Enzyme method

专利摘要:
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a RecD helicase. The helicase controls the movement of the target polynucleotide through the pore.
公开号:AU2012360244A1
申请号:U2012360244
申请日:2012-12-28
公开日:2014-07-24
发明作者:Andrew John Heron；Ruth Moysey；Szabolcs SOEROES
申请人:Oxford Nanopore Technologies PLC；
IPC主号:C12Q1-68

专利说明:
WO 2013/098562 PCT/GB2012/053274 1 ENZYME METHOD Field of the invention The invention relates to a new method of characterising a target polynucleotide. The 5 method uses a pore and a RecD helicase. The helicase controls the movement of the target polynucleotide through the pore. Background of the invention There is currently a need for rapid and cheap polynucleotide (e.g. DNA or RNA) 10 sequencing and identification technologies 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 quantity of specialist fluorescent chemicals for signal detection. Transmembrane pores (nanopores) have great potential as direct, electrical biosensors for 15 polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as a potential DNA sequencing technology. When a potential is applied across a nanopore, there is a change in the current flow when an analyte, such as a nucleotide, resides transiently in the barrel for a certain period of time. Nanopore detection of the nucleotide gives a current change of known signature and duration. In 20 the "Strand Sequencing" method, a single polynucleotide strand is passed through the pore and the identity of the nucleotides are derived. Strand Sequencing can involve the use of a nucleotide handling protein to control the movement of the polynucleotide through the pore. Summary of the invention 25 The inventors have demonstrated that a RecD helicase can control the movement of a polynucleotide through a pore especially when a potential, such as a voltage, is applied. The helicase is capable of moving a target polynucleotide in a controlled and stepwise fashion against or with the field resulting from the applied voltage. Surprisingly, the helicase is capable of functioning at a high salt concentration which is advantageous for characterising the 30 polynucleotide and, in particular, for determining its sequence using Strand Sequencing. This is discussed in more detail below. Accordingly, the invention provides a method of characterising a target polynucleotide, comprising: WO 2013/098562 PCT/GB2012/053274 2 (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) taking one or more measurements as the polynucleotide moves with respect to the 5 pore wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide. The invention also provides: - a method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore and a RecD helicase and thereby forming a sensor 10 for characterising the target polynucleotide; - use of a RecD helicase to control the movement of a target polynucleotide through a pore; - a kit for characteri sing a target polynucleotide comprising (a) a pore and (b) a RecD helicase; and 15 - an analysis apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of a RecD helicase; - a method of characterising a target polynucleotide, comprising: (a) contacting the target polynucleotide with a RecD helicase such that the RecD helicase controls the movement of the target polynucleotide; and 20 (b) taking one or more measurements as the RecD helicase controls the movement of the polynucleotide wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide; - use of a RecD helicase to control the movement of a target polynucleotide during characterisation of the polynucleotide; 25 - use of a RecD helicase to control the movement of a target polynucleotide during sequencing of part or all of the polynucleotide; - an analysis apparatus for characterising target polynucleotides in a sample, characterised in that it comprises a RecD helicase; and - a kit for characterising a target polynucleotide comprising (a) an analysis apparatus for 30 characterising target polynucleotides and (b) a RecD helicase. Description of the Figures Fig. 1. A) Example schematic of use of a helicase to control DNA movement through a nanopore. A ssDNA substrate with an annealed primer containing a cholesterol-tag is added to 35 the cis side of the bilayer. The cholesterol tag binds to the bilayer, enriching the substrate at the WO 2013/098562 PCT/GB2012/053274 3 bilayer surface. Helicase added to the cis compartment binds to the 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. The DNA is pulled through the pore under the force of the applied potential until a helicase, bound to the DNA, contacts the top of 5 the pore, preventing further uncontrolled DNA translocation. After this the helicase proceeds to move the DNA through the nanopore in a controlled fashion. The schematic shows two possible methods of introducing the DNA to the nanopore: in one mode (top section) the helicase moves the captured DNA into the nanopore in the direction of the applied field, and in the other mode (lower section) the helicase pulls the captured DNA 10 out of the nanopore against the direction of the applied field. When moved with the applied field the DNA is moved to the trans side of the membrane. In both upper and lower sections the arrows on the trans side indicate the direction of motion of the DNA and the arrows on the cis side indicate direction of motion of the helicase with respect to the DNA. When moved against the field, the DNA is moved back to the cis side of the membrane,and the DNA may translocate 15 completely to the cis side if the helicase does not dissociate. Through substrate design, such as use of suitable leaders, one or both methods can be used at a time. The RecD family of helicases move in the 5'-3' direction along the DNA. Therefore, moving the DNA with the field requires 5'down capture of the DNA, and moving the DNA against the field requires 3' down DNA capture. B) The DNA substrate design used in the Example. 20 Fig. 2. Helicase is able to move DNA through a nanopore in a controlled fashion, producing stepwise changes in 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 MgCl 2 ). Top) Section of current vs. time acquisition of Tral 400mer DNA 25 events. The open-pore current is ~100 pA. DNA is captured by the nanopore under the force of the applied potential (+140 mV). DNA with enzyme attached results in a long block (at ~25 pA in this condition) that shows stepwise changes in current as the enzyme moves the DNA through the pore. Middle) An enlargement of one of the helicase-controlled DNA events, showing DNA enzyme capture, and stepwise current changes as the DNA is pulled through the pore. Bottom) 30 Further enlargement of the stepwise changes in current as DNA is moved through the nanopore. Fig. 3. Further examples of Tral Eco (SEQ ID 61) helicase controlled 400mer DNA (400mer DNA SEQ ID NOs: 172, 173 and 174) movement events through an MspA-B2(8) nanopore. Bottom) An enlargement of a section of the event showing the stepwise changes in current from the different sections of DNA as the strand moves through the nanopore.
WO 2013/098562 PCT/GB2012/053274 4 Fig. 4. Fluorescence assay for testing enzyme activity. A) A custom fluorescent substrate was used to assay the ability of the helicase to displace hybridised dsDNA. 1) The fluorescent substrate strand (50 nM final) has a 5' ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strand has a carboxyfluorescein base at the 3' end, and the hybridised 5 complement has a black-hole quencher (BHQ-1) base at the 5' end. When hybridised the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 pM of a capture strand that is complementary to the shorter strand of the fluorescent substrate is included in the assay. 2) In the presence of ATP (1 mM) and MgCl 2 (10 mM), helicase (100 nM) added to the substrate binds to the 5' tail of the fluorescent 10 substrate, moves along the major strand, and displaces the complementary strand as shown. 3) Once the complementary strand with BHQ-1 is fully displaced the fluorescein on the major strand fluoresces. 4) Excess of capture strand preferentially anneals to the complementary DNA to prevent re-annealing of initial substrate and loss of fluorescence. B) Graph of the initial rate of RecD helicase activity in buffer solutions (RecD Nth and Dth SEQ IDs 28 and 35, 100 mM 15 Hepes pH 8.0, 1 mM ATP, 10 mM MgCl 2 , 50 nM fluorescent substrate DNA, 1 pM capture DNA) containing different concentrations of KCl from 100 mM to 1 M. 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, 400mer DNA SEQ ID NOs: 172, 172 and 173, 100 nM TrwC Cba SEQ ID 65, 1 mM DTT, 1 mM ATP, 1 mM MgCl 2 ). Top) Section of 20 current vs. time acquisition of TrwC Cba 400mer DNA events. The open-pore current is -100 pA. DNA is captured by the nanopore under the force of the applied potential (+140 mV). DNA with enzyme attached results in a long block (at ~25 pA in this condition) that shows stepwise changes in current as the enzyme moves the DNA through the pore. Bottom) The bottom traces show enlarged sections of the DNA events, showing the stepwise sequence dependent current 25 changes as the DNA is pulled through the pore. Fig. 6. Example of current trace showing helicase controlled DNA movement using a TrwC (Atr) (SEQ ID NO: 144) helicase. Fig. 7. Example of current trace showing helicase controlled DNA movement using a TrwC (Sal) (SEQ ID NO: 140) helicase. 30 Fig. 8. Example of current trace showing helicase controlled DNA movement using a TrwC (Ccr) (SEQ ID NO: 136) helicase. Fig. 9. Example of current trace showing helicase controlled DNA movement using a TrwC (Eco) (SEQ ID NO: 74) helicase. Fig. 10. Example of current trace showing helicase controlled DNA movement using a 35 TrwC (Oma) (SEQ ID NO: 106) helicase.
