method and apparatus for characterizing a target polynucleotide, kit, and, using a helicase

专利摘要:
METHOD AND APPARATUS TO CHARACTERIZE A TARGET POLYNUCLEOTIDE, KIT, AND USE OF A HELICASEThe invention relates to a new method for distinguishing a target polynucleotide. The method uses a pore and a RecD helicase. Helicase controls the movement of the target polynucleotide through the pore.
公开号:BR112014016112A2
申请号:R112014016112-7
申请日:2012-12-28
公开日:2020-07-14
发明作者:Ruth Moysey；Andrew John Heron；Szabolcs Soeroes
申请人:Oxford Nanopore Technologies Limited；
IPC主号:

专利说明:

[0001] [0001] The invention relates to a new method of distinguishing a target polynucleotide. The method uses a pore and a RecD helicase. Helicase controls the movement of the target polynucleotide through the pore. Fundamentals of the invention
[0002] [0002] There is currently a need for fast and inexpensive polynucleotide sequencing and identification technologies (for example 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 special fluorescent chemicals for signal detection.
[0003] [0003] Transmembrane pores (nanopores) have enormous potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology.
[0004] [0004] When a potential is applied through a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, transiently resides in the tube for a certain period of time. The detection of the nucleotide's nanopore gives a change in signature current and known duration. In the “Filament Sequencing” method, a single polynucleotide filament is passed through the pore and the identity of the nucleotides is derived. Filament Sequencing may involve the use of a nucleotide handling protein to control the movement of the polynucleotide through the pore. Summary of the invention
[0005] [0005] It has been shown that a RecD helicase can control the
[0006] [0006] Accordingly, the invention provides a method of distinguishing a target polynucleotide, comprising: (a) contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore; and (b) obtaining one or more measurements as the polynucleotide moves with respect to the pore in which the measurements are indicative of one or more characteristics of the target polynucleotide and thereby distinguishing the target polynucleotide.
[0007] [0007] The invention also provides: - a method of forming a sensor to distinguish a target polynucleotide, which comprises forming a complex between a pore and a RecD helicase and thereby forming a sensor to distinguish the target polynucleotide; - use of a RecD helicase to control the movement of a target polynucleotide through a pore; - a kit for distinguishing a target polynucleotide comprising (a) a pore and (b) a RecD helicase; and - an analysis apparatus for distinguishing a target polynucleotide in a sample, which comprises a plurality of pores and a
[0008] [0008] Fig. 1. A) Schematic example of using a helicase to control the movement of DNA through a nanopore. An SssDNA substrate with an annealed primer that contains a cholesterol tag is added to the cis side of the bilayer. The cholesterol label binds to the bilayer, enriching the substrate on the bilayer surface. 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. Under an applied voltage, the DNA substrate is captured by the nanopore. DNA is pulled through the pore under the forces of potential
[00010] [00010] Fig. 2. The helicase is capable of moving DNA through a nanopore in a controlled manner, producing staggered changes in the current as the DNA moves through the nanopore (MspA- (B2) 8). Example helicase-DNA events (140 mV, 400 mM NaCl, 10 mM Hepes, pH 8.0, 0.60 nM 400 mer DNA (SEQ ID NO: 172, 173 and 174), 100 nM Tral Eco (SEQ ID NO: 61), 1 mM DTT, 1 mM ATP, 1 mM MegCl>). Top) Current acquisition section vs. time of 400 mer DNA Tral events. The open pore current is -100 pA. DNA is
[00011] [00011] Fig. 3. Other examples of 400 mere DNA movement events controlled by the Tral Eco helicase (SEQ ID 61) (400 mere DNA SEQ ID NOs: 172, 173 and 174) through an MspA-B2 nanopore (8). Background) An enlargement of a section of the event that shows the staggered changes in the current of different sections of DNA as the filament moves through the nanopore.
[00012] [00012] Fig. 4. Fluorescence assay to test enzyme activity. A) A custom-made fluorescent substrate was used to test the helicase's ability to displace hybridized dsDNA. 1) The filament of the fluorescent substrate (50 nM final) has a 5 'ssSDNA projection, and a 40-base section of hybridized dsDNA. The larger upper filament has a carboxyfluorescein base at the 3rd end, and the hybridized complement has a black hole extinguishing base (BHQ-1) at the 5th end. When hybridized, fluorescein fluorescence is extinguished by local BHQ-1, and the substrate is essentially non-fluorescent. 1 µl of a capture filament that is complementary to the shorter filament of the fluorescent substrate is included in the assay. 2) In the presence of ATP (1 mM) and MegCl, (10 mM), helicase (100 nM) added to the substrate binds to the 5th tail of the fluorescent substrate, moves along the major filament and displaces the complementary filament as shown. 3) Since the BHQ-I complement filament is completely displaced by fluorescein in the
[00013] [00013] Fig. 5. Examples of helicase-controlled DNA events using a different Tral helicase, TrwC Cba (+140 mV, 10 mM Hepes, PH 8.0, 0.6 nM, 400 mere DNA SEQ ID NOs : 172, 172 and 173, 100 nM TrwC Cba SEQ ID 65, 1 mM DTT, 1 mM ATP, | mM MgClb). Top) Current acquisition section vs. time of DNA events of 400 mere TrwC Cba. The open pore current is -100 pA. DNA is captured by the nanopore under the forces of the applied potential (+140 mV). Bound enzyme DNA results in a long block (at —25 pA in this condition) that shows staggered changes in the current as the enzyme moves DNA through the pore. Background) The background traces show enlarged sections of the DNA events, showing the sequence-dependent current changes staggered as the DNA is pulled through the pore.
[00014] [00014] Fig. 6. Example of a current trace that shows helicase-controlled DNA movement using a TrwC (Atr) helicase (SEQ ID NO: 144).
[00015] [00015] Fig. 7. Example of a current trace showing helicase-controlled DNA movement using a TrwC (Sal) helicase (SEQ ID NO: 140).
[00016] [00016] Fig. 8. Example of a current trace that shows helicase-controlled DNA movement using a TrwC (Cer) helicase (SEQ ID NO: 136).
[00017] [00017] Fig. 9. Example of a current trace that shows the movement
[00018] [00018] Fig. 10. Example of a current trace showing movement. of helicase-controlled DNA using a TrwC (Oma) helicase (SEQ ID: NO: 106). á [00019] Fig. 11. Example of a current trace that shows movement: of helicase-controlled DNA using a TrwC (Me) helicase (SEQ ID NO: 86). The lower trace shows an expanded region of helicase-controlled DNA movement.
[00020] [00020] Fig. 12. Example of a current trace showing helicase-controlled DNA movement using a TrwC (Mph) helicase (SEQ ID NO: 94). The lower trace shows an expanded region of helicase-controlled DNA movement. String Listing Description
[00021] [00021] SEQ ID NO: | shows the optimized polynucleotide sequence in the codon encoding the mutant MspA monomer MS-B 1. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, DI 18R, DI 34R and E139K.
[00022] [00022] SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B 1 mutant of the MSspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, DI 18R, DI34R and E139K.
[00023] [00023] SEQ ID NO: 3 shows the polynucleotide sequence that encodes a sub-unit of α-hemolysin E111N / K147N (a-HL-NN; Stoddart et al., PNAS, 2009; 106 (19): 7702-7707).
[00024] [00024] SEQ ID NO: 4 shows the amino acid sequence of a subunit of a-HL-NN. SEQ ID NOs: 5 to 7 show the amino acid sequences of MspB, C and D.
[00025] [00025] SEQ ID NO: 8 shows the sequence of motif I equal to
[00026] [00026] SEQ ID NOs: 9, 10 and 11 show motif I sequences equal to extended RecD. SEQ ID NO: 12 shows the sequence of the reason: from RecD. : [00027] SEQ ID NOs: 13, 14 and 15 show the sequences of the extended RecD motif I. SEQ ID NO: 16 shows the sequence of motif V equal to RecD.
[00028] [00028] SEQ ID NO: 17 shows the sequence of RecD motif V.
[00029] [00029] SEQ ID NOs: 18 to 45 show the amino acid sequences of the RecD helicases in table 5. SEQ ID NOs: 46 to 53 show the motif III motif sequences.
[00030] [00030] SEQ ID NOs: 54 to 60 show the motif III sequences of MobQ.
[00031] [00031] SEQ ID NOs: 61 to 171 show the amino acid sequences of the Tral helicase and helicases of the Tral subgroup shown in table 7.
[00032] [00032] SEQ ID NOs: 172 to 182 show the strings used in the Examples. Detailed Description of the Invention
[00033] [00033] It should be understood that applications other than the products and methods disclosed can be made to order for specific needs in the art. It should also be understood that the terminology used here is only for the purpose of describing the particular embodiments of the invention, and is not intended to be limiting.
[00034] [00034] In addition, as used in this specification and the appended claims, the singular forms "one", "one", "o" and "a" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "one pore" includes two or more of such pores, reference to "one helicase" includes two or more of such helicases, reference to
[00035] [00035] All publications, patents and patent applications here E cited, either above or below, are hereby incorporated by reference P in their entirety. : Methods of Invention Ú [00036] The invention provides a method of distinguishing a target polynucleotide. The method comprises contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore. One or more characteristics of the target polynucleotide are then measured as the polynucleotide moves with respect to the pore using standard methods known in the art. One or more characteristics of the target polynucleotide are preferably measured as the polynucleotide moves through the pore. 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 may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example of this is the use of a salt gradient through an amphiphilic layer. A salt gradient is disclosed in Holden et al., J Am Chem Soc. July 11, 2007; 129 (27): 8650-5.
