Experiments were performed at a constant potential of +140 mV. Results and discussion
[00207] The addition of the DNA substrate - helicase to MspA nanopores as shown in Figure 10 produces characteristic current blocks as the helicase controls the DNA translocation through the pore. Figure 11 shows traces from the example event that indicate how the position of the 900 mers varied as the Mbu homolog of Hel308 helicase controlled DNA strand translocation through the MspA pore. This helicase was found to mediate control of DNA translocation, however, when the helicase detached from the DNA, the strand was observed to move backwards through the pore due to the force exerted by the externally applied potential. In the case of the Mbu homologue of Hel308 helicase, the 900mer filament slipped back in a large number of positions (approximately 100-200 bases) each time a helicase came off. These rapid changes in position are indicated in figure 11 by dotted circles. For this experiment, where Mbu homolog of Hel308 helicase was used as the molecular engine, 32% of all detected events were found to have read the full length 900-mer chain sequence. Figure 12 shows event traces from similar examples that indicate how the position of the 900 mers varied as the Tga homologue of Hel308 helicase controlled DNA strand translocation through the MspA pore. This enzyme exhibited a greater tendency to bind internally, than the Mbu homolog, because when the Tga helicase detaches (indicated by a color change from black to gray in Figure 12), the DNA strand moves back through. of the pore for a relatively short distance (< 50 bases). For this experiment, where the Tga homologue of Hel308 helicase was used as the molecular engine, 74% of all detected events were found to have read the full length 900-mer filament sequence. This means that the Tga homolog of helicase can provide increased reading lengths of single-stranded DNA compared to the Mbu homolog of helicase due to its greater tendency to bind internally. Example 6
This Example illustrates that by employing the Tga homolog of helicase Hel308 it is possible to control the translocation of the 5 kb strand of DNA.
A similar experimental procedure was followed as described in Example 5. It was observed that employing Hel308 Tga, it was possible to detect the controlled translocation of the complete 5 kB strand of DNA through MS-(B1-G75S-G77S-L88N-Q126R )8. In an identical experiment using Hel308 Mbu, it was not possible to detect the translocation of a complete 5kB filament. Example 7
This example compares the processability of helicase enzyme Hel308 Mbu (SEQ ID NO: 10) with Hel308 Mok (SEQ ID NO: 29) using a fluorescence-based assay.
A custom fluorescent substrate was used to test the ability of the helicase to displace hybridized dsDNA (Figure 3). The fluorescent substrate (50 nM final) has a 3' ssDNA overhang, and 80 and 33 base pair sections of hybridized dsDNA (Figure 13 section A, SEQ ID NO: 70). The lower main "template" filament is hybridized to an 80 nt "blocker" filament (SEQ ID NO: 71), adjacent to its 3' overhang, and 33 nt fluorescent probe, labeled at its 5' and 3' ends with carboxyfluorescein (FAM) and black-hole fire extinguisher (BHQ -1) bases, respectively (SEQ ID NO:72). When hybridized, FAM is distant from BHQ-1 and the substrate is essentially fluorescent. In the presence of ATP (1 mM) and MgCl2 (10 mM ), the helicase (20 nM ) binds to the 3' overhang of the substrate (SEQ ID NO: 70), moves along the lower filament, and begins to displace the 80 nt blocker filament (SEQ ID NO: 71 ), As shown in Figure 13 section B. If processive, the helicase displaces the fluorescent probe (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM), at its end 5' and a black-hole fire extinguisher (BHQ-1 ), at its 3' end) as well (Figure 13 section C). The fluorescent probe is designed in such a way that its 5' and 3' ends are self-complementary and thus form a kinetically stable hairpin once displaced, preventing the probe from re-annealing with the template filament (Figure 13 section D). After hairpin product formation, FAM is brought into the vicinity of BHQ-1 and its fluorescence is quenched. The process enzyme, able to displace the 80 mer (SEQ ID NO: 71) and fluorescent (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) "blockers" at its 5' end a black-hole extinguisher (BHQ-1) at its 3' end) will therefore lead to a decrease in fluorescence over time. However, if the enzyme has a processability of less than 80 nt, it would be unable to displace the fluorescent filament (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5' end in a blackhole extinguisher ( BHQ-1) at its 3' end) and then the "blocker" filament (SEQ ID NO:71) would reanneal to the main lower filament (Figure 13 section E, SEQ ID NO:70).
[00212] Additional custom fluorescent substrates were also used for control purposes. The substrate used as a negative control was identical to that described in Figure 13, but lacking the 3' overhang (Figure 14 section A, (SEQ ID NOs: 71, 72 (labeled with a carboxyfluorescein (FAM ) ) at its 5' end a “black-hole” fire extinguisher (BHQ-1 ), at its end 3') and 73) ). A substrate similar to that depicted in Figure 13 but missing the 80 base pair section, used as a positive control for active, but not necessarily processive, helicases (Figure 14 section B, (SEQ ID NO: 72 (labeled with a carboxyfluorescein () FAM ), at its 5' end a “black-hole” fire extinguisher (BHQ-1 ), at its 3' end) and 74) ).
[00213] Figure 15 shows a graph of the time-dependent fluorescence changes when testing Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase (SEQ ID NO: 29) against the processability substrate shown in Figure 13 in buffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl2, 50 nM fluorescent substrate DNA (SEQ ID NOs: 70, 71 and 72 (labeled with carboxyfluorescein (FAM)) at its 5 end ' and a black-hole quencher (BHQ-1) at its 3' end.) The decrease in fluorescence exhibited by Hel308 Mok denotes the increased processability of these complexes as compared to Hel308 Mbu (SEQ ID NO: 10). Figure 16 shows positive controls demonstrating that all helicases were indeed active, as denoted by a decrease in fluorescence for all samples.