WO 2013/098562 PCT/GB2012/053274 5 Fig. 11. Example of current trace showing helicase controlled DNA movement using a TrwC (Afe) (SEQ ID NO: 86) helicase. The lower trace shows an expanded region of the helicase controlled DNA movement. Fig. 12. Example of current trace showing helicase controlled DNA movement using a 5 TrwC (Mph) (SEQ ID NO: 94) helicase. The lower trace shows an expanded region of the helicase controlled DNA movement. Description of the Sequence Listing SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the MS-B 1 10 mutant MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D1 18R, D134R and E139K. SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B 1 mutant of the MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. 15 SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit of a-hemolysin E111N/K147N (a-HL-NN; Stoddart et al., PNAS, 2009; 106(19): 7702-7707). SEQ ID NO: 4 shows the amino acid sequence of one subunit of ao-HL-NN. SEQ ID NOs: 5 to 7 shows the amino acid sequences of MspB, C and D. SEQ ID NO: 8 shows the sequence of the RecD-like motif I. 20 SEQ ID NOs: 9, 10 and 11 show the sequences of the extended RecD-like motif I. SEQ ID NO: 12 shows the sequence of the RecD motif I. SEQ ID NOs: 13, 14 and 15 show the sequences of the extended RecD motif I. SEQ ID NO: 16 shows the sequence of the RecD-like motif V. SEQ ID NO: 17 shows the sequence of the RecD motif V. 25 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 sequences of the MobF motif III. SEQ ID NOs: 54 to 60 show the sequences of the MobQ motif III. SEQ ID NOs: 61 to 171 show the amino acid sequences of the Tral helicase and Tral subgroup helicases shown in Table 7. 30 SEQ ID NOs: 172 to 182 show the sequences used in the Examples. Detailed description of the invention It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology WO 2013/098562 PCT/GB2012/053274 6 used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for 5 example, reference to "a pore" includes two or more such pores, reference to "a helicase" includes two or more such helicases, reference to "a polynucleotide" includes two or more such polynucleotides, and the like. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. 10 Methods of the invention The invention provides a method of characterising a target polynucleotide. The method comprises contacting the target polynucleotide with a transmembrane pore and a RecD helicase such that the target polynucleotide moves moves through the pore and the RecD helicase 15 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 across the pore. As 20 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 using a salt gradient across an amphiphilic layer. A salt gradient is disclosed in Holden et al., J Am Chem Soc. 2007 Jul 11;129(27):8650-5. 25 In some instances, the current passing through the pore as the polynucleotide moves with respect to the pore is used to determine the sequence of the target polynucleotide. This is Strand Sequencing. The method has several advantages. First, the inventors have surprisingly shown that RecD helicases have a surprisingly high salt tolerance and so the method of the invention may be 30 carried out at high salt concentrations. In the context of Strand Sequencing, a charge carrier, such as a salt, is necessary to create a conductive solution for applying a voltage offset to capture and translocate the target polynucleotide and to measure the resulting sequence-dependent current changes as the polynucleotide moves with respect to the pore. Since the measurement signal is dependent on the concentration of the salt, it is advantageous to use high salt 35 concentrations to increase the magnitude of the acquired signal. High salt concentrations WO 2013/098562 PCT/GB2012/053274 7 provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations. For Strand Sequencing, salt concentrations in excess of 100 mM are ideal, for example salt concentrations in excess of 400mM, 600mM or 800mM. The inventors have surprisingly shown that RecD 5 helicases will function effectively at very high salt concentrations such as, for example, 1 M. The invention encompasses helicases which function effectively at salt concentrations in excess of IM, for example 2M. Second, when a voltage is applied, RecD helicases can surprisingly move the target polynucleotide in two directions, namely with or against the field resulting from the applied 10 voltage. Hence, the method of the invention may be carried out in one of two preferred modes. Different signals are obtained depending on the direction the target polynucleotide moves with respect to the pore, ie in the direction of or against the field. This is discussed in more detail below. Third, RecD helicases typically move the target polynucleotide through the pore one 15 nucleotide at a time. RecD helicases can therefore function like a single-base ratchet. This is of course advantageous when sequencing a target polynucleotide because substantially all, if not all, of the nucleotides in the target polynucleotide may be identified using the pore. Fourth, RecD helicases are capable of controlling the movement of single stranded polynucleotides and double stranded polynucleotides. This means that a variety of different 20 target polynucleotides can be characterised in accordance with the invention. Fifth, RecD helicases appear very resistant to the field resulting from applied voltages. The inventors have seen very little movement of the polynucleotide under an "unzipping" condition. Unzipping conditions will typically be in the absence of nucleotides, for example the absence of ATP. When the helicase is operating in unzipping mode it acts like a brake 25 preventing the target sequence from moving through the pore too quickly under the influence of the applied voltage. This is important because it means that there are no complications from unwanted "backwards" movements when moving polynucleotides against the field resulting from an applied voltage. Sixth, RecD helicases are easy to produce and easy to handle. Their use therefore 30 contributed to a straightforward and less expensive method of sequencing. The method of the invention is for characterising a target polynucleotide. A polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the target 35 polynucleotide can be oxidized or methylated. One or more nucleotides in the target WO 2013/098562 PCT/GB2012/053274 8 polynucleotide may be damaged. One or more nucleotides in the target polynucleotide may be modified, for instance with a label or a tag. The target polynucleotide may comprise one or more spacers. A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. 5 The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine, guanine, thymine, uracil and cytosine. The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be 10 attached on the 5' or 3' side of a nucleotide. Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine 15 monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMIP, dGMP or dCMP. A nucleotide may be abasic (i.e. lack a nucleobase). The polynucleotide may be single stranded or double stranded. At least a portion of the 20 polynucleotide is preferably double stranded. The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The target polynucleotide can comprise one strand of RNA hybridized to one strand of DNA. The polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), 25 locked nucleic acid (LNA) or other synthetic polymers with nucleotide side chains. The whole or only part of the target polynucleotide may be characterised using this method. The target polynucleotide can be any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotide 30 pairs, 5000 or more nucleotide pairs in length or 100000 or more nucleotide pairs in length. The target polynucleotide is present in any suitable sample. The invention is typically carried out on a sample that is known to contain or suspected to contain the target polynucleotide. Alternatively, the invention may be carried out on a sample to confirm the identity of one or more target polynucleotides whose presence in the sample is known or 35 expected.
WO 2013/098562 PCT/GB2012/053274 9 The sample may be a biological sample. The invention may be carried out in vitro on a sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaean, prokaryotic or eukaryotic and typically belongs to one the five kingdoms: plantae, animalia, fungi, monera and protista. The invention may be carried out 5 in vitro on a sample obtained from or extracted from any virus. The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. 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 mammal animal such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets 10 such as cats or dogs. Alternatively a sample of plant origin is typically obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton. The sample may be a non-biological sample. The non-biological sample is preferably a 15 fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests. The sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be 20 typically stored prior to assay, preferably below -70'C. A transmembrane pore is a structure that crosses the membrane to some degree. It permits ions, such as hydrated ions, driven by an applied potential to flow across or within the membrane. The transmembrane pore typically crosses the entire membrane so that ions may flow from one side of the membrane to the other side of the membrane. However, the 25 transmembrane pore does not have to cross the membrane. It may be closed at one end. For instance, the pore may be a well in the membrane along which or into which ions may flow. Any membrane may be used in accordance with 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, which have both at least 30 one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer. The amphiphilic layer is typically a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, 35 lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel WO 2013/098562 PCT/GB2012/053274 10 recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International 5 Application No. PCT/GB08/000563 (published as WO 2008/10212 1), International Application No. PCT/GB08/004127 (published as WO 2009/077734) and International Application No. PCT/GB2006/001057 (published as WO 2006/100484). Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in the Example. Lipid bilayers are commonly formed by the method of Montal and Mueller 10 (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface. The method of Montal & Mueller is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for protein pore 15 insertion. Other common methods of bilayer formation include tip-dipping, painting bilayers and patch-clamping of liposome bilayers. In a preferred embodiment, the lipid bilayer is formed as described in International Application No. PCT/GB08/004127 (published as WO 2009/077734). In another preferred embodiment, the membrane is a solid state layer. A solid-state layer 20 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 manufactured 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 3
N