[00037] [00037] In some cases, the current that passes through the pore as the polynucleotide moves with respect to the pore is used to determine the sequence of the target polynucleotide. This is the Filament Sequencing.
[00038] [00038] The method has several advantages. First, surprisingly it was shown that RecDs helicases have a high salt tolerance
[00039] [00039] Second, when a voltage is applied, the RecDs helicases can surprisingly move the target polynucleotide in two directions, namely with or against the field that results from the applied voltage. Consequently, the method of the invention can be carried out in one of two preferred ways. Different signals are obtained depending on the direction the target polynucleotide moves in relation to the pore, that is, in the direction of the field or against it. This is discussed in more detail below.
[00040] [00040] Third, RecDs helicases typically move the target polynucleotide through the pore one nucleotide at a time. RecDs helicases therefore can function as a single base ratchet. This is of course advantageous when sequencing a
[00041] [00041] Fourth, the RecDs helicases are able to control the. movement of single - stranded polynucleotides and polynucleotides of. double filament. This means that a variety of different target polynucleotides can be distinguished according to the invention.
[00043] [00043] Sixth, the RecDs helicases are easy to produce and easy to handle. Its use therefore contributed to a direct and less expensive method of sequencing.
[00044] [00044] The method of the invention is to distinguish a target polynucleotide. A polynucleotide, such as a nucleic acid, is a macromolecule that comprises two or more nucleotides. The polynucleotide or nucleic acid can comprise any combination of any nucleotides. Nucleotides can be naturally occurring or artificial. One or more nucleotides in the target polynucleotide can be oxidized or methylated. One or more nucleotides in the target 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.
[00045] [00045] 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. The nucleotide dj 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 linked on the 5th or 3rd side of a nucleotide.
[00046] [00046] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), cytidine monophosphate cyclic adenosine (CAMP), cyclic guanosine monophosphate (CGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythidine monophosphate (dTMP), deoxyuridine monophosphate (dUM) and dUMP (dUM). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
[00047] [00047] A nucleotide can be abasic (ie a nucleobase is missing).
[00048] [00048] The polynucleotide can be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded.
[00049] [00049] 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 nucleotide side chains .
[00050] [00050] The whole or only part of the target polynucleotide can be distinguished 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. minus 250, minus 300, minus 400 or minus 500 nucleotide pairs in length. The polynucleotide can be 1000 or more 'nucleotide pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length.
[00051] [00051] The target polynucleotide is present in any suitable sample. The invention is typically performed on a sample that is known to contain or is suspected to contain the target polynucleotide. Alternatively, the invention can be carried out on a sample to confirm the identity of one or more target polynucleotides whose presence in the sample is known or expected.
[00052] [00052] The sample can be a biological sample. The invention can be carried out in vitro on a sample obtained or extracted from any organism or microorganism. The organism or microorganism is typically Archean, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: vegetable, animal, fungi, monera and protist. The invention can be carried out in vitro on a sample obtained or extracted from any virus. The sample is preferably a fluid sample. The sample typically comprises a patient's body fluid. The sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the sample is human in origin, but alternatively it may be from another mammalian animal such as commercial farm animals such as horses, cattle, sheep or pigs or alternatively it may be from pets such as cats or dogs. Alternatively a sample of plant origin is typically obtained from a commercial crop, such as a cereal, vegetable, fruit or vegetable, for example
[00055] [00055] A transmembrane pore is a structure that crosses the membrane to some degree. It allows ions, such as hydrated ions, to be triggered by an applied potential to flow through or within the membrane. The transmembrane pore typically crosses the entire membrane so that ions can flow from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not have to cross the membrane. It can be closed at one end. For example, the pore can be a reservoir in the membrane through which or into which ions can flow.
[00056] [00056] Any membrane can be used according to the invention. Suitable 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 at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically a planar lipid bilayer or a sustained bilayer.
[00057] [00057] The amphiphilic layer is typically a lipid bilayer. i Lipid bilayers are models of cell membranes and serve as. excellent platforms for a range of experimental studies. Per . For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single channel recording. Alternatively, the lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a sustained bilayer or a liposome. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International Application No. PCT / GB08 / 000563 (published as WO 2008/102121), International Application No. PCT / GBO08 / 004127 (published as WO 2009/077734) and International Application No. PCT / GB2006 / 001057 (published as WO 2006/100484).
[00058] [00058] Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Example. Lipid bilayers are usually formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is charged at the interface of the aqueous / air solution passes each side of an opening that is perpendicular to this interface.
[00059] [00059] The Montal & Mueller method is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for pore insertion into protein. Other common bilayer formation methods include tip dipping, painted bilayers and liposome bilayer patch-clamp.
[00060] [00060] In a preferred embodiment, the lipid bilayer is formed as described in International Application No. PCT / GB08 / 004127 (published as WO 2009/077734).
[00061] [00061] 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 Si; N, a, ALIO ;, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon or elastomers such as two-component curing silicone rubber, and glasses. Solid-state layers can be formed from monatomic layers, such as graphene, or layers that are only a few atoms thick. Suitable graphene layers are disclosed in International Application No. PCT / US2008 / 010637 (published as WO 2009/035647).
[00062] [00062] The method is typically performed using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated, naturally occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted into it. The method is typically carried out using an artificial amphiphilic layer, such as an artificial lipid bilayer. The layer may comprise another transmembrane and / or membrane intraproteins as well as other molecules besides the pore. The appropriate devices and conditions are discussed below. The method of the invention is typically carried out in vitro.
[00063] [00063] The polynucleotide can be attached to 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 above), the polynucleotide is preferably attached to the membrane via a polypeptide present in the membrane or an anchor
[00065] [00065] The connection can be stable or transient. For certain applications, the transitory nature of the connection is preferred. If a stable binding molecule was attached directly to each of the 5th or 3rd ends of a polynucleotide, then some data will be lost since the distinction processing cannot continue until the end of the polynucleotide due to the distance between the bilayer and the active site helicase. If the bond is transient, then when the randomly bonded end becomes free of the bilayer, then the polynucleotide can be processed until completion. The chemical groups that form stable or transient bonds with the membrane are discussed in more detail below. The polynucleotide can be transiently attached to an amphiphilic layer, such as a lipid bilayer using cholesterol or an acyl grease chain. Any chain of acyl grease having a length of 6 to 30 carbon atoms, such as hexadecanoic acid, can be used.
[00066] [00066] In preferred embodiments, the polynucleotide is attached to an amphiphilic layer. The binding of polynucleotides to the bilayers of
[00067] [00067] 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 binding chemicals give a set of binding options for polynucleotides. Each different modification group ties the polynucleotide in a slightly different way and the bond is not always permanent so as to give different residence times for the polynucleotide to the bilayer. The advantages of the transient link are discussed above.
[00068] [00068] Polynucleotide binding can also be achieved by several other means as long as a reactive group can be added to the polynucleotide. The addition of reactive groups to each end of the DNA has been previously reported. A thiol group can be added to the 5th of ssSDNA using the polynucleotide kinase and ATPyS (Grant, GP and PZ Qin (2007). “A facile method for attaching nitroxide spin labels at the 5th 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 modified oligonucleotides for the 3rd ssSDNA (Kumar, A., P. Tchen, er al. (1988). “ Nonradioactive
[00070] [00070] A common technique for amplifying sections of genomic DNA is to use the polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, several copies of the same DNA section can be generated, where for each 5º copy of each strand in the duplex it 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 binding.
[00071] [00071] The transmembrane pore is preferably a transmembrane protein pore. A pore of transmembrane protein is a
[00072] [00072] The transmembrane protein pore can be a monomer or an oligomer. The pore is preferably composed of several repeating subunits, such as 6, 7, 8 or 9 subunits. The pore is preferably a hexameric, heptameric, octameric or nonameric pore.
[00073] [00073] The transmembrane protein pore typically comprises a tube or channel through which ions can flow. The pore subunits typically surround a central axis and contribute filaments to a transmembrane tube or channel A or a helix bundle or transmembrane channel.
[00074] [00074] The transmembrane protein pore tube or channel typically comprises amino acids that facilitate interaction with analyte, such as nucleotides, polynucleotides or nucleic acids. These amino acids are preferably located close to a tube or channel constriction. The transmembrane protein 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.
[00076] [00076] The transmembrane protein pore is preferably derived from Msp, preferably from MspA. Such a pore will be oligomeric and typically comprises 7, 8, 9 or 10 monomers derived from Msp. The pore can be a homo-oligomeric pore derived from Msp that comprises identical monomers. Alternatively, the pore may be an Msp-derived hetero-oligomeric pore that comprises at least one monomer that differs from the others. Preferably, the pore is derived from MspA or a homologous or analogous to it.
[00077] [00077] A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2 or a variant thereof. SEQID NO: 2 is the MS- (B1) 8 mutant of the monomer MspA. It includes the following mutations: D90N, D91N, D93N, DI 18R, DI34R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence that
[00078] [00078] Over the entire 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 the amino acid identity. More preferably, the variant 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 minus 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 with respect to the entire sequence. There can be at least 80%, for example at least 85%, 90% or 95%, of amino acid identity in a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids (“difficult homology ”).