权利要求:
Claims (16)
[0001]
1. Method for characterizing a target polynucleotide, characterized in that it comprises: (a) contacting the target polynucleotide with a transmembrane pore and a Hel308 helicase so that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and (b) measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby characterizing the target polynucleotide.
[0002]
2. Method according to claim 1, characterized in that the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the target polynucleotide sequence, (iv) the secondary structure of the target polynucleotide and (v) whether or not the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers.
[0003]
3. Method according to any one of claims 1 to 2, characterized in that the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement and the electrical measurement is a current measurement, a measurement of impedance, a tunneling measurement or a field effect transistor (FET) measurement.
[0004]
4. Method according to claim 1, characterized in that the method comprises: (a) contacting the target polynucleotide with a transmembrane pore and a Hel308 helicase so that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and (b) measuring current passing through the pore during one or more interactions to measure one or more characteristics of the target polynucleotide and thereby characterize the target polynucleotide.
[0005]
5. Method according to any one of the preceding claims, characterized in that the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the helicase and wherein at least a portion of the polynucleotide is of double filament.
[0006]
6. Method according to any one of the preceding claims, characterized in that the pore is a transmembrane protein pore or a solid state pore and the transmembrane protein pore is selected from a hemolysin, leucocidin, porin A of Mycobacterium smegmatis (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisserial autotransporter lipoprotein (NalP) and WZA.
[0007]
7. Method according to claim 6, characterized in that the transmembrane protein is (a) formed from eight identical subunits as shown in SEQ ID NO: 2 or (b) α-hemolysin formed from seven identical subunits as shown in SEQ ID NO: 4.
[0008]
8. Method according to any one of the preceding claims, characterized in that the Hel308 helicase comprises the amino acid motif Q-X1-X2-GRAGR (SEQ ID NO: 8), where X1 is C, M or L and X2 is any amino acid residue preferably where X2 is A, F, M, C, V, L, I, S, T or P.
[0009]
9. Method according to any one of the preceding claims, characterized in that (a) the Hel308 helicase is one of the helicases shown in Table 4 or 5 or (b) the Hel308 helicase comprises the sequence shown in any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58.
[0010]
10. Method according to any one of the preceding claims, characterized in that the Hel308 helicase is capable of binding to the target polynucleotide in an internal nucleotide.
[0011]
11. Method according to any one of the preceding claims, characterized in that the method is carried out using a salt concentration of at least 0.3 M or at least 1.0 M.
[0012]
12. Method according to claim 11, characterized in that the salt is KCl.
[0013]
13. Method for forming a sensor to characterize a target polynucleotide, characterized in that it comprises forming a complex between a transmembrane pore and a Hel308 helicase and thus forming a sensor to characterize the target polynucleotide, preferably in which the complex is formed by (a ) contacting the pore and the helicase in the presence of the target polynucleotide and (b) applying a potential across the pore, where the potential is a voltage potential or a chemical potential and preferably where the complex is formed by covalently bonding the pore to the helicase.
[0014]
14. Use of a Hel308 helicase, characterized in that it is to control the movement of a target polynucleotide through a transmembrane pore.
[0015]
15. Kit for characterizing a target polynucleotide, characterized in that it comprises (a) a transmembrane pore and (b) a Hel308 helicase.
[0016]
16. Analysis apparatus for characterizing target polynucleotides in a sample, characterized in that it comprises a plurality of transmembrane pores and a plurality of Hel308 helicases, preferably wherein the analysis apparatus comprises: a sensor device that is capable of supporting the plurality of pores and operable to perform polynucleotide characterization using pores and helicases; at least one reservoir to hold the material to carry out the characterization; a fluidic system configured to controllably deliver material from at least one reservoir to the sensor device; and a plurality of containers for receiving the respective samples, the fluidic system being configured to selectively deliver the samples from the containers to the sensor device.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-30| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-06-16| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-12-01| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-01| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-06-01| B09W| Decision of grant: rectification|Free format text: RETIFICACAO DA PUBLICACAO DEVIDO A INCORRECOES NO TITULO DO RELATORIO DE EXAME TECNICO E NO QUADRO 1 DO PARECER DE DEFERIMENTO. |

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2021-06-22| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 18/10/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161549998P| true| 2011-10-21|2011-10-21|

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US61/549,998|2011-10-21|

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