4 , A1 2 0 3 , and SiO, organic and inorganic polymers such as polyamide, plastics such 25 as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses. The solid state layer may 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). The method is typically carried out using (i) an artificial amphiphilic layer comprising a 30 pore, (ii) an isolated, naturally-occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically carried out using an artificial amphiphilic layer, such as an artificial lipid bilayer. The layer may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below. The method of the invention is typically carried out in vitro. 35 The polynucleotide may be coupled to the membrane. This may be done using any WO 2013/098562 PCT/GB2012/053274 11 known method. If the membrane is an amphiphilic layer, such as a lipid bilayer (as discussed in detail above), the polynucleotide is preferably coupled to the membrane via a polypeptide present in the membrane or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, fatty acid, sterol, carbon nanotube or amino acid. 5 The polynucleotide may be coupled directly to the membrane. The polynucleotide is preferably coupled to the membrane via a linker. Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs) and polypeptides. If a polynucleotide is coupled directly to the membrane, then some data will be lost as the characterising run cannot continue to the end of the polynucleotide due to the distance between 10 the membrane and the helicase. If a linker is used, then the polynucleotide can be processed to completion. If a linker is used, the linker may be attached to the polynucleotide at any position. The linker is preferably attached to the polynucleotide at the tail polymer. The coupling may be stable or transient. For certain applications, the transient nature of the coupling is preferred. If a stable coupling molecule were attached directly to either the 5' or 15 3' end of a polynucleotide, then some data will be lost as the characterising run cannot continue to the end of the polynucleotide due to the distance between the bilayer and the helicase's active site. If the coupling is transient, then when the coupled end randomly becomes free of the bilayer, then the polynucleotide can be processed to completion. Chemical groups that form stable or transient links with the membrane are discussed in more detail below. The 20 polynucleotide may be transiently coupled to an amphiphilic layer, such as a lipid bilayer using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to 30 carbon atoms, such as hexadecanoic acid, may be used. In preferred embodiments, the polynucleotide is coupled to an amphiphilic layer. Coupling of polynucleotides to synthetic lipid bilayers has been carried out previously with 25 various different tethering strategies. These are summarised in Table 1 below. Table 1 Attachment group Type of coupling Reference Thiol Stable Yoshina-Ishii, C. and S. G. Boxer (2003). "Arrays of mobile tethered vesicles on supported lipid bilayers." J Am Chem Soc 125(13): 3696-7. Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). "Behavior of giant vesicles with anchored DNA molecules." Biophys J 92(12): 4356-68 Cholestrol Transient Pfeiffer, I. and F. Hook (2004). "Bivalent cholesterol based coupling of oligonucletides to lipid membrane assemblies." J Am Chem Soc 126(33): 10224-5 Lipid Stable van Lengerich, B., R. J. Rawle, et al. "Covalent attachment of lipid vesicles to a fluid-supported WO 2013/098562 PCT/GB2012/053274 12 bilayer allows observation of DNA-mediated vesicle interactions." Langmuir 26(11): 8666-72 Polynucleotides may 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 attachment chemistries give a suite of attachment 5 options for polynucleotides. Each different modification group tethers the polynucleotide in a slightly different way and coupling is not always permanent so giving different dwell times for the polynucleotide to the bilayer. The advantages of transient coupling are discussed above. Coupling of polynucleotides can also be achieved by a number of other means provided that a reactive group can be added to the polynucleotide. The addition of reactive groups to 10 either end of DNA has been reported previously. A thiol group can be added to the 5' of ssDNA using polynucleotide kinase and ATP7S (Grant, G. P. and P. Z. Qin (2007). "A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids." Nucleic Acids Res 35(10): e77). A more diverse selection of chemical groups, such as biotin, thiols and fluorophores, can be added using terminal transferase to incorporate modified oligonucleotides to the 3' of ssDNA 15 (Kumar, A., P. Tchen, et al. (1988). "Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169(2): 376-82). Alternatively, the reactive group could be considered to be the addition of a short piece of DNA complementary to one already coupled to the bilayer, so that attachment can be achieved via hybridisation. Ligation of short pieces of ssDNA have been reported using T4 RNA ligase I 20 (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992). "Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc Natl Acad Sci U S A 89(20): 9823 5). Alternatively either ssDNA or dsDNA could be ligated to native dsDNA and then the two strands separated by thermal or chemical denaturation. To native dsDNA, it is possible to add either a piece of ssDNA to one or both of the ends of the duplex, or dsDNA to one or both ends. 25 Then, when the duplex is melted, each single strand will have either a 5' or 3' modification if ssDNA was used for ligation or a modification at the 5' end, the 3' end or both if dsDNA was used for ligation. If the polynucleotide is a synthetic strand, the coupling chemistry can be incorporated during the chemical synthesis of the polynucleotide. For instance, the polynucleotide can be synthesized using a primer a reactive group attached to it. 30 A common technique for the amplification of sections of genomic DNA is using polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a number of copies of the same section of DNA can be generated, where for each copy the 5' of each strand in the duplex will be a synthetic polynucleotide. By using an antisense primer that has a WO 2013/098562 PCT/GB2012/053274 13 reactive group, such as a cholesterol, thiol, biotin or lipid, each copy of the target DNA amplified will contain a reactive group for coupling. The transmembrane pore is preferably a transmembrane protein pore. A transmembrane protein pore is a protein structure that crosses the membrane to some degree. It permits ions 5 driven by an applied potential to flow across or within the membrane. A transmembrane protein pore is typically a polypeptide or a collection of polypeptides that permits ions, such as analyte, to flow from one side of a membrane to the other side of the membrane. However, the transmembrane protein pore does not have to cross the membrane. It may be closed at one end. For instance, the transmembrane pore may form a well in the membrane along which or into 10 which ions may flow. The transmembrane protein pore preferably permits analytes, such as nucleotides, to flow across or within the membrane. The transmembrane protein pore allows a polynucleotide, such as DNA or RNA, to be moved through the pore. The transmembrane protein pore may be a monomer or an oligomer. The pore is preferably made up of several repeating subunits, such as 6, 7, 8 or 9 subunits. The pore is 15 preferably a hexameric, heptameric, octameric or nonameric pore. The transmembrane protein pore typically comprises a barrel or channel through which the ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane P barrel or channel or a transmembrane a-helix bundle or channel. The barrel or channel of the transmembrane protein pore typically comprises amino acids 20 that facilitate interaction with analyte, such as nucleotides, polynucleotides or nucleic acids. These amino acids are preferably located near a constriction of the barrel or channel. 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. These amino acids typically facilitate the interaction between the pore and nucleotides, 25 polynucleotides or nucleic acids. Transmembrane protein pores for use in accordance with the invention can be derived from -barrel pores or a-helix bundle pores. P-barrel pores comprise a barrel or channel that is formed from -strands. Suitable Q-barrel pores include, but are not limited to, p-toxins, such as a-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, 30 such as Mycobacterium smegmatis porin (Msp), for example MspA, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP). a-helix bundle pores comprise a barrel or channel that is formed from a-helices. Suitable a-helix bundle pores include, but are not limited to, inner WO 2013/098562 PCT/GB2012/053274 14 membrane proteins and ca outer membrane proteins, such as WZA and ClyA toxin. The transmembrane pore may be derived from Msp or from a-hemolysin (a-HL). 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 5 Msp. The pore may be a homo-oligomeric pore derived from Msp comprising identical monomers. Alternatively, the pore may be a hetero-oligomeric pore derived from Msp comprising at least one monomer that differs from the others. Preferably the pore is derived from MspA or a homolog or paralog thereof. A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2 or a 10 variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. It includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. The ability of a variant to form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an 15 amphiphilic layer along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into membranes, such as amphiphilic layers. For example, subunits may be suspended in a purified form in a solution containing a lipid bilayer such that it diffuses to the lipid bilayer and is inserted by binding to the lipid bilayer and assembling into a functional state. Alternatively, subunits may 20 be directly inserted into the membrane using the "pick and place" method described in M.A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057 (published as WO 2006/100484). 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 amino acid identity. More 25 preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous 30 amino acids ("hard homology"). Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux el al (1984) Nucleic Acids Research 12, p387 395). The PILEUP and BLAST algorithms can be used to calculate homology or line up 35 sequences (such as identifying equivalent residues or corresponding sequences (typically on their WO 2013/098562 PCT/GB2012/053274 15 default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). 5 SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant may comprise any of the mutations in the MspB, C or D monomers compared with 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 may comprise one or more of the following substitutions present in MspC: A96G, N102E and A138P. The 10 variant may comprise one or more of the following mutations present in MspD: Deletion of GI, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, 149H, 168V, D91G, A96Q, N102D, S103T, V1041, S136K and G141A. The variant may comprise combinations of one or more of the mutations and substitutions from Msp B, C and D. The variant preferably comprises the mutation L88N. The variant of SEQ ID NO: 2 has the mutation L88N in addition to all the 15 mutations of MS-B1 and is called MS-B2. The pore used in the invention is preferably MS (B2)8. Amino acid substitutions may be made to 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 20 structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be 25 selected in accordance with the properties of the 20 main amino acids as defined in Table 2 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 3. Table 2 - Chemical properties of amino acids 30 Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral Glu polar, hydrophilic, charged (-) Gln polar, hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+) WO 2013/098562 PCT/GB2012/053274 16 Gly aliphatic, neutral Ser polar, hydrophilic, neutral His aromatic, polar, hydrophilic, Thr polar, hydrophilic, neutral charged (+) Ile aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trp aromatic, hydrophobic, neutral Len aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic Table 3- Hydropathy scale Side Chain Hydropathy 5 Ile 4.5 Val 4.2 Leu 3.8 Phe 2.8 10 Cys 2.5 Met 1.9 Ala 1.8 Gly -0.4 Thr -0.7 15 Ser -0.8 Trp -0.9 Tyr -1.3 Pro -1.6 His -3.2 20 Glu -3.5 Gln -3.5 Asp -3.5 Asn -3.5 Lys -3.9 25 Arg -4.5 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 may be deleted, or more. 30 Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore forming activity. Fragments may be at least 50, 100, 150 or 200 amino acids in length. Such fragments may be used to produce the 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, fragments include all of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 35 2. One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The WO 2013/098562 PCT/GB2012/053274 17 extension may be quite short, for example from I to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below. 