[00079] [00079] Standard methods 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, for example used i. 23/78 in their default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate: homology or sequence alignment (such as identifying equivalent i residues or corresponding sequences (typically in your |: 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: 403-10. The software to 'perform the BLAST analysis is publicly available through the National Center for Biotechnology Information (http: //www.ncbi.nlm.nih.gov/).
[00080] [00080] SEQ ID NO: 2 is the MS- (B1) 8 mutant of the MSpA monomer. The variant can 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, the variant may comprise the following substitution present in MspB: A138P. The variant can comprise one or more of the following substitutions present in MspC: A96G, NI02E and A138P. The variant may comprise one or more of the following mutations present in MspD: Deletion of Gl, L2V, ESQ, L8V, DI3G, W21A, D22E, K47T, 149H, I68V, D91G, A96Q, NI102D, S103T, V1041, SI36K and GI41A . The variant may comprise combinations of one or more of the Msp B, Ce D mutations and substitutions. The variant preferably comprises the L88N mutation. The variant of SEQ ID NO: 2 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.
[00081] [00081] Amino acid substitutions can be manufactured for 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 amino acids introduced may have polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge similar to those
[00082] [00082] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues can be deleted, or more.
[00083] [00083] Variants can include fragments of SEQ ID NO: 2. Such fragments retain pore-forming activity. The fragments can be: at least 50, 100, 150 or 200 amino acids in length. Such fragments can be used to produce pores. A fragment "preferably comprises the pore-forming domain of SEQ ID NO: 2. 'Fragments must include one of residues 88, 90, 91, 105, 118 and 134 of SEQ' ID NO: 2. Typically, the fragments include all residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2.
[00084] [00084] One or more amino acids can be alternatively or additionally added to the polypeptides described above. An extension can be provided at the amino or carboxy terminal of the amino acid sequence of SEQ ID NO: 2 or variant of polypeptide or fragment thereof. The length 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 an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below.
[00085] [00085] 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 ability to form pore of Msp, which contains a tube B, is provided by the leaves f in each subunit. A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form B leaves. One or more modifications can be made to the regions of SEQ ID NO: 2 that form B leaves as 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 helices a and / or loop regions.
[00086] [00086] Msp-derived monomers can be modified to aid in their identification or purification, for example by adding: histidine residues (a hist 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 polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag in a native or engineered position in the pore. An example of this would be to react a gel-changing reagent with an engineered cysteine outside the pore. This has been demonstrated as a method for separating hetero-oligomers from hemolysin (Chem Biol. Jul 1997; 4 (7): 497-505).
[00087] [00087] The Msp-derived monomer can be labeled with a developer tag. The developer tag can be any suitable tag that allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, for example 1251, ** S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.
[00088] [00088] The Msp-derived monomer can also be produced 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 to produce such proteins or peptides.
[00089] [00089] The Msp-derived monomer contains one or more specific modifications to facilitate nucleotide discrimination. The Msp-derived monomer can also contain other non-specific modifications as long as they do not interfere with pore formation. Various non-specific side chain modifications are known in the art and can be manufactured for the side chains of the Msp-derived monomer. Such modifications include, for example, reductive alkylation of amino acids
[00091] [00091] The transmembrane protein pore is also preferably derived from α-hemolysin (α-HL). The pore of wild-type a-HL is formed from seven identical monomers or subunits (that is, the same is heptameric). The sequence of an α-hemolysin-NN monomer or subunit is shown in SEQ ID NO: 4. The pore of the transmembrane protein 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 a constriction of the a-HL tube or channel.
[00092] [00092] 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 (homoheptamer) or different (heteroheptamer).
[00093] [00093] A variant of SEQ ID NO: 4 is a protein that has a
[00094] [00094] The variant may include modifications that facilitate covalent bonding or interaction with the helicase. The variant preferably comprises one or more reactive cysteine residues that facilitate binding to helicase. For example, the variant may include a cysteine in 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. The preferred variants comprise a replacement of the residue in positions 8, 9, 17, 237, 239 and 287 of SEQID NO: 4 with cysteine (ABC, T9C, NI7C, K237C, S8239C or E287C). The variant is preferably any of the variants described in International Application No. 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).
[00095] [00095] The variant can also include modifications that facilitate any interaction with nucleotides.
[00096] [00096] The variant can be a naturally occurring variant that is naturally expressed by an organism, for example by a Staphilococcus bacterium. Alternatively, the variant can be expressed in vitro or recombinantly by bacteria such as Escherichia coli. Variants also include naturally occurring variants produced by recombinant technology. In the entire length of the sequence of í; 29/78 amino acid of SEQ ID NO: 4, a variant will preferably be at least 50% homologous to this sequence based on the amino acid identity. More: preferably, the variant polypeptide can be at least 55%, at least: at least 60%, at least 65%, at least 70%, at least 75%, at least | : minus 80%, at least 85%, at least 90% and more preferably at Ú at least 95%, 97% or 99% homologous based on the amino acid identity with the amino acid sequence of SEQ ID NO: 4 with respect to the sequence entire. There can be at least 80%, for example at least 85%, 90% or 95%, of amino acid identity in a stretch of 200 or more, for example 230, 250, 270 or 280 or more, contiguous amino acids (“difficult homology ”). Homology can be determined as discussed above.
[00097] [00097] 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.
[00098] [00098] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 4 can be further deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues can be deleted, or more.
[00099] [00099] Variants can be fragments of SEQ ID NO: 4. Such fragments retain the activity that forms pore. The 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.
[000100] [000100] One or more amino acids can be alternatively or additionally added to the polypeptides described above. An extension can be provided at the amino or carboxy terminal of the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. THE
[000102] [000102] A variant of SEQ ID NO: 4 preferably includes one or more modifications, such as substitutions, additions or deletions, within its a-helices and / or loop regions. The amino acids that form a-helices and loops are discussed above.
[000103] [000103] The variant can be modified to assist in its identification or purification as discussed above. The pores derived from a-HL can be manufactured as discussed above with reference to the pores derived from Msp.
[000104] [000104] In some embodiments, the transmembrane protein pore is chemically modified. The pore can be chemically modified in any way and anywhere. The transmembrane protein pore is preferably chemically modified by the binding
[000105] [000105] For example, the pore can be chemically modified by the bonding of a dye or a fluorophore.
[000106] [000106] 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 are preferably chemically modified as discussed above.
[000107] [000107] The reactivity of cysteine residues can be enhanced by modifying adjacent residues. For example, the basic groups of flanking residues of arginine, histidine or lysine will change the pKa of the thiol group of cysteines to that of the more reactive S 'group. The reactivity of cysteine residues can be protected by thiol protecting groups such as dTNB. These can be reacted with one or more pore cysteine residues before a ligand is attached.
[000108] [000108] The molecule (with which the pore is chemically modified) can be linked directly to the pore or linked via a linker as disclosed in International Order Nos. PCT / GB09 / 001690 (published as WO 2010/004273), PCT / GB09 / 001679 (published as WO 2010/004265) or PCT / GBI0 / 000133 (published as WO 2010/086603).
[000109] [000109] Any RecD helicase can be used according to the invention. The structures of the RecD helicases are known in the art (FEES IL. April 2008; 275 (8): 1835-51. Epub 9 March 2008. O: 32/78 RecD ATPase activity is essential for the growth of Pseudomonas syringae from | Antarctica Lz4W at low temperature Satapathy AK, Pavankumar TL,: Bhattacharjya S, Sankaranarayanan R, Ray MK; EMS Microbiol Rev. May 2009; 33 (3): 657-87. The diversity of conjugative relaxases and its , application in plasmid rate. Garcillan-Barcia P, Francia MV, de la Cruz F; Biol Chem. April 8, 2011; 286 (14): 12670-82. Epub February 2, 2011. Functional characterization of the multidomain Plasmid Tral relaxase-helicase (Cheng Y, McNamara 131E Miley MJ, Nash RP, Redinbo MR).
[000110] [000110] RecD helicase typically comprises the amino acid motif X1-X2-X3-G-X4-X5-X6-X7 (hereinafter referred to as motif as RecD 1; SEQ ID NO: 8), where X1 is G , S or A, X2 is any amino acid, X3 is P, A, S or G, X4 is T, A, V, Sou C, XS is Gou A, X6 is K orReX7 is TousS. X1 is preferably G. X2 is preferably G, 1, Y or A. X2 is more preferably G. X3 is preferably P or A. X4 is preferably T, A, V or C. X4 is preferably T, V or C. X5 is preferably G. X6 is preferably K. X7 is preferably T or S. To the Helicase RecD preferably comprises Q- (X8) 6-18-X1-X2-X3-G-X4-X5- X6-X7 (hereinafter called the motif equal to extended RecD 1; SEQ ID NOs: 9, 10 and 11 where there are 16, 17 and 18 X8s respectively), where X1 to X7 are as defined above and X8 is any amino acid. There are preferably 16 X8 residues (i.e. (X8) 16) on the motif as extended RecD I (SEQ ID NO: 9). Suitable sequences for (X8) ,, can be identified in SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42 and 44.
[000111] [000111] The RecD helicase preferably comprises the amino acid motif GGPG-Xa-GK-Xb (hereinafter called motif RecD I; SEQ 1D NO: 12) where Xa is T, Vou Ce Xbé Tou S. Xa is preferably T. Xb is preferably T. The Rec-D helicase preferably comprises the sequence GGPGTGKT (SEQ ID NO: 19; see Table
[000112] [000112] RecD helicase typically comprises the amino acid motif X1-X2- X3-X4-X5- (X6); - QX7 (hereinafter called motif V equal to RecD; SEQ ID NO: 160, where X1 is Y , WouF, X2 is A, T, S, M, C or V, X3 is any amino acid, X4 is T, Nou S, X5 is A, T, G, S, Vou, X6 is any amino acid and X7 is G or S X1 is preferably Y. X2 is preferably A, M, C or V. X2 is more preferably A. X3 is preferably 1, M or L. X3 is more preferably I or L, X4 is preferably T or S. X4 is more preferably T. X5 is preferably A, V or I. X5 is more preferably V or 1. X5 is more preferably V. (X6) 3 is preferably HKS, HGA or HRS (X63 is more preferably HKS. X7 is preferably G. A RecD helicase preferably comprises the amino acid motif Xa-Xb-Xc-Xd-Xe-HK-SQG (hereinafter motif V of RecD; SEQ ID NO: 17), where Xa is Y, WouF. Xbé A, M , Cou V, Xc is 1, MouL. Xd is TouSe Xe is V or L Xa is preferably Y. Xb is preferably A. Xd is pref preferably T. Xe is preferably V. The RecD helicase preferably comprises (1) motifs equal to RecD I and V (SEQ ID NOs: 8 and 12), (2) motif 1 of RecD and motif V equal to RecD (SEQ ID NOs : 12 and 16), 3) RecD motifs I and V (SEQ ID NOs: 12 and 17), (4) prolonged RecD motif I and RecD motif V (SEQ ID NOs: 9, 10 or 11 and 16), (5) prolonged RecD motif I and RecD motif V (SEQ ID NOs: 13, 14
[000113] [000113] The preferred I RecD motifs are shown in table 5 below, the motifs | Preferred RecD equals are shown in Table 7 below. Preferred RecD-like V motifs are shown in Tables 5 and 7 below.
[000114] [000114] The RecD helicase is preferably one of the helicases shown in table 4 below or a variant thereof. Table 4 - Preferred RecD helicases and their accession numbers 1 NP295625.1 - exodesoxyribonuciease V RecD subunit [Deinococcus radiodurans RI) 2 VP604297.1 - helicaseRecD / TraA [Deinococcus acoutromalis DSM 11300] 3 - YPO027863433 VCD115] 4 3EISA chain A, Structure of Deinococcus N-Terminal Truncation - VPOOS2561441 helicase, RecD / TraA family [Deinococcus proteolyticus MRP] 6 VPO04170918.1 - helicase, RecD / TraA family [Deinococcus maricopensis DSM 2121138 XSH38118] X - 11 , family RecD / TraA [Deinococcus proteolyticus MRP] 8 - YPOO3SSS838,1 helicase, family RecD / TraA [Cyanothece sp. PCC 7822] 9 ZPOSS79275,1 - helicase, family RecD / TraA [Prevotella multisaccharivorax DSM 17128] —VP0023776921 helicase, family RecD / TraA [Cyanothece sp. PCC 7424] 1) VPOO0ISIGBIS! helicaseda family RecD / TraA [Acaryochloris marina MBIC11017] 2 VPOO03318882,1 - helicase, family RecD / TraA [Sphaerobacter thermophilus DSM 20745] 13 YPOO04671137.1 hypothetical protein SNE A07690 [Simkania neaevensis Z) 4 YP375364 - YP375364 Chlorobium luteolum DSM 273]> gbjABB24321,1 | 15º YPOO0Z4189081 helicaseda family RecD / TraA [Methylobacterium chlorometanicum CM4] 16 —VPO03065757,1 helicase (Methylobacterium extorpuens DM4]> embjCAX21689,1 | 17 - ZPOOSIS989,1 helicaseRecD / Traà [Crocosphaera helicase3] / TraA [Ktedonobacter racemifer DSM 449631 19 - ZPOB486910.1 helicase, family RecD / TraA [Metilomicrobium album BG $ 1 —YPO02015362.1 helicaseda family RecD / Traà [Prosthecochloris aestuarii DSM 271] 21 YPOOII30786io helicasa Chibrida family RecD DSM 265] 22 —VPO029612581 helicase (Methylobacterium extorouens AM1]> gb | ACS37981.1 | 23 ZPO87729021 helicase, family RecD / TraA [Thiocapsa marina 5811]> gbjEGV16093.1 | 24 - YPOOI637509.1 helical family Pal] - ZPO20628241 helicase, family RecD / TraA [Rickettsiella grilli]> gb / EDP46829, 1) 26 - ZPOST687T53,1 helicase, family RecD / TraA [Thiocapsa marina 5811]> gblEGV20712,1 | 27 - VPO001922739,1 helicase, family RecD / TraA [Methylobacterium populi BJOO1] 28 —YPO02018300.1 helicase, family RecD / TraA [Pelodictyon phacoclathratiforme BU-1] 29 —ZPO6MASITII helicase, family RecD / Traà [Victivallis vadensis ATCC BAA-S482] - ZPOST7 helicase, family RecD / TraA [Thiocapsa marina 5811]> eb [EGV17897,1 | 31 - ZPOST698991 helicase, family RecD / TraA [Thiocapsa marina 5811]> 0 [EGV18833,1 | 32 —ZP03727363] - exodesoxyribonuclease V [Opitutaceae bacterium TAV2) 33 - ZP050277971 helicase, RecD / TraA family [PCC 7420 microcoleus chthonoplastes] 34 - VPOOIS2I445.1 helicaseda RecD / TraA family [AcaryochD1103M3103M3D2] Symp3D3D3D3D3D3D3D3D3D3D3D3D3D3D3DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDF autotrophicum EIRM2]> gbjACN17985, | 36 —VPOO3IGS6IS! helicase, family RecD / TraA [Candidatus Accumulibacter phosfatis] 37 —ZPOI7322651 - helicase RecD / TraA [Cyanothece sp. CCYO110]> RbIEAZ88318.1 | 38 YP901533.1 - helicaseda family RecD / TraA [Pelobacter propionicus DSM 2379] 39 —VPOO4I21205.1 helicase, family RecD / TraA [Desulfovibrio aespocensis Aspo-2] 4th YP9I1313,1 - helicaseda family RecD / TraA [Chlorobium phacobacter 26] 41 - YPO024240081 helicase family RecD / TraA [Methylobacterium chlorometanicum] 42 YP320143,1 helicase RecD / TraA [Anabaena variabilis ATCC 29413] 4s YPO00I603050,1 - exodesoxyrribonuclease [Gluconacetobacter diazotrophice PAZ 5 Octadecabacter antarcticus 307] 45 - YPOO3A45I64,1 helicase, family RecD / TraA [Allochromatium vinosum DSM 180) 46 NP4901771 - exodesoxyribonuclease V, alpha chain [Nostoc sp. PCC 7120] 47 NP 923575.1 exodesoxyribonuclease V alpha chain [Gloeobacter violaceus PCC 7421] 48 —YPOOI6OI2A41 exodesoxyribonuclease V alpha chain [Gluconacetobacter diazotrophicus]
[000115] [000115] The RecD helicase is most preferably one of the helicases shown in table 5 below or a variant thereof. The RecD helicase most preferably comprises the sequence of one of the helicases shown in table 5, i.e. one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37.39, 41.42 and 44 , or a variant thereof. Table 5 - Most preferred RecD helicases% of | The reason V equals | SEO Name | Source Reference NCBI | Identity for | "The Ss No) Reco | | cD2 Dra *) | (SEQIDNO: | ReeD acarvochtoris | NCBI reference | WAVTIH 18 2 S Sequence: 29.8 GGPGTGKT (19)) - KSQG Ama | Marim YP 001521445,1 20 NCBI reference YALTVH a | or aa | “Ps Sequence GGPGTGKS (22) | RAQG | YP 002786343,1: 23 24 | RecD - Deinococeus | E eeEn GGPGTGKS (22) ie Pp cia: 2Dge | geotheromalis | = vpesoa2971 | Rep | Halangum | Reference CNBI YAISVA aloe | Ochraceum String: 30 GGPGVGKT (26) - KSQG DSM YP 0032701181 27)
[000116] [000116] All strings in the Table above comprise a V motif equal to RecD (as shown). Only SEQ ID NOs: 18, 25.28, 30.35, 37.39 and 42 comprise a RecD V motif (as shown).
[000117] [000117] The RecD helicase is preferably a Tral helicase or a Tral subgroup helicase. The Tral helicases and the Tral subgroup helicases can contain two domains of the RecD helicase, a relaxase domain and a C-terminal domain. The helicase of the Tral subgroup is preferably a TrwC helicase. The Tral helicase or helicase of the Tral subgroup is preferably one of the helicases shown in table 6 below or a variant thereof.