5 As discussed above, a variant is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 2 that are responsible for pore formation. The pore forming ability of Msp, which contains a p-barrel, is provided by p-sheets in each subunit. A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form B-sheets. 10 One or more modifications can be made to the regions of SEQ ID NO: 2 that form 1-sheets 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 c-helices and/or loop regions. The monomers derived from Msp may be modified to assist their identification or 15 purification, for example by the addition of histidine residues (a hist tag), aspartic acid residues (an asp tag), a streptavidin tag or a flag tag, or by the addition of a signal sequence to promote their 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 onto a native or engineered position on the pore. An example of this would be to react a gel-shift reagent to a cysteine 20 engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 Jul; 4(7):497-505). The monomer derived from Msp may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. I, 35S, enzymes, antibodies, 25 antigens, polynucleotides and ligands such as biotin. The monomer derived from Msp may also be produced using D-amino acids. For instance, the monomer derived from Msp may comprise a mixture of L-amino acids and D amino acids. This is conventional in the art for producing such proteins or peptides. The monomer derived from Msp contains one or more specific modifications to facilitate 30 nucleotide discrimination. The monomer derived from Msp may also contain other non-specific modifications as long as they do not interfere with pore formation. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the monomer derived from Msp. Such modifications include, for example, reductive alkylation of WO 2013/098562 PCT/GB2012/053274 18 amino acids by reaction with an aldehyde followed by reduction with NaBH 4 , amidination with methylacetimidate or acylation with acetic anhydride. The monomer derived from Msp can be produced using standard methods known in the art. The monomer derived from Msp may be made synthetically or by recombinant means. For 5 example, the pore may be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing pores are discussed in International Application Nos. PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB1O/000133 (published as WO 2010/086603). Methods for inserting pores into membranes are discussed. 10 The transmembrane protein pore is also preferably derived from a-hemolysin (a-HL). The wild type a-HL pore is formed of seven identical monomers or subunits (i.e. it is heptameric). The sequence of one monomer or subunit of a-hemolysin-NN is shown in SEQ ID NO: 4. The transmembrane protein pore preferably comprises seven monomers each comprising the sequence shown in SEQ ID NO: 4 or a variant thereof. Amino acids 1, 7 to 21, 31 to 34, 45 15 to51,63 to66,72,92 to97, 104to 111, 124to 136, 149to 153, 160to 164, 173 to206,210to 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 barrel or channel of a-iL. In such embodiments, a pore comprising seven proteins or monomers each comprising 20 the sequence shown in SEQ ID NO: 4 or a variant thereof are preferably used in the method of the invention. The seven proteins may be the same (homoheptamer) or different (heteroheptamer). A variant of SEQ ID NO: 4 is a protein that has an amino acid sequence which varies from that of SEQ ID NO: 4 and which retains its pore forming ability. The ability of a variant to 25 form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an amphiphilic layer, such as a lipid bilayer, along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into amphiphilic layers, such as lipid bilayers. Suitable methods are discussed above. 30 The variant may include modifications that facilitate covalent attachment to or interaction with the helicase. The variant preferably comprises one or more reactive cysteine residues that facilitate attachment to the helicase. For instance, the variant may include a cysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45, 50, 51, 237, 239 and 287 and/or on the amino or carboxy terminus of SEQ ID NO: 4. Preferred variants comprise a substitution of the residue at 35 position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 with cysteine (A8C, T9C, N17C, K237C, WO 2013/098562 PCT/GB2012/053274 19 S239C or E287C). The variant is preferably any one 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). The variant may also include modifications that facilitate any interaction with 5 nucleotides. The variant may be a naturally occurring variant which is expressed naturally by an organism, for instance by a Staphylococcus bacterium. Alternatively, the variant may be expressed in vitro or recombinantly by a bacterium such as Escherichia coli. Variants also include non-naturally occurring variants produced by recombinant technology. Over the entire 10 length of the amino acid sequence of SEQ ID NO: 4, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 4 over the entire 15 sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 200 or more, for example 230, 250, 270 or 280 or more, contiguous amino acids ("hard homology"). Homology can be determined as discussed above. Amino acid substitutions may 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. 20 Conservative substitutions may be made as discussed above. One or more amino acid residues of the amino acid sequence of SEQ ID NO: 4 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more. Variants may be fragments of SEQ ID NO: 4. Such fragments retain pore-forming 25 activity. Fragments may 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. One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminus or carboxy terminus of 30 the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. The extension may be quite short, for example from I to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to a pore or variant. As discussed above, a variant of SEQ ID NO: 4 is a subunit that has an amino acid 35 sequence which varies from that of SEQ ID NO: 4 and which retains its ability to form a pore. A WO 2013/098562 PCT/GB2012/053274 20 variant typically contains the regions of SEQ ID NO: 4 that are responsible for pore formation. The pore forming ability of u-HL, which contains a P-barrel, is provided by P-strands in each subunit. A variant of SEQ ID NO: 4 typically comprises the regions in SEQ ID NO: 4 that form p-strands. The amino acids of SEQ ID NO: 4 that form p-strands are discussed above. One or 5 more modifications can be made to the regions of SEQ ID NO: 4 that form O-strands as long as the resulting variant retains its ability to form a pore. Specific modifications that can be made to the D-strand regions of SEQ ID NO: 4 are discussed above. 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. Amino acids that 10 form a-helices and loops are discussed above. The variant may be modified to assist its identification or purification as discussed above. Pores derived from ac-HL can be made as discussed above with reference to pores derived from Msp. In some embodiments, the transmembrane protein pore is chemically modified. The pore 15 can be chemically modified in any way and at any site. The transmembrane protein pore is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The 20 transmembrane protein pore may be chemically modified by the attachment of any molecule. For instance, the pore may be chemically modified by attachment of a dye or a fluorophore. Any number of the monomers in the pore may be chemically modified. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers is preferably chemically modified as discussed above. 25 The reactivity of cysteine residues may be enhanced by modification of the adjacent residues. For instance, the basic groups of flanking arginine, histidine or lysine residues will change the pKa of the cysteines thiol group to that of the more reactive S group. The reactivity of cysteine residues may be protected by thiol protective groups such as dTNB. These may be reacted with one or more cysteine residues of the pore before a linker is attached. 30 The molecule (with which the pore is chemically modified) may be attached directly to the pore or attached via a linker as disclosed in International Application Nos. 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).
WO 2013/098562 PCT/GB2012/053274 21 Any RecD helicase may be used in accordance with the invention. The structures of RecD helicases are known in the art (FEBS J, 2008 Apr;275(8): 183 5-5 1. Epub 2008 Mar 9. ATPase activity of RecD is essential for growth of the Antarctic Pseudomonas syrinPae Lz4W at low temperature. Satapathy AK, Pavankumar TL, Bhattacharjya S, Sankaranarayanan R, Ray 5 MK EMS NMicrobiol Rev. 2009 May; 33(3):657-87. The diversity of conjiugative relaxases and its application in plasmid classification. Garcilln-Barcia IP, Francia MV, de la Cruz F; J Biol Chern. 2011 Apr 8;286(14):12670-82. Epub 2011 Feb 2. Functional characterization of the nmitidomain F plasmid Tral relaxase-helicase. Cheng Y, McNamara DE, Mi ley MJ, Nash RP, Redinbo MvR). 10 The RecD helicase typically comprises the amino acid motif XI-X2-X3-G-X4-X5-X6 X7 (hereinafter called the RecD-like motif I; SEQ ID NO: 8), wherein X1 is G, S or A, X2 is any amino acid, X3 is P, A, S or G, X4 is T, A, V, S or C, X5 is G or A, X6 is K or R and X7 is T or S. XI is preferably G. X2 is preferably G, I, 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. X.5 is preferably G. X6 is 15 preferably K. X7 is preferably T or S The Reed) helicase preferably comprises Q-(X8)16Is-XI X2-X3-G-XI-X5-X6-X7 (hereinafter called the extended RecD-ike motif I; SEQ ID NOs: 9,10 and II where there are 16, 17 and 18 X8s respectively), wherein Xl to X7 are as defined above and X8 is any amino acid. There are preferably 16 X8 residues (i.e. (X8) 16 ) in the extended RecD-like notifI (SEQ ID NO 9), Suitable sequences for (X8)6 can be identified in SEQ ID 20 NOs: 1,21,24,25,28,30,32,35 39 41,42and44. The R.ecD helicase preferably comprises the amino acid motif G-G-P-G-Xa-G-K-Xb (hereinafter called the RecD motif I; SEQ ID NO: 12) wherein Xa is T', V or C and Xb is Tor S. Xa is preferably T. Xb is preferably T. The Rec-D helicase preferably comprises the sequence G-G~P-G-TG-KT (SEQ ID NCY 19 ee Table 5). The Reel) helicase more preferably 25 comprises the amino acid motif Q-(X8) 1 s-G-G-P-G-Xa-G-K-Xb (hereinafter called the extended RecD motif ; SF0 ID NOs: 13, 14 and 15 where there are 16, 17 and 18 X8s respectively), wherein Xa and Xb are as defined above and X8 is any amino acid. There are preferably 16 X8 residues (i e. (X8),) in the extended RecD motif I (SEQ ID NO: 13). Suitable sequences for (X8) can be identified in SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 30 42 and 44. The ReeD helicase typically comprises the amino acid motif X1-X2-X3-X4-X5-(X6)3Q X (hereinafter called the RecD-like motif V; SEQ ID NO: 16), wherein Xi is Y, W or F, N2 is A. T, S, M, C or V, X3 is any amino acid, X4 is T, N or S, X5 is A, T, G, S, V or I, X6 is any amino acid and X7 is G or S. XI is preferably Y. X2 is preferably A, M, C or V. X2 is more 35 preferably A, X3 is preferably 11, M4 or IL X3 is more preferably I or L. X4 is preferably T or S.
WO 2013/098562 PCT/GB2012/053274 22 X4 is more preferably T. X5 is preferably A, V or f X5 is more preferably V or I X5 is most preferably V. (X6) 3 is preferably H-K-S,. H-M--A, H-G-A or H-R-S. (X6) 3 is more preferably H K-S. X7 is preferably G. The RecD helicase preferably comprises the amino acid motif Xa-Xb Xc-Xd-Xe-H-K-S-Q-G (hereinafter called the ReeD motif V SEQ ID NO: 17), wherein Xa is 5 Y, W or F, Xb is A, M, C or V, Xc is I, M or L, Xd is T or S and Xe is V or I. Xa is preferably Y. Xb is preferably A. Xd is preferably T. Xe is preferaly V. T1he ReeD helicase preferably comprises (1) RecD-like motifs I and V (SEQ ID NOs: 8 and 12), (2) ReeD motif I and ReeD like motif V (SEQ I1)D NOs: 12 and 16), (3) RecD motifs I and V (SEQ I) NOs: 12 and 17), (4) extended ReeD-like motif I and RecD-like motif V (SEQ ID NOs: 9, 10 or II and 16), (5) 10 extended Recl) motif I and RecD-like motif V (SEQ 11) NOs: 13, 14 or 15 and 16) or (6) extended RecD motif I and RecD motif V (SEQ ID NOs: 13, 14 or 15 and 17). Preferred ReeD motifs I are shown in Table 5 below Preferred RecD-like motifs I are shown in Table 7 below. Preferred RecD-like motifs V are shown in Tables 5 and 7 below. The RecD helicase is preferably one of the helicases shown in Table 4 below or a variant 15 thereof WO 2013/098562 PCT/GB2012/053274 23 Table 4 - Preferred RecD helicases and their Accession numbers 1 NP 295625.1 exodeoxyribonuclease V subunit RecD [Deinococcus radiodurans RI] 2 YP 604297.1 helicase RecD/TraA [Deinococcus Reothermalis DSM 113001 3 YP 002786343.1 exodeoxyribonuclease V subunit aloha rDeinococcus deserti VCD 1151 4 3E1S A Chain A, Structure Of An N-Terminal Truncation Of Deinococcus 5 YP 004256144.1 helicase, RecD/TraA family [Deinococcus proteolvticus MIRP1 6 YP 004170918.1 helicase, RecD/TraA family [Deinococcus maricopensis DSM 212111 7 YP 004256838.1 helicase, RecD/TraA family [Deinococcus proteolvticus MRP1 8 YP 003885838.1 helicase, RecD/TraA family [Cvanothece so. PCC 78221 9 ZP 08579275.1 helicase. RecD/TraA family [Prevotella multisaccharivorax DSM 171281 10 YP 002377692 1 helicase, RecD/TraA family [Cvanothece so. PCC 74241 11 YP3 001519318.1 RecD/TraA family helicase [Acarvochloris marina MBIC 110171 12 YP3 003318882.1 helicase, RecD/TraA family [Sphaerobacter thermophilus DSM 207451 13 YP 004671137.1 hvothetical protein SNE A07690 rSimkania nesevensis Zi 14 YP 375364.1 helicase RecD/TraA [Chlorobium luteolum DSM 2731 >RblABB24321. 1 15 YP 002418908.1 RecD/TraA family helicase [Methylobacterium chloromethanicum CM41 16 YP 003065757.1 Helicase rMethylobacterium extorauens DM41 >emblCAX21689. 1 17 ZP 00518989.1 Helicase RecD/TraA rCrocosphaera watsonii WH 85011 18 ZP 06973397.1 helicase, RecD/TraA family [Ktedonobacter racemifer DSM 449631 19 ZP 08486910.1 helicase, RecD/TraA family [Methylomicrobium album BG81 20 YP 002015362.1 RecD/TraA family helicase [Prosthecochloris aestuarii DSM 2711 21 YP 001130786.1 RecD/TraA family helicase [Chlorobium phaeovibrioides DSM 2651 22 YP 002961258.1 Helicase rMethylobacterium extorauens AMI] >blACS37981.1| 23 ZP 08772902.1 helicase, RecD/TraA family [Thiocapsa marina 58111 >blEGV16093. 11 24 YP 001637509.1 RecD/TraA family helicase [Methylobacterium extorcauens PAl] 25 ZP 02062824.1 helicase, RecD/TraA family [Rickettsiella grvllil >blEDP46829.1| 26 ZP 08768753.1 helicase, RecD/TraA family [Thiocaosa marina 58111 >ablEGV20712. 11 27 YP 001922739.1 helicase, RecD/TraA family [Methylobacterium voouli BJ0011 28 YP 002018300.1 helicase, RecD/TraA family [Pelodictvon phaeoclathratiforme BU-11 29 ZP 06245171.1 helicase, RecD/TraA family [Victivallis vadensis ATCC BAA-5481 30 ZP 08771217.1 helicase, RecD/TraA family [Thiocapsa marina 58111 >blEGV17897.11 31 ZP 08769899.1 helicase, RecD/TraA family [Thiocapsa marina 58111 >blEGV18833.11 32 ZP 03727363 1 Exodeoxvribonuclease V [Opitutaceae bacterium TAV21 33 ZP 05027797 1 helicase. RecD/TraA family [Microcoleus chthonoDlastes PCC 74201 34 YPI 001521445.1 RecD/TraA family helicase [Acarvochloris marina MBIC110171 35 YP 002606149.1 RecD3 rDesulfobacterium autotrophicum HIRM21 >gblACN17985.11 36 YP 003165615.1 helicase, RecD/TraA family [Candidatus Accumulibacter ohosphatis 37 ZP 01732265.1 Helicase RecD/TraA rCvanothece so. CCYO1101 >QblEAZ88318.1| WO 2013/098562 PCT/GB2012/053274 24 38 YP 901533.1 RecD/TraA family helicase [Pelobacter propionicus DSM 23791 39 YP 004121205.1 helicase, RecD/TraA family [Desulfovibrio aespoeensis Aspo-21 40 YP 9113131 RecD/TraA family helicase [Chlorobium phaeobacteroides DSM 2661 41 YP 002424008.1 RecD/TraA family helicase [Methvlobacterium chloromethanicum 42 YP 320143.1 helicase RecD/TraA [Anabaena variabilis ATCC 294131 43 YP 001603050 1 exodeoxyribonuclease [Gluconacetobacter diazotrophicus PAl 51 44 ZP 05054956.1 helicase, RecD/TraA family [Octadecabacter antarcticus 3071 45 YP 003445164.1 helicase, RecD/TraA family rAllochromatium vinosum DSM 1801 46 NP 490177.1 exodeoxyribonuclease V, alpha chain [Nostoc sp. PCC 71201 47 NP 9235751 exodeoxyribonuclease V alpha chain [Gloeobacter violaceus PCC 74211 48 YP 001601244.1 exodeoxyribonuclease V alpha chain [Gluconacetobacter diazotrophicus 49 YP 004748470.1 exodeoxyribonuclease V subunit alpha [Acidithiobacillus caldus SM-11 50 Y P 004863326.1 helicase , RecD/TraA family rCandidatus Chloracidobacterium 51 YP 001520750 1 RecD/TraA family helicase [Acaryochloris marina MBIC110171 52 YP 003197384.1 helicase, RecD/TraA family [Desulfohalobium retbaense DSM 56921 53 ZP 08900128.1 helicase, RecD/TraA family protein rGluconacetobacter oboediens 54 YP 002275391 .1 helicase, RecD/TraA family [Gluconacetobacter diazotrophicus PAl 51 55 YP 003156740.1 RecD/TraA family helicase [Desulfomicrobium baculatum DSM 40281 56 ZP 08821817.1 helicase, RecD/TraA family [Thiorhodococcus drewsii AZll 57 ZP 01731986.1 Helicase RecD/TraA rCyanothece sp. CCYO1101 >gblEAZ88625.11 58 YP 001943002.1 RecD/TraA family helicase [Chlorobium limicola DSM 2451 59 ZP 08318929.1 hypothetical protein SXCC 04894 rGluconacetobacter sp. SXCC-11 60 YP 002017890.1 helicase, RecD/TraA family [Pelodictyon phaeoclathratiforme BU-1] 61 ZP 07972826.1 RecD/TraA family helicase [Synechococcus sp. CBO1011 62 YP 003189342.1 DNA helicase RecD/TraA [Acetobacter pasteurianus IFO 3283-011 63 YP 001959197.1 RecD/TraA family helicase [Chlorobium phaeobacteroides BS11 64 ZP 05064957.1 helicase, RecD/TraA family [Octadecabacter antarcticus 2381 65 YP 001772290 1 RecD/TraA family helicase [Methylobacterium sp. 4-461 66 YP 001998378.1 RecD/TraA family helicase [Chlorobaculum parvum NCIB 83271 67 YP 001869949.1 RecD/TraA family helicase [Nostoc punctiforme PCC 731021 68 ZP 08109907.1 helicase, RecD/TraA family [Desulfovibrio sp. ND1321 69 ZP 06965850.1 helicase, RecD/TraA family [Ktedonobacter racemifer DSM 449631 70 ZP 05428586.1 helicase, RecD/TraA family [Clostridium thermocellum DSM 23601 71 ZP 05404007.1 helicase, RecD/TraA family [Mitsuokella multacida DSM 205441 72 YP 002992028.1 helicase, RecD/TraA family [Desulfovibrio salexigens DSM 26381 73 ZP 02190744.1 Helicase RecD/TraA alpha proteobacterium BAL1991 74 ZP 08959149.1 RecD/TraA family helicase [Halomonas sp. HAL11 >gblEHA16215.1| 75 YP 003709145.1 exodeoxyribonuclease V, alpha subunit [Waddlia chondrophila WSU 76 YP 003528424.1 helicase, RecD/TraA family [Nitrosococcus halophilus Nc41 77 ZP 02191403.1 Helicase RecD/TraA [alpha proteobacterium BAL1991 78 YP 004802608.1 helicase, RecD/TraA family [Streptomyces sp. SirexAA-E 79 CCB91170.1 uncharacterized