[000118] [000118] The Tral helicase or a helicase of the Tral subgroup typically comprises a RecD motif I as defined above (SEQ ID NO: 8) and / or a V motif equal to RecD as defined above (SEQ ID NO: 16). The Tral helicase or a Tricas subgroup helicase preferably comprises both a RecD motif I (SEQ ID NO: 8) and a RecD motif V (SEQ ID NO: 16). Tral helicase or helicase
[000119] [000119] Tral helicase or Tricas subgroup helicase are most preferably one of the helicases shown in table 7 below or a variant thereof. The Tral helicase or helicase of the Tral subgroup more preferably comprises the sequence of one of the helicases shown in table 7, i.e. one of SEQ ID NOs: 61, 65, 69, 73, 74, 78, 82, 86, 90, 94 , 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168, or a variant the same. Table 7 - Tral helicase and helicases of the most preferred Tral subgroup SEQID: *% of Reason | equal | Reason V equal Reason II! Mob NO: Cepe Name NCBI Reference Identity aRecD a RecD FouQ Ê with Tral Eco | (SEQ ID NO:) | (SEQ ID NO:) | (SEQ ID NO: GYAGV YAITA DIS Tral sequence under, NCBI Reference: El [ | Eco Escherichia coli | np 0614831 Genbank | o Pray AAQ98619.1 64)
[000120] [000120] SEQ ID NOs: 78 and 106 comprise a MobQ motif III, while the other sequences in table 7 comprise a MobF motif III.
[000121] [000121] Tral helicase preferably comprises the sequence
[000123] [000123] In the entire length of the amino acid sequence of any of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and
[000124] [000124] Homology is determined as described above. The variant may differ from the wild type sequence in any of the modes discussed above with reference to SEQ ID NOs: 2 and 4.
[000125] [000125] In particular, variants may include fragments of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168. Such fragments retain polynucleotide binding activity. The fragments can be at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 650, at least about 700, at least about of 800, at least about 900 or at least about 1000 amino acids in length. The length
[000127] [000127] Amino acid substitutions can be made to the amino acid sequence of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69 , 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151 , 152, 156, 160, 164 and 168, for example up to 1, 2, 3,4, 5,10, 20 or 30 substitutions. Substitutions are preferably conservative substitutions as discussed above. One or more substitutions can be made at the amino acid positions K555,
[000128] [000128] A variant, such as a fragment, of any of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69 , 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151 , 152, 156, 160, 164 and 168 preferably comprise RecD motif I (or RecD motif I) and / or RecD motif V (or RecD motif V) of the relevant wild-type sequence. A variant, such as a fragment, of any of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises motif I equal to RecD (or motif I of RecD) and motif V equal to RecD (or motif V of RecD) of the relevant wild-type sequence. For example, a variant of SEQ ID NO: 18 preferably comprises the motif I of Rec (D GGPGTGKT (SEQ ID NO: 19) and the motif V of Rec / D WAVTIHKSQG (SEQ ID NO: 20). equal to RecD (or RecD I and V motifs) for each of SEQ ID NOs: 18, 21, 24, 25,28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 69.73, 74, 78, 82, 86, 90, 94, 98,
[000129] [000129] Helicase can be covalently attached to the pore. Helicase is preferably not covalently attached to the pore. The application of a voltage to the pore and helicase typically results in the formation of a sensor that is capable of sequencing target polynucleotides. This is discussed in more detail below.
[000130] [000130] Any of the proteins described here, ie the RecDs pores or helicases of the transmembrane protein, can be modified to aid in their identification or purification, for example by adding histidine residues (a his tag), acid residues aspartic (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 where the polypeptide it does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag in a native or engineered position in the pore or helicase. An example of this would be to react a gel changing reagent to an engineered cysteine outside the pore. This has been demonstrated as a method to separate hetero-oligomers from hemolysin (Chem Biol. July 1997; 4 (7): 497-505).
[000131] [000131] The pore and / or helicase can be labeled with a label of
[000132] [000132] Proteins can be manufactured synthetically or by recombinant means *. For example, the pore and / or helicase can be synthesized by in vitro translation and transcription (IVTT). The pore and / or helicase amino acid sequence 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 following synthetic or recombinant production.
[000133] [000133] The pore and / or helicase can also be produced using D-amino acids. For example, the pore or helicase can comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art to produce such proteins or peptides.
[000134] [000134] The pore and / or helicase may also contain other non-specific modifications as long as they do not interfere with the formation of the pore or function of the helicase. Various non-specific side chain modifications are known in the art and can be made for the side chains of the protein (s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBHA, amidination with methyl acetimidate or acylation with acetic anhydride.
[000135] [000135] The pore and helicase can be produced using standard 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 host cell; 61/78 bacterial using standard techniques in the industry. 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. i These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Is Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory: Press, Cold Spring Harbor, NY.
[000136] [000136] The pore and / or helicase can be produced on a large scale following purification by any liquid protein chromatography system from organisms that produce protein or after recombinant expression. typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system.
[000137] [000137] 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 (1) from the length of the target polynucleotide, (ii) from the identity of the target polynucleotide, (iii) from the sequence of the target polynucleotide, (iv) from the secondary structure of the target polynucleotide and (v) if the target polynucleotide is modified or not. Any combination of (i) to (v) can be measured according to the invention.
[000138] [000138] For (1), the length of the polynucleotide can be measured using the number of interactions between the target polynucleotide and the pore.
[000139] [000139] For (ii), the polynucleotide identity can be measured in several ways. The identity of the polynucleotide can be measured in conjunction with measuring the sequence of the target polynucleotide or without measuring the sequence of the target polynucleotide. The first is direct; the polynucleotide is sequenced and thus identified. The latter can be done in several ways. For example, the presence of a particular motif in the polynucleotide
[000140] [000140] For (iii), the polynucleotide sequence can be determined: as previously described. Suitable 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.
[000141] [000141] For (iv), the 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 the current that flows through the pore. This allows single-stranded and double-stranded polynucleotide regions to be distinguished.
[000142] [000142] For (v), the presence or absence of any modification can be measured. The method preferably comprises determining whether the target polynucleotide is modified or not by methylation, oxidation, 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 based on the current that flows through the pore during its interaction with each nucleotide.
[000143] [000143] A variety of different types of measurements can be made. These include without limitation: electrical measurements and optical measurements. Possible electrical measurements include: current measurements, impedance measurements, tunneling measurements (Ivanov AP et al., Nano Lett. 12
[000145] [000145] In a preferred embodiment, the method comprises: (a) contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore; and (b) measuring the current passing through the pore as the polynucleotide moves with respect to the pore in which the current is indicative of one or more characteristics of the target polynucleotide and thereby distinguishing the target polynucleotide.
[000146] [000146] The methods can be performed using any device 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 device that is suitable for feeling the transmembrane pore. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier which separates the chamber into two sections. The barrier has an opening in which the membrane containing the pore is formed.
[000147] [000147] The methods can be performed using the device described
[000148] [000148] The methods may involve measuring the current passing. through the pore as the polynucleotide moves with respect to the pore. Therefore, the device can also comprise an electrical circuit capable of: applying a potential and measuring an electrical signal through the membrane and pore. C The methods can be performed using a patch-clamp or a 'voltage clamp'. The method preferably involves the use of a voltage clamp.
[000149] [000149] The methods of the invention may involve measuring a current that passes through the pore as the polynucleotide moves with respect to the pore. Suitable conditions for measuring ionic currents through the pores of the transmembrane protein are known in the art and disclosed in the Example. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically +2 V to -2 V, typically from -400 mV to + 400mV. The voltage used is preferably in a range having a selected lower limit of -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, - 20 mV and O mV and an independently selected upper limit +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 of 100 mV to 240 mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase the discrimination between different nucleotides by a pore by using an increased applied potential.
[000150] [000150] The methods are typically carried out in the presence of any of the charge carriers, such as metal salts, for example alkali metal salt, halide salt, for example chloride salts, such as alkali metal chloride salt. Cargo carriers can include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3- chloride
[000151] [000151] The 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 method of the invention. Typically, the buffer is HEPES. Another suitable buffer is the Tris-HCl buffer. The methods are typically performed at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8 , 7 or 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.
[000152] [000152] The methods can be performed from 0ºC to 100ºC, from 15ºC to 95ºC, from 16ºC to 90ºC, from 17ºC to 85ºC, from 18ºC to 80ºC, from 19ºC to 70ºC, or from 20ºC to 60ºC. The methods are typically performed at room temperature. The methods are optionally carried out at a temperature that supports the enzyme function, such as around 37ºC.
[000153] [000153] The method is typically performed in the presence of free nucleotides or free nucleotide analogs and an enzyme cofactor that facilitates
[000154] [000154] The target polynucleotide can be contacted with the RecD helicase and the pore in any order. It is preferred that when the target polynucleotide is contacted with the RecD helicase and the pore, the target polynucleotide first forms a complex with the helicase. When the voltage is applied through the pore, the target polynucleotide / helicase complex then
[000156] [000156] The method of the invention most preferably involves a pore derived from MspA and a helicase comprising the sequence shown in SEQ ID NO: 61 or a variant thereof. Any of the embodiments discussed above with reference to MspA and SEQ ID NO: 61 can be used in combination. Other methods
[000157] [000157] The invention also provides a method of forming a sensor to distinguish a target polynucleotide. The method comprises forming a complex between a pore and a RecD helicase. The complex can be formed by contacting the pore and helicase in the presence of the target polynucleotide and then applying a potential across 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 attaching the pore to the helicase. Methods for covalent bonding are known in the
[000158] [000158] The present invention also provides kits for distinguishing a target polynucleotide. The kits comprise (a) a pore and (b) a RecD helicase. Any of the embodiments discussed above with reference to the method of the invention also apply to kits.
[000159] [000159] The kit can further comprise the components of a membrane, such as the phospholipids needed to form an amphiphilic layer, such as a lipid bilayer.
[000160] [000160] The kits of the invention may additionally comprise one or more other reagents or instruments that enable any of the above-mentioned embodiments to be performed. Such reagents or instruments include one or more of the following: suitable buffer (s) (aqueous solutions), means for obtaining a sample from an individual (such as a vessel or an instrument comprising a needle), means for amplifying and / or expressing polynucleotides, a membrane as defined above, or voltage or contact clamping apparatus. The reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit, optionally, may also comprise instructions for allowing the kit to be used in the method of the invention or details with respect to which patients the method can be used. The kit can optionally comprise nucleotides.
[000162] [000162] The apparatus is preferably adjusted to carry out the method of the invention. The apparatus preferably comprises: a sensor device that is capable of supporting the membrane and the plurality of pores and is operable to perform the polynucleotide distinction using the pores and helicases; at least one reservoir to contain material to perform the distinction; a fluidic system configured to controllably supply 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 deliver the samples selectively from the containers to the sensor device. The apparatus can 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). Characterization without a pore
[000163] [000163] In some embodiments, the target polynucleotide is distinguished, such as partially or completely sequenced, using one is S 70/78 helicase RecD, but without using a pore. In particular, the invention also provides a method of distinguishing a target polynucleotide which comprises contacting the target polynucleotide with a RecD helicase such that the RecD helicase controls the movement of the target polynucleotide. In this method, the target polynucleotide is preferably not contacted with a pore, such as a transmembrane pore. The method involves obtaining one or more measurements: as the RecD helicase controls the movement of the polynucleotide and thus distinguishes the target polynucleotide. The measurements are indicative of one or more characteristics of the target polynucleotide. Any of such measurements can be considered in accordance with the invention. They include without limitation: electrical measurements and optical measurements. These are discussed in detail above. Any of the embodiments discussed above with reference to the pore-based method of the invention can be used in the method which lacks a pore. For example, any of the RecDs helicases discussed above can be used.
[000164] [000164] The invention also provides an analysis apparatus comprising a RecD helicase. The invention also provides a Kkit for distinguishing a target polynucleotide comprising (a) an analysis apparatus for distinguishing a target polynucleotide and (b) a RecD helicase. This apparatus and kits preferably do not comprise a pore, such as a transmembrane pore. Suitable devices are discussed above.
[000165] [000165] The Examples that follow illustrate the invention. Example 1
[000166] [000166] This example illustrates the use of a Tral helicase (Tral Eco; SEQ ID NO: 61) to control the movement of intact DNA strands through a nanopore. The general method and the substrate used throughout this example is shown in Fig. | and described in the figure legend. Materials and methods
[000167] [000167] The primers were designed to amplify a fragment
[000168] [000168] The design of the DNA substrate used in all the experiments described here is shown in Fig. IB. The DNA substrate consists of a section of PhiX ssSDNA, with a 5º 50T leader to help capture by the nanopore (SEQ ID NO: 172). The annealing to this filament just after the leader 50T is an initiator (SEQ ID NO: 173) that contains a cholesterol tag in 3rd to enrich the DNA on the surface of the bilayer, and thus improves the capture efficiency. An additional primer (SEQ ID NO: 174) is used for the 3rd end of the filament to assist in capturing the filament from the 3 "end.
[000169] [000169] Buffered solution: 400 mM NaCl, 10 mM Hepes, pH 8.0, 1 MM ATP, 1 mM MgCh ,, 1 mM DTT
[000170] [000170] Nanoporo: Ecoli MS (B2) J8 MspA ONLP3476 MS- (L88N / D9ON / D91N / D93N / D1 18R / D134R / E139K) 8
[000171] [000171] Enzyme: Tral Eco (SEQ ID NO: 61; ONLP3572, 4.3 µM) i 23.3 nl -> 100 nM final. . [000172] Electrical measurements were acquired from the unique MSpA nanopores inserted in the lipid bilayers of 1,2-diphitanoyl-glycero-3 'phosphocholine (Avanti Polar Lipides). The bilayers were formed through C openings of -100 µm in diameter in PTFE films of 20 µm thickness (in customized Delrin chambers) using the Montal-Mueller technique, separated into two 1 ml buffered solutions. All experiments were carried out in the established buffered solution. The single channel currents were measured in Axopatch 200B amplifiers (Molecular Devices) equipped with 1440A digitizers. The Ag / AgCl electrodes were connected to the buffer solutions so that the cis compartment (to which both the nanopore and the enzyme / DNA are added) is connected to the ground of the Axopatch preamplifier, and the trans compartment is connected to the active electrode of the preamplifier. After obtaining a single pore in the bilayer, the DNA polynucleotide and helicase were added to 50 µl of buffer and pre-incubated for 5 mins (DNA = 12.0 nM, Enzyme = 2 µM). This pre-incubation mixture was added to 950 μl of buffer in the cis compartment of the electrophysiology chamber to start capturing the helicase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 0.6 nM, Enzyme = 0, 1 one). Helicase's ATPase activity was initiated as required by the addition of bivalent metal (1 mM MgCl)) and NTP (1 mM ATP) to the cis compartment. The experiments were carried out at a constant potential of +140 mV. Results and Debate
[000173] [000173] The addition of Helicase-DNA substrate to the MSspA nanopores as shown in Fig. 1 produces characteristic chain blocks as shown in Figs. 2 and 3. DNA without bound helicase interacts
[000175] [000175] In the implementation shown in Fig. 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 filament. Salt Tolerance
[000176] [000176] Nanoporous filament sequencing experiments of this type generally require ionic salt. Ionic salts are needed to create a conductive solution to apply a displacement voltage to capture and displace DNA, and to measure current changes depending on the resulting sequence as the DNA passes through the nanopore. Since the measurement signal is dependent on the concentration of the ions, it is advantageous to use ionic salts in high concentration 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 400 mM KCl and above are preferred.
[000177] [000177] However, many enzymes (including some helicases and DNA motor proteins) do not tolerate high salt conditions. Under high salt conditions, enzymes break down 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 approximately 100
[000179] [000179] Most helicases move along single-stranded polynucleotide substrates in a unidirectional manner, moving a specific number of bases for each metabolized NTPase. The movement of helicase can be explored in different ways to release DNA through the nanopore in a controlled manner. Fig. 1 illustrates two basic modes, “forward” and “reverse” operation. In the advanced mode, the DNA is fed into the pore by the helicase in the same direction as the DNA would move under the forces of the applied field. This direction is shown by the trans arrows. For Tral, which is a 5-3 "helicase, this requires capturing the 5th end of the DNA in 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, ie, moving from cis to trans. The reverse mode requires capturing the 3rd end of the DNA, after the helicase proceeds to pull the passed DNA through a nanopore back opening against the applied potential field, ie , move from trans to cis Fig. 1 shows these two modes of operation using Tral Eco.
[000180] [000180] This example illustrates the salt tolerance of the RecDs helicases
[000181] [000181] A custom-made fluorescent substrate was used to test the helicase's ability to displace hybridized dsDNA (Fig. 4A). As shown in 1) of Fig. 44, the filament of the fluorescent substrate (50 'nM final) has a ssSDNA 5 projection, and a 40-base section of hybridized "dsDNA. The larger upper filament has a] carboxyfluorescein base in the 3rd end, and the hybridized complement has a black hole extinguishing base (BHQ-1) at the 5th end. When hybridized the fluorescein fluorescence is extinguished by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 nM of a filament capture that is complementary to the shorter filament of the fluorescent substrate is included in the assay. As shown in 2), in the presence of ATP (1 mM) and MegCl> (10 mM), helicase (100 nM) added to the substrate binds at the 5th tail of the fluorescent substrate, it moves along the larger filament, and displaces the complementary filament as shown, as shown in 3), since the BHQ-1 complementary filament is completely displaced fluorescein in the larger fluorescent filament. As shown in 4), an excess of capture filament preferably anneal to complementary DNA to prevent annealing of the substrate and loss of fluorescence.
[000182] [000182] Substrate DNA: SEQ ID NO: 175 with a carboxyfluorescein near the 3rd end and SEQ ID NO: 176 with a Black Hole Extinguisher-1 at the 5th end
[000183] [000183] Capture DNA: SEQ ID NO: 177
[000184] [000184] The graph in Fig. 4B shows the initial activity rate of two RecDs helicases (RecD Nth and Dth, SEQ IDs 28 and 35) in buffer solutions (100 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCb ,, 50 nM fluorescent substrate DNA, 1 μM capture DNA) containing different concentrations of 100 mM KCI at 1 M. Helicase works at 1 M.