protein yrrC [Waddlia chondrophila 2032/991 80 YP 289811.1 helicase RecD/TraA [Thermobifida fusca YX] >gblAAZ55788.1| 81 ZP 07015918.1 helicase, RecD/TraA family [Desulfonatronospira thiodismutans ASO3 82 YP 004766648.1 helicase rMegasphaera elsdenii DSM 204601 >emb|CCC73821.1| 83 ZP 04708454.1 putative exodeoxyribonuclease V [Streptomyces roseosporus NRRL 84 YP 001039578.1 RecD/TraA family helicase [Clostridium thermocellum ATCC 274051 85 YP 594664.1 exonuclease V subunit alpha rLawsonia intracellularis PHE/MN1-001 86 NP 662288.1 exodeoxyribonuclease V, alpha subunit, putative rChlorobium tepidum 87 ZP 08423994.1 helicase, RecD/TraA family [Desulfovibrio africanus str. Walvis Bay] 88 YP 007688.1 putative exodeoxyribonuclease V [Candidatus Protochlamydia 89 YP 002953244.1 helicase RecD/TraA family protein rDesulfovibrio magneticus RS-1] 90 ADW05584.1 helicase, RecD/TraA family [Streptomyces flavogriseus ATCC 333311 91 ZP 01385982 1 Helicase RecD/TraA rChlorobium ferrooxidans DSM 130311 92 YP 0017169651 RecD/TraA family helicase [Candidatus Desulforudis audaxviator 93 ADL25833.1 helicase, RecD/TraA family [Fibrobacter succinogenes subsp. 94 YP 002480970.1 helicase, RecD/TraA family [Cyanothece sp. PCC 74251 95 YP 004516136.1 helicase, RecD/TraA family rDesulfotomaculum kuznetsovii DSM 96 ZP 08778308.1 exodeoxyribonuclease [Candidatus Odyssella thessalonicensis L131 WO 2013/098562 PCT/GB2012/053274 25 97 ZP 06825719.1 RecD/TraA family helicase rStreptomyces so. SPB741 >gblEDY42267.2| 98 ZP 05293745.1 Exodeoxyribonuclease V alpha chain [Acidithiobacillus caldus ATCC 99 YP 480657.1 helicase RecD/TraA [Frankia sp. CcI31 >gblABD10928.11 Helicase 100 ZP 07017628.1 helicase, RecD/TraA family rDesulfonatronospira thiodismutans ASO3-11 101 YP 379155.1 helicase RecD/TraA [Chlorobium chlorochromatii CaD31 102 YP 004897355.1 helicase rAcidaminococcus intestini RyC-MR951 >gblAEQ23215.11 103 ZP 03311944. 1 hypothetical protein DESPIG 01864 [Desulfovibrio piger ATCC 290981 104 YP 004783252.1 RecD/TraA family helicase rAcidithiobacillus ferrivorans SS31 105 ZP 03928493.1 helicase [Acidaminococcus so. D211 >gblEEH89723.1 helicase 106 ZP 06530901.1 RecD/TraA family helicase rStreptomvces lividans TK241 107 ZP 01667371.1 helicase, RecD/TraA family [Thermosinus carboxydivorans Norl 108 ZP 08942446. 1 helicase, RecD/TraA family [Thiorhodovibrio sp. 9701 >gblEGZ54636.11 109 NP 626969.1 deoxyribonuclease rStreptomyces coelicolor A3(2)] >emblCAB66276. 1 110 ADU73817.1 helicase, RecD/TraA family [Clostridium thermocellum DSM 13131 111 YP 001157093.1 RecD/TraA family helicase rSalinispora tropica CNB-4401 112 ZP 02929767.1 Putative exodeoxyribonuclease [Verrucomicrobium spinosum DSM 41361 113 ZP 08455023.1 Putative exodeoxyribonuclease V rStreptomvces so. Tu60711 114 YP 003022840.1 helicase, RecD/TraA family [Geobacter sp. M211 >gblACT19082.11 115 YP 003549103.1 helicase, RecD/TraA family [Coraliomargarita akaiimensis DSM 452211 116 YP 001530229.1 RecD/TraA family helicase rDesulfococcus oleovorans Hxd31 117 YP 004461132.1 helicase, RecD/TraA family [Tepidanaerobacter so. Rell 118 ZP 08943153.1 helicase, RecD/TraA family [Thiorhodovibrio so. 9701 >gblEGZ54097. 11 119 ZP 06560617.1 helicase, RecD/TraA family [Megasphaera genomosp. tyoe 1 str. 28L] 120 YP 002138036.1 helicase, RecD/TraA family [Geobacter bemidiiensis Beml 121 YP 001300657.1 exonuclease V subunit alpha rBacteroides vulgatus ATCC 84821 122 ZP 07303897.1 exodeoxyribonuclease V, alpha subunit rStreptomyces viridochromogenes 123 YP 003399141.1 helicase, RecD/TraA family [Acidaminococcus fermentans DSM 207311 124 YPl 389216.1 RecD/TraA family helicase rDesulfovibrio alaskensis G201 125 ZP 01085074.1 Helicase RecD/TraA [Synechococcus so. WH 57011 >gblEAQ75130.1| 126 ZP 07271541.1 exodeoxyribonuclease V, alpha subunit rStreptomyces so. SPB781 127 ZP 02731419. 1 helicase, RecD/TraA family protein rGemmata obscuriglobus UQM 22461 128 ZP 08287369. 1 deoxyribonuclease [Streptomyces griseoaurantiacus M0451 129 CBX272 15 1 hypothetical protein N47 A12440 [uncultured Desulfobacterium sp.] 130 YP 001530553.1 RecD/TraA family helicase rDesulfococcus oleovorans Hxd31 131 ZP 06707995.1 RecD/TraA family helicase rStreotomyces so. e141 >gblEFF91117.1| 132 ZP 06917215. 1 exodeoxyribonuclease V, alpha subunit rStreptomyces sviceus ATCC 133 YP 002955020.1 helicase RecD/TraA family protein [Desulfovibrio magneticus RS-11 134 ZP 07985895.1 Putative exodeoxyribonuclease V rStreptomyces so. SA3 actF1 135 YP 003103858.1 helicase, RecD/TraA family [Actinosynnema mirum DSM 438271 136 YP 001826337.1 putative exodeoxyribonuclease V rStreptomyces griseus subsp. guiseus 137 NP 826506.1 exodeoxyribonuclease V rStreptomyces avermitilis MA-46801 138 ZP 01048465.1 Helicase RecD/TraA [Nitrobacter so. Nb-311A1 >gblEAQ33584.11 139 YP 003761194.1 helicase, RecD/TraA family [Nitrosococcus watsonii C-1 131 140 YP 003681742.1 RecD/TraA family helicase [Nocardiopsis dassonvillei subsp. dassonvillei 141 ZP 08944617.1 helicase, RecD/TraA family [Thiorhodovibrio sp. 9701 >gblEGZ52593.11 142 ZP 08803068.1 DNA-binding protein [Streptomyces zinciresistens K421 143 ZP 07740397.1 helicase, RecD/TraA family [Aminomonas paucivorans DSM 122601 144 YP 003250238.1 helicase, RecD/TraA family [Fibrobacter succinogenes subsp. 145 ZP 01903329.1 Helicase RecD/TraA [Roseobacter so. AzwK-3bl >gb EDM71427.11 146 ZP 03641678.1 hypothetical protein BACCOPRO 00005 rBacteroides coprophilus DSM 147 ZP 06578909.1 exodeoxyribonuclease V [Streptomyces ghanaensis ATCC 146721 148 Y1 004652609.1 protein yrrC [Parachlamydia acanthamoebae UV71 >emblCCB86755. 1 149 ZP 06299454.1 hypothetical protein pah c032o017 rParachlamydia acanthamoebae str. 150 YPl 001771030.1 RecD/TraA family helicase rMethylobacterium so. 4-461 151 ZP 08291769 1 exodeoxyribonuclease V alpha chain [Chlamydophila psittaci Cal101 152 CCB74558.I Exodeoxyribonuclease V [Streptomyces cattleya NRRL 80571 153 ZP 08077054. 1 helicase, RecD/TraA family [Phascolarctobacterium so. YIT 120671 154 ZP 07297625. 1 RecD/TraA family helicase rStreptomyces hygroscopicus ATCC 536531 155 ZP 08030087. 1 helicase, RecD/TraA family [Selenomonas artemidis F03991 WO 2013/098562 PCT/GB2012/053274 26 156 YP 003300321.1 helicase, RecD/TraA family [Thermomonospora curvata DSM 431831 157 YP 001535192.1 RecD/TraA family helicase [Salinispora arenicola CNS-2051 158 NP 829514.1 RecD/TraA family helicase [Chlamydophila caviae GPICl 159 ZP 08843685.1 RecD/TraA family helicase [Desulfovibrio sp. 6 1 46AFAAl 160 ZP 07331538.1 helicase, RecD/TraA family [Desulfovibrio fructosovorans JJl 161 YP 003491438.1 DNA-binding protein [Streptomyces scabiei 87.221 >emblCBG72898.11 162 ZP 08073657.1 helicase, RecD/TraA family [Methylocvstis sp. ATCC 492421 163 ZP 07829281.1 helicase, RecD/TraA family [Selenomonas sp. oral taxon 137 str. F04301 164 YP 899880.1 RecD/TraA family helicase [Pelobacter propionicus DSM 23791 165 YP 343034.1 helicase RecD/TraA [Nitrosococcus oceani ATCC 197071 166 YP 004817633.1 helicase, RecD/TraA family [Streptomyces violaceusniger Tu 41131 167 BAJ31218.1 putative helicase RecD/TraA family protein rKitasatospora setae KM 168 YP 578071.1 helicase RecD/TraA [Nitrobacter hamburgensis X141 >gblABE63611.11 169 ZP 01873510.1 ATP-dependent exoDNAse (exonuclease V), alpha subunit-helicase 170 EFE27709.1 helicase, RecD/TraA family [Filifactor alocis ATCC 358961 171 YP 220018.1 putative exodeoxyribonuclease [Chlamvdophila abortus S26/31 172 ZP 07327131.1 helicase, RecD/TraA family [Acetivibrio cellulolyticus CD21 173 YP 002480862.1 helicase, RecD/TraA family [Desulfovibrio desulfuricans subsp. 