[000185] [000185] In this example, a different Tral helicase was used, namely TrwC Cba (SEQ ID NO: 65). All experiments were performed as previously described in Example 1 under the same experimental conditions (pore = MspA B2, DNA = 400 mere SEQ ID NO: 172, 173 and CU 174, buffer = 400 mM KCl, 10 mM Hepes pH 8.0, 1 mM DTT, 1 µM ATP, 1 mM MgCl;). Fig. 5 shows two typical examples of DNA events controlled by helicase using this enzyme. Example 4
[000186] [000186] In this example several different TrwC helicases (TrwC (Atr) (SEQ ID NO: 144), TrwC (Sal) (SEQ ID NO: 140), TrwC (Cer) (SEQ ID NO: 136) and TrwC (Eco) (SEQ ID NO: 74)) were investigated for their ability to control DNA movement (SEQ ID NOs: 178, 179 (with / iSpl8 // iSpl8 // iSp18 // ISp18 // ISp18 // ISpl8 / TT / —3CholTEG / at the 3 ”end) and 180) through an MSspA nanopore (MS- (G758 / G77S / L88N / D90N / D91N / D93N / D1 18R / Q126R / D134R / E139K) 8, ie 8 x SEQ ID NO: 2 with G75S / G77S / L88N / Q126R Materials and Methods
[000187] [000187] Buffered solution: 625 mM KCl, 75 mM K Ferrocyanide, 25 mM K Ferricyanide, 100 mM Hepes at pH 8.0 for TrwC (Atr), TrwC (Eco) and TrwC (CcR), and at pH 9.0 for TrwC (Salt).
[000188] [000188] Enzyme: TrwC (Atr) (100 nM) or TrwC (Salt) (100 nM) or TrwC (Cer) (100 nM) or TrwC (Eco) (100 nM) all in a final concentration of 100 nM
[000189] [000189] Electrical measurements were acquired from single MspA nanopores inserted in bilayers of 1,2-diphitanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipides) as described in Example 1, except that platinum electrodes were used instead of Ag / AgCl. After obtaining a single pore in the bilayer, MgCl, (10 mM) and dTTP (5 mM, for
[000190] [000190] The movement of DNA controlled by the helicase was observed for each of the investigated helicases. The example strokes are shown in Figures 6 to 9 respectively. Example 5
[000191] [000191] In this example, several different TrwC helicases (TrwC (Oma) (SEQ ID NO: 106), TrwC (Afe) (SEQ ID NO: 86), and TrwC (Mph) (SEQ ID NO: 94)) as to its ability to control the movement of DNA (SEQ ID NOs: 172 to 174 for TrwC (Oma), and SEQ ID NO: 181 hybridized to SEQ ID NO: 182 (with a cholesterol tag at the 3rd end) for TrwC (Afe) and TrwC (Mph)) through an MspA nanopore (MS (G75S / G77S / L88N / D90N / D91N / D93N / D1 18R / Q126R / D134R / E139K) 8 ie 8 x SEQ ID NO: 2 with G758 / G77S / L88N / Q126R.
[000192] [000192] Buffered solution: 625 mM KCl, 75 mM K Ferrocyanide, 25 mM K Ferricyanide, 100 mM Hepes. pH 8.0
[000193] [000193] Enzyme: TrwC (Oma), TrwC (Afe), and TrwC (Mph) all in a final concentration of 100 nM
[000194] [000194] Electrical measurements were acquired from single i MSpA nanopores inserted in bilayers of 1,2-diphitanoyl-glycero-3-phosphocholine 'lipid (Avanti Polar Lipids) as described in Example 1, except that platinum electrodes were used instead of Ag / AgCl. After obtaining 'a single pore in the bilayer, MgCl, (10 mM) was added to the cis i chamber and a control experiment was conducted for 5 mins at an applied' potential of 120 mV. 0.15 nM final DNA polynucleotide (SEQ ID NOs: 172 to 174 (as in Example 1) for TrwC (Oma), or SEQ ID NO: 181 hybridized to 182 by TrwC (Afe) and TrwC (Mph)) and Final 100 nM of the appropriate helicase (TrwC (Oma), TrwC (Afe), and TrwC (Mph) were added to the cis chamber and another control experiment was conducted for 10 mins at an applied potential of +120 mV. The ATPase activity of the helicase was initiated by the addition of ATP (lI mM) to the cis compartment of the electrophysiology chamber.The experiments were carried out at a constant potential of +120 mV.
[000195] [000195] The movement of DNA controlled by helicase was observed for each of the investigated helicases. Example strokes are shown in Figures 10 to 12 respectively.

权利要求:
Claims (21)
[1]
1. Method for characterizing a target polynucleotide, characterized by the fact that it comprises: (a) contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves through the pore and the RecD helicase controls the movement of the target polynucleotide through the pore; and (b) obtaining one or more measurements as the polynucleotide moves with respect to the pore in which the measurements are indicative of one or more characteristics of the target polynucleotide and thus characterizing the target polynucleotide.
[2]
2. Method according to claim 1, characterized in that the one or more characteristics are selected from (1) the length of the target polynucleotide, (11) the identity of the target polynucleotide, (111) the sequence of the target polynucleotide, (iv) the secondary structure of the target polynucleotide and (v) whether the target polynucleotide is modified or not, optionally in which the target polynucleotide is modified by methylation, oxidation, damage, with one or more proteins or with one or more labels , labels or spacers.
[3]
3. Method according to claim | or 3, characterized by the fact that the one or more characteristics of the target polynucleotide are measured by electrical measurement and / or optical measurement, and in which the electrical measurement is a current measurement, an impedance measurement, a tunnel formation measurement or a field effect transistor (FET) measurement.
[4]
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.
[5]
Method according to any one of the preceding claims, characterized in that the at least a portion of the polynucleotide is double-stranded.
[6]
6. Method according to any of the preceding claims, characterized in that the pore is a pore of transmembrane protein or a pore of solid state, optionally wherein the pore of transmembrane protein is selected from a hemolysin, leucocidine, porcine A from Mycobacterium smegmatis (MspA), porin F external membrane (OmpFP), porin G from external membrane (OmpG), phospholipase A from external membrane, Neisseria autotransporter lipoprotein (NalP) and WZA.
[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 is a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 2 based on amino acid identity to the entire sequence and retains pore activity, or (b) a-hemolysin formed from seven identical subunits as shown in SEQ ID NO: 4 or is a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on the amino acid identity to the entire sequence and retains pore activity.
[8]
8. Method according to any of the preceding claims, characterized in that the RecD helicase comprises: - the amino acid motif X1-X2-X3-G-X4-X5-X6-X7 (SEQ ID NO: 8), where X1 is G, Sou A, X2 is any amino acid, X3 is P, A, S or G, X4A is T, A, V, Sou C, X5 is Gou A, XG is Kou Re X7 is Tou S; and / or - the amino acid motif X1-X2-X3-X4-X5- (X6) 3-Q-X7 (SEQ ID NOs: 9, 10 and 11), where X1 is Y, WouF, X2 is A, T , S, M, CouV, X3 is any amino acid, X4 is Tou N, X5 is A, T, G, S, V or 1, X6 is any amino acid and X7 is G or S; optionally wherein the RecD helicase comprises the reasons: (a) GGPGTGKT (SEQ ID NO: 19) and / or WAVTIHKSQG (SEQ ID NO: 20); (b) GGPGTGKS (SEQ ID NO: 22) and / or YALTVHRAQG (SEQ ID NO: 23); (0) GGPGVGKT (SEQ ID NO: 26) and / or YAISVHKSQG (SEQ ID NO: 27); (d) GGPGTGKT (SEQ ID NO: 19) and / or YCISVHKSQG (SEQ ID NO: 29); (e) GGPGVGKT (SEQ ID NO: 26) and / or YAATIHKSQG (SEQ ID NO: 31); (OD GGPGCGKS (SEQ ID NO: 33) and / or YAMTIHRSQG (SEQ ID NO: 34); (8) GGPGTGKS (SEQ ID NO: 22) and / or YAVSIHKSQG (SEQ ID NO: 36); (h) GGPGVGKT ( SEQ ID NO: 26) and / or YATSVHKSQG (SEQ ID NO: 38); () GGPGTGKT (SEQ ID NO: 19) and / or YAVSVHKSQG (SEQ ID NO: 40); (6) GGPGVGKT (SEQ ID NO: 26) ) and / or YATSIHKSQG (SEQ ID NO: 43); or (À) GGPGTGKS (SEQ ID NO: 22) and / or YALTVHRGQG (SEQ ID NO: 45).
[9]
9. Method according to any of the preceding claims, characterized by the fact that the RecD helicase is one of the helicases shown in tables 4 or 5 or a variant thereof, optionally in which the RecD helicase comprises: (a) the sequence shown in any of the SEQ ID NOs:
18, 21.24, 25.28, 30, 32, 35, 37, 39, 41, 42 and 44; or (b) a variant thereof having at least 25% homology to the relevant sequence based on the amino acid identity to the entire sequence and which retains helicase activity.
[10]
10. Method according to any one of claims 1 to 8, characterized in that the RecD helicase is a Tral helicase or Tral subgroup, optionally in which the Tral helicase or TralI subgroup helicase additionally comprises: - the reason amino acid H- (X1); - X2-R- (X3) s.12-H-X4-H (SEQ ID NOs: 46 to 53), where X1 and X3 are any amino acid and X2 and X4 are independently selected any amino acid except D, F, Ke R; or - the amino acid motif G-X1-X2-X3-X4-X5-X6-X7-H- (X8) s-12-H-X9 (SEQ ID NOs: 54 to 60), where X1, X2, X3, X5, X6, X7 and X9 are independently selected from any amino acid except D, E, K and R, X4 is Dou E and X8 is any amino acid.
[11]
11. Method according to claim 10, characterized in that the Tral helicase or Tral subgroup helicase comprises the following reasons: (a) GYAGVGKT (SEQ ID NO: 62), YAITAHGAQG (SEQ ID NO: 63) and HDTSRDQEPQLHTH (SEQ ID NO: 64); (b) GIAGAGKS (SEQ ID NO: 66), YALNVHMAQG (SEQ ID NO: 67) and HDTNRNQEPNLHFH (SEQ ID NC: 68); (0) GAAGAGKT (SEQ ID NO: 70), YCITIHRSQG (SEQ ID NO: 71) and HEDARTVDDIADPQLHTH (SEQ ID NO: 72); (d) GFAGTGKS (SEQ ID NO: 75), YATTVHSSQG (SEQ ID NO: 76) and HETSRERDPQLHTH (SEQ ID NO: 77); (e) GRAGAGKT (SEQ ID NO: 79), YATTIHKSQG (SEQ ID NO: 80) and GMVADWVYHDNPGNPHIH (SEQ ID NO: 81);
(f) GAAGTGKT (SEQ ID NO: 83), YASTAHKSQG (SEQ ID NO: 84) and HSTSRAQDPHLHSH (SEQ ID NO: 85);
(8) GHAGAGKT (SEQ ID NO: 87), YAGTITHRNQG (SEQ ID NO: 88) and HASSREQDPQIHSH (SEQ ID NO: 89);
(h) GLAGTGKT (SEQ ID NO: 91), YAVTSHSSQG (SEQ ID NO: 92) and HDTARPVNGYAAPQLHTH (SEQ ID NO: 93);
(i) GPAGAGKT (SEQ ID NO: 95), YAITAHRAQG (SEQ ID NO: 96) and HYDSRAGDPQLHTH (SEQ ID NO: 97);
(6) GWAGVGKT (SEQ ID NO: 99), YTAVTADHMQG (SEQ ID NO: 100) and HLCGRLDPQIHNH (SEQ ID NO: 101);
(k) GVAGAGKT (SEQ ID NO: 103), YALTIDSAQG (SEQ ID NO: 104) and HMTSGDGSPHLHVH (SEQ ID NO: 105);
(1) GYAGTGKS (SEQ ID NO: 107), YAATIHKAQG (SEQ ID NO: 108) and GMIADLVYNVHWDIGEDGKAKPHAH (SEQ ID NO: 109);
(m) GIAGAGKS (SEQ ID NO: 66), YALNAHMAQG (SEQ ID NO: 67) and HDTNRNQEPNLHFH (SEQ ID NO: 111);
(n) GVAGAGKS (SEQ ID NO: 115), TALNAHMAQG (SEQ ID NO: 67) and HDTNRNQEPNAHFH (SEQ ID NO: 116);
(o) GGAGVGKS (SEQ ID NO: 118), YAINVHIAQG (SEQ ID NO: 119) and HDVSRNNDPQLHVH (SEQ ID NO: 120);
(p) GIAGAGKS (SEQ ID NO: 66), YTALNMHMAQG (SEQ ID NO: 122) and HDTSRALDPQGHIH (SEQ ID NO: 123);
(q) GVAGAGKS (SEQ ID NO: 115), TALNAHMAQG (SEQ ID NO: 67) and HDTSRALDPQGHIH (SEQ ID NO: 123);
(&) GRAGTGKT (SEQ ID NO: 126), FASTAHGAQG (SEQ ID NO: 127) and HLASRNLDPQLHSH (SEQ ID NO: 128);
(s) GYAGTGKT (SEQ ID NO: 130), TAMTSHAAQG (SEQ ID NO: 131) and HDISRDKDPQLHTH (SEQ ID NO: 132);
(t) GLAGTGKT (SEQ ID NO: 91), YAQTVHASQG (SEQ ID
NO: 134) and HNTSRDLDPQTHTH (SEQ ID NO: 135); (u) GFAGTAKT (SEQ ID NO: 137), YVQTAFAAQG (SEQ ID NO: 138) and HETSRAQDPQLHTH (SEQ ID NO: 139); (v) GYAGTAKT (SEQ ID NO: 141), YVYDTAFAAQG (SEQ ID NO: 142) and HGTISRAQDPQLHTH (SEQ ID NO: 143); (w) GYAGTAKT (SEQ ID NO: 141), YASTAFAAQG (SEQ ID NO: 145) and HGTISRALDPQLHSH (SEQ ID NO: 146); (x) GSAGSGKT (SEQ ID NO: 148), YAVTSYSAQG (SEQ ID NO: 149) and HDTARPVGGYAAPQLHTH (SEQ ID NO: 150); (y) GEAGTGKT (SEQ ID NO: 153), YAHTSYKEQG (SEQ ID NO: 154) and HETNRENEPQLHNH (SEQ ID NO: 155); (2) GYAGVAKT (SEQ ID NO: 157), YVLTNYKVQG (SEQ ID NO: 158) and QPSSRANDPALHTH (SEQ ID NO: 159); (aa) GSAGTGKT (SEQ ID NO: 161), YSLTANRAQG (SEQ ID NO: 162) and HSSMSRAGDPEMHNH (SEQ ID NO: 163); (bb) AGAGTGKT (SEQ ID NO: 165), YAGTIVYAAQG (SEQ ID NO: 166) and HYTTREGDPNIHTH (SEQ ID NO: 167); or (co) APAGAGKT (SEQ ID NO: 169), YAVTVHAAQG (SEQ ID NO: 170) and HETSRAGDPHLHTH (SEQ ID NO: 171).
[12]
12. Method according to claim 10 or 11, characterized in that the Tral helicase or the Tral subgroup is one of the helicases shown in tables 6 or 7 or a variant thereof, optionally in which the Tral helicase or helicase of the The Tral subgroup comprises (a) the sequence shown in any of SEQ ID NOs: 61, 65, 69, 73, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 112, 113, 114 , 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 or (b) a variant thereof having at least 10% homology with the sequence relevant based on amino acid identity to the entire sequence and retains helicase activity.
[13]
13. 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 and the salt is optionally KCl.
[14]
14. Method for forming a sensor to characterize a target polynucleotide, the method characterized by the fact that it comprises forming a complex between a pore and a RecD helicase and thereby forming a sensor to characterize the target polynucleotide.
[15]
15. Method according to claim 14, characterized by the fact that the complex is formed by (a) contact the pore and helicase in the presence of the target polynucleotide and applying a voltage potential or a chemical potential through the pore or (b ) covalent connection of the pore to the helicase.
[16]
16. Kit to characterize a target polynucleotide, characterized by the fact that it comprises (a) an analysis apparatus to characterize target polynucleotides and a RecD helicase or (a) a pore and a RecD helicase; optionally wherein the kit additionally comprises a chip comprising an amphiphilic layer.
[17]
17. Analysis apparatus for characterizing a target polynucleotide in a sample, the apparatus characterized by the fact that it comprises (a) a RecD helicase or (b) a plurality of pores and a plurality of a RecD helicase.
[18]
18. Analysis apparatus according to claim 17, characterized by the fact that the analysis apparatus comprises: a sensor device that is capable of sustaining the plurality of pores and is operable to carry out the polynucleotide characterization using the pores and helicases ; at least one reservoir to contain the material to carry out the characterization; a fluidic system configured to controllably supply 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 deliver the samples selectively from the containers to the sensor device.
[19]
19. Method for characterizing a target polynucleotide, the method characterized by the fact that it comprises: (a) contacting the target polynucleotide with a RecD helicase such that the RecD helicase controls the movement of the target polynucleotide; and (b) obtaining one or more measurements as the RecD helicase controls the movement of the polynucleotide in which the measurements are indicative of one or more characteristics of the target polynucleotide and thus characterize the target polynucleotide.
[20]
20. Method according to claim 19, characterized in that: (a) one or more characteristics are selected from (1) the length of the target polynucleotide, (11) the identity of the target polynucleotide, (111) the sequence the target polynucleotide, (iv) the secondary structure of the target polynucleotide and (v) whether the target polynucleotide is modified or not; (b) the target polynucleotide is modified by methylation, oxidation, damage, with one or more proteins or with one or more labels, tags or spacers and / or at least a portion of the target polynucleotide is double-stranded; (c) the one or more characteristics of the target polynucleotide are measured by electrical measurement and / or optical measurement; (d) the RecD helicase is one of the helicases shown in tables 4 or 5 or a variant thereof and / or the RecD helicase is a Tral helicase or Tricas subgroup helicase, and / or the RecD comprises: - the amino acid motif X1 -X2-X3-G-X4-X5-X6-X7 (SEQ
ID NO: 8), where X1 is G, I am A, X2 is any amino acid, X3 is P, A, S or G, X4A is T, A, V, Sou C, X5 is Gou A, XG is Kou Re X7 is Tou S; and / or - the amino acid motif X1-X2-X3-X4-X5- (X6) 3-Q-X7 (SEQ ID NOs: 9, 10 and 11), where X1 is Y, WouF, X2 is A, T , S, M, CouV, X3 is any amino acid, X4 is Tou N, X5 is A, T, G, S, V or 1, X6 is any amino acid and X7 is G or S; or (e) the method is carried out using a salt concentration of at least 0.3 M and the salt is optionally KCI.
[21]
21. Use of a RecD helicase, characterized by the fact that it is to control the movement of a target polynucleotide through a pore.

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JP6228128B2|2017-11-08|

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CA2861808A1|2013-07-04|

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CA2861808C|2021-02-23|

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法律状态:
2020-07-28| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-01-26| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-09-08| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-01-04| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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