174 EGK69360.1 putative exodeoxvribonuclease V subunit alpha [Chlamydophila abortus 175 YP 001967390.1 helicase RecD/TraA [Rickettsia monacensisl >gblABQ85878.11 helicase 176 ZP 03754577. 1 hypothetical protein ROSEINA2194 03004 [Roseburia inulinivorans 177 ZP 04608382. 1 helicase [Micromonospora sp. ATCC 391491 >gblEEP74312.1| helicase 178 YP 001509194.1 RecD/TraA family helicase [Frankia sp. EANIpec1 >gblABW14288.11 179 ZP 06771382.1 Exodeoxyribonuclease V [Streptomyces clavuligerus ATCC 270641 180 ZP 08838047.1 RecD/TraA family helicase [Bilophila sp. 4 1 301 >gblEGW42429.11 181 CBE67477.1 Helicase, RecD/TraA family [NC 10 bacterium 'Dutch sediment'l 182 YP 001950493.1 helicase, RecD/TraA family [Geobacter lovlevi SZ] >gblACD93973.ll 183 ZP 02192076.1 Helicase RecD/TraA ralpha proteobacterium BAL1991 >gblEDP61161.11 184 ZP 07943852.1 RecD/TraA family helicase [Bilophila wadsworthia 3 1 61 185 YP 003652363.1 helicase, RecD/TraA family [Thermobispora bispora DSM 438331 186 ZP 05005893.1 exodeoxyribonuclease V rStreptomyces clavuligerus ATCC 270641 187 ZP 05065242.1 helicase, RecD/TraA family [Octadecabacter antarcticus 2381 188 CBL24549.1 helicase, putative, RecD/TraA family [Ruminococcus obeum A2-1621 189 YP 002499923.1 RecD/TraA family helicase [Methylobacterium nodulans ORS 20601 190 YP 002432033.1 helicase, RecD/TraA family [Desulfatibacillum alkenivorans AK-011 191 ZP 07286835. I exodeoxyribonuclease V, alpha subunit [Streptomyces sp. C1 192 YP 001105553.1 helicase RecD/TraA [Saccharopolvspora erythraea NRRL 23381 193 YP 003639317.1 helicase, RecD/TraA family [Thermincola so. JR1 >gblADG81416.11 194 CBK63520.1 helicase, putative, RecD/TraA family [Alistives shahii WAL 83011 195 ZP 07940306.1 RecD/TraA family helicase [Bacteroides sp. 4 1 36] >gblEFV24451.1| 196 ZP 08905541.1 helicase RecD/TraA family protein [Desulfovibrio sp. FW1012B1 197 CBL07724.1 helicase, putative, RecD/TraA family [Roseburia intestinalis M50/11 198 ZP 03729282.1 helicase, RecD/TraA family [Dethiobacter alkaliphilus AHT 11 199 YP 001220283.1 RecD/TraA family helicase [Acidiphilium cryptum JF-51 200 ZP 05382244.1 exodeoxyribonuclease V alpha chain [Chlamydia trachomatis D(s)29231 201 YP 001654372.1 exodeoxyribonuclease V alpha chain rChlamydia trachomatis 434/Bul 202 ZP 04743359.1 helicase, RecD/TraA family [Roseburia intestinalis L1-821 203 ZP 08626355.1 helicase, RecD/TraA family protein rAcetonema longum DSM 65401 204 YP 004197836.1 helicase, RecD/TraA family [Geobacter sp. M181 >gblADW12560.11 205 ZP 06415577.1 helicase, RecD/TraA family [Frankia so. EUNifl >gblEFC81619.ll 206 YP 001618568.1 exodeoxyribonuclease V [Sorangium cellulosum 'So ce 56'] 207 ZP 05000079.1 exodeoxyribonuclease V [Streptomyces sp. Mgll >gblEDX24590.11 208 ZP 05346027.3 helicase, RecD/TraA family [Brvantella formatexigens DSM 144691 209 ADI 10122.1 exodeoxyribonuclease V rStreptomyces bingchenggensis BCW-11 210 YP 001220030.1 RecD/TraA family helicase [Acidiphilium cryotum JF-51 211 YP 515278.1 ATP-dependent dsDNA/ssDNA exodeoxyribonuclease V alpha 212 ZP 04658601.1 exodeoxyribonuclease V alpha subunit rSelenomonas flueggei ATCC 213 YP 002887661.1 exodeoxyribonuclease V alpha chain rChlamvdia trachomatis 214 ADI-117723.l exodeoxyribonuclease V alpha chain [Chlamydia trachomatis G/97681 WO 2013/098562 PCT/GB2012/053274 27 215 YP 327831.1 exodeoxyribonuclease V alpha chain rChlamvdia trachomatis A/HAR 216 ZP 06604245.1 RecD/TraA family helicase [Selenomonas noxia ATCC 435411 217 YP 004819301.1 helicase, RecD/TraA family rThermoanaerobacter wiegelii Rt8.B1] 218 ZP 05353404.1 exodeoxyribonuclease V alpha chain rChlamvdia trachomatis 62761 219 YP 003340096.1 exodeoxyribonuclease V [Streptosporangium roseum DSM 430211 220 YP 003965592.1 Helicase RecD/TraA rPaenibacillus polymyxa SC21 >gblADO59524.11 221 CCA55 822.1 RecD DNA helicase YrrC rStreptomyces venezuelae ATCC 107121 222 NPI 296681.1 exodeoxyribonuclease V, alpha subunit [Chlamydia muridarum Nigg] 223 YP 001126584 1 exodeoxyribonuclease V subunit alpha rGeobacillus thermodenitrificans 224 YP 004584034.1 RecD/TraA family helicase [Frankia symbiont of Datisca glomeratal 225 ZP 08710208.1 helicase, RecD/TraA family [Megasphaera sp. UPII 135-El 226 NP 219535.1 exodeoxyribonuclease V alpha chain rChlamydia trachomatis D/UW 227 ZP 05899746.1 helicase, RecD/TraA family [Selenomonas sputigena ATCC 351851 228 ZP 08502002.1 RecD/TraA family helicase [Centipeda periodontii DSM 27781 229 ZP 06590805.1 exodeoxyribonuclease V [Streptomyces albus J10741 >gblEFE81266.11 230 YP 003116724.1 helicase, RecD/TraA family [Catenulispora acidiphila DSM 449281 231 YP 002953905.1 helicase RecD/TraA family protein [Desulfovibrio magneticus RS-l] 232 ZP 06250970.1 helicase, RecD/TraA family [Prevotella copri DSM 182051 233 ZP 08634959.1 RecD/TraA family helicase [Acidiphilium sp. PM1 >gblEGO93242.11 234 YP 846029. 1 RecD/TraA family helicase [Syntrophobacter fumaroxidans MPOB1 235 YP 003476334.1 helicase, RecD/TraA family [Thermoanaerobacter italicus Ab9] 236 YP 001665762.1 RecD/TraA family helicase [Thermoanaerobacter pseudethanolicus 237 YP 001662092.1 RecD/TraA family helicase [Thermoanaerobacter sp. X5141 238 ZP 01462693.1 helicase, RecD/TraA family [Stigmatella aurantiaca DW4/3-11 239 Z P 07396437.1 RecD/TraA family helicase [Selenomonas sp. oral taxon 149 str. 240 ZP 0821116.3. 1 helicase, RecD/TraA family [Thermoanaerobacter ethanolicus JW 2001 241 YP 003953494.1 exodeoxyribonuclease v alpha chain [Stigmatella aurantiaca DW4/3-11 242 YP 003676322.1 RecD/TraA family helicase [Thermoanaerobacter mathranii subsp. 243 YP 003252104.1 helicase, RecD/TraA family [Geobacillus so. Y412MC611 244 YP 001918465.1 helicase, RecD/TraA family [Natranaerobius thermophilus JW/NM-WN 245 ZP 08880276.1 helicase RecD/TraA [Saccharopolvsvora spinosa NRRL 183951 246 ZP 03991439.1 possible exodeoxyribonuclease V alpha subunit rOribacterium sinus 247 AAG23283.1 probable exodeoxyribonuclease V [Saccharopolvspora spinosal 248 YP 148414.1 ATP-dependent exonuclease V [Geobacillus kaustophilus HTA426] 249 YP 714380.1 putative exodeoxyribonuclease V [Frankia alni ACN14a] 250 CA J74974.1 similar to exodeoxyribonuclease V alpha subunit [Candidatus Kuenenia 251 YP 001936773.1 exodeoxyribonuclease V alpha subunit [Orientia tsutsugamushi str. 252 YP 001248319.1 helicase RecD/TraA, ATP-dependent exoDNAse (exonuclease V) 253 YP 004377643.1 RecD/TraA family helicase [Chlamydophila decorum E581 254 CBL20603.1 helicase, putative, RecD/TraA family [Ruminococcus sp. SR1/51 255 YP 004421448.1 helicase RecD/TraA [Candidatus Rickettsia amblyommii AaR/SCl 256 ZP 04857179.1 conserved hypothetical protein [Ruminococcus sp. 5 1 39B FAA] 257 YP 003670549.1 helicase, RecD/TraA family [Geobacillus sp. C56-T31 >gblADI25972.11 258 YP 003824777.1 helicase, RecD/TraA family [Thermosediminibacter ocean DSM 166461 259 ZP 08812773.1 hypothetical protein DOT 4190 [Desulfosporosinus sp. OTl 260 ZP 07757395.1 helicase, RecD/TraA family [Megasphaera micronuciformis F03 591 261 ZP 08131496.1 helicase, RecD/TraA family [Clostridium sp. D51 >gblEGB91345.11 262 ZP 04698234. 1 helicase, RecD/TraA family [Rickettsia endosymbiont of Ixodes 263 AEH95290.1 Putative helicase rAplvsina aerophoba bacterial symbiont clone 264 YP 002464026.1 helicase, RecD/TraA family [Chloroflexus aggregans DSM 94851 265 YP 461625.1 exodeoxyribonuclease V subunit alpha rSyntrophus aciditrophicus SB] 266 YP 010116.1 RecD/TraA family helicase [Desulfovibrio vulgaris str. Hildenboroughl 267 YP 001634449.1 RecD/TraA family helicase [Chloroflexus aurantiacus J-10-fl] 268 YP 004371330 1 helicase, RecD/TraA family [Desulfobacca acetoxidans DSM 111091 269 ZP 02432140.1 hypothetical protein CLOSCI 02385 [Clostridium scindens ATCC 270 YP 001546377.1 RecD/TraA family helicase [Herpetosiphon aurantiacus DSM 7851 271 Z P 01995753.1 hypothetical protein DORLON 01748 rDorea longicatena DSM 138141 272 ZP 08602931.1 RecD/TraA family helicase [Lachnospiraceae bacterium 5 1 57FAAl 273 YP 003409821.1 helicase, RecD/TraA family [Geodermatophilus obscurus DSM 431601 WO 2013/098562 PCT/GB2012/053274 28 274 YP 004839529.1 helicase, RecD/TraA family protein rRoseburia hominis A2-1831 275 ZP 08864377.1 helicase, RecD/TraA family rDesulfovibrio sp. A21 >gblEGY27050. 11 276 ZP 05733053.1 helicase, RecD/TraA family rDialister invisus DSM 154701 277 YP 003270118.1 helicase, RecD/TraA family rHaliangium ochraceum DSM 143651 278 YP 001717534.1 RecD/TraA family helicase [Candidatus Desulforudis audaxviator 279 YP 003317021.1 helicase, RecD/TraA family [Thermanaerovibrio acidaminovorans DSM 280 NP 622165.1 exonuclease V subunit alpha rThermoanaerobacter tengcongensis MB41 281 ZP 05346627.1 helicase, RecD/TraA family rBryantella formatexigens DSM 144691 282 ZP 04451325.1 hypothetical protein GCWU000182 00609 rAbiotrophia defectiva ATCC 283 NP 623674.1 exonuclease V subunit alpha rThermoanaerobacter tengcongensis MB41 284 ZP 02037832.1 hypothetical protein BACCAP 03451 rBacteroides capillosus ATCC 285 EGS35366.1 helicase, RecD/TraA family rFinegoldia magna SY403409CC0010504171 286 ZP 05092205.1 helicase, RecD/TraA family rCarboxydibrachium pacificum DSM 126531 287 ZP 08419913.1 helicase, RecD/TraA family rRuminococcaceae bacterium D161 288 YP 001692807.1 ATP-dependent exodeoxyribonuclease subunit alpha [Finegoldia magna 289 ZP 07268899.1 helicase, RecD/TraA family [Finegoldia magna ACS-171-V-Col31 290 ZP 06598130.1 helicase, RecD/TraA family [Oribacterium sp. oral taxon 078 str. F02621 291 YPl 003807812.1 helicase, RecD/TraA family [Desulfarculus baarsii DSM 20751 292 ZP 02233368.1 hypothetical protein DORFOR 00200 [Dorea formicigenerans ATCC 293 ZP 02037912. 1 hypothetical protein BACCAP 03531 [Bacteroides capillosus ATCC 294 ZP 07202886. 1 helicase, RecD/TraA family [delta proteobacterium NaphS21 295 YP 003152507.1 helicase, RecD/TraA family [Anaerococcus prevotii DSM 20548] 296 ZP 04861948.1 helicase, RecD/TraA family [Clostridium botulinum D str. 18731 297 ZP 07398794.1 RecD/TraA family helicase [Peptoniphilus duerdenii ATCC BAA-16401 298 YP 867272,1 RecD/TraA family helicase [Magnetococcus sp. MC-11 299 YP 003852761.1 helicase, RecD/TraA family [Thermoanaerobacterium 300 ZP 07959337.1 RecD/TraA family Helicase [Lachnospiraceae bacterium 8 1 57FAAl 301 YP 004020312.1 helicase, RecD/TraA family [Frankia sp. EuIlcl >gblADP84442.11 302 ZP 05055731.1 helicase, RecD/TraA family [Verrucomicrobiae bacterium DG12351 303 YP 002936648.1 helicase, RecD/TraA family [Eubacterium rectale ATCC 336561 304 ZP 02620578.1 helicase, RecD/TraA family [Clostridium botulinum C str. Eklund] 305 CBK80090.1 helicase, putative, RecD/TraA family [Coprococcus catus GD/71 306 ZP 08865335.1 hypothetical protein DA2 1615 rDesulfovibrio sp. A21 >gblEGY26242.11 307 YP 004309919.1 helicase, RecD/TraA family [Clostridium lentocellum DSM 54271 308 YP 004471588.1 helicase, RecD/TraA family [Thermoanaerobacterium xylanolyticum LX 309 CBL21608.1 helicase, putative, RecD/TraA family [Ruminococcus sp. SRI/51 310 YP 003844493.1 helicase, RecD/TraA family [Clostridium cellulovorans 743B] 311 ZP 07321245. 1 helicase, RecD/TraA family [Finegoldia magna BVS033A41 312 YP 699450.1 RecD/TraA family helicase [Clostridium perfringens SM1011 313 ZP1 03781777.1 hypothetical protein RUMHYD 01213 [Blautia hydrogenotrophica DSM 314 YP 00243 5820.1 helicase, RecD/TraA family [Desulfovibrio vulgaris str. 'Miyazaki F'] 315 ZP 02042737.1 hypothetical protein RUMGNA 03541 [Ruminococcus gnavus ATCC 316 YP 004003526.1 helicase, recd/traa family [Caldicellulosiruptor owensensis OL1 317 ZP 04666257.1 helicase [Clostridiales bacterium 1 7 47 FAA] >gblEEQ62058. 1 318 YP 004025165.1 helicase, recd/traa family [Caldicellulosiruptor kronotskyensis 20021 319 YT 003937377.1 DNA-binding protein rClostridium sticklandii DSM 5191 320 ZP 03777575.1 hypothetical protein CLOHYLEM 04627 [Clostridium hylemonae DSM 321 ZP 02089268.1 hypothetical protein CLOBOL 06837 [Clostridium bolteae ATCC BAA 322 ZP 06946532.1 RecD/TraA family helicase [Finegoldia magna ATCC 535161 323 ZP 03762681. 1 hypothetical protein CLOSTASPAR 06723 [Clostridium asparagiforme 324 ZP 08933657.1 RecD/TraA family helicase [Peptoniphilus indolicus ATCC 294271 325 YP 003759289.1 UvrD/REP helicase [Dehalogenimonas lykanthroporepellens BL-DC-91 326 ZP 02865223.1 helicase, RecD/TraA family [Clostridium perfringens C str. JGS14951 327 YP 002315104.1 ATP-dependent exoDNAse (exonuclease V) subunit alpha - helicase 328 YP 003820655.1 helicase, RecD/TraA family [Clostridium saccharolyticum WM1] 329 NP 563091.1 helicase, RecD/TraA family [Clostridium perfringens str. 131 330 ZP 02631593.1 helicase, RecD/TraA family [Clostridium perfringens E str. JGS19871 331 YP 754748.1 exodeoxyribonuclease V rSyntrophomonas wolfei subsp. wolfei str. 332 YP 002574552.1 RecD/TraA family helicase [Caldicellulosiruptor bescii DSM 67251 WO 2013/098562 PCT/GB2012/053274 29 333 ZP 02641429.1 helicase, RecD/TraA family [Clostridium perfringens NCTC 82391 334 YP 004121351.1 ATP-dependent RecD/TraA family DNA helicase [Desulfovibrio 335 EGC82456.1 helicase, RecD/TraA family [Anaerococcus prevotii ACS-065-V-Coll3l 336 YP 004199699.1 ATP-dependent RecD/TraA family DNA helicase rGeobacter so. M181 337 ZP 08616225. 1 RecD/TraA family helicase [Lachnospiraceae bacterium 1 4 56FAA] 338 ZP 06113685. 1 helicase, RecD/TraA family [Clostridium hathewayi DSM 134791 339 ZP 03799911.1 hypothetical protein COPCOM 02174 rCoprococcus comes ATCC 340 YP 003841513.1 helicase, RecD/TraA family [Caldicellulosiruptor obsidiansis OB471 341 YP 004464342.1 RecD/TraA family ATP-dependent DNA helicase [Mahella australiensis 342 YP 696854.1 RecD/TraA family helicase [Clostridium perfringens ATCC 131241 343 ZP 03 167580.1 hypothetical protein RUMLAC 01253 rRuminococcus lactaris ATCC 344 YP 847893 1 hypothetical protein Sfum 3789 [Syntrophobacter fumaroxidans MPOB] 345 ZP 05430222.1 helicase, RecD/TraA family [Clostridium thermocellum DSM 23601 346 ZP 022 11142.1 hypothetical protein CLOBAR 00740 [Clostridium bartlettii DSM 347 YP 388414.2 UvrD/REP helicase rDesulfovibrio alaskensis G201 >gblABB38719.2| 348 YP 003807790 1 ATP-dependent RecD/TraA family DNA helicase rDesulfarculus baarsii 349 YP 003993640.1 helicase, recd/traa family [Caldicellulosiruptor hydrothermalis 1081 350 YPl 001038644.1 ATP-dependent RecD/TraA family DNA helicase rClostridium 351 ZP 06597516.1 helicase, RecD/TraA family [Oribacterium sp. oral taxon 078 str. F02621 352 YP 004799933.1 helicase, RecD/TraA family [Caldicellulosiruptor lactoaceticus 6A1 353 YP 001179036.1 RecD/TraA family helicase [Caldicellulosiruptor saccharolyticus DSM 354 ZP 03759537. 1 hypothetical protein CLOSTASPAR 03561 [Clostridium asparagiforme 355 ZP 04564978.1 exodeoxyribonuclease subunit V alpha rMollicutes bacterium D71 356 YP 001557372.1 RecD/TraA family helicase [Clostridium phytofermentans ISDgl 357 ZP 02094462.1 hypothetical protein PEPMIC 01228 [Parvimonas micra ATCC 332701 358 ZP 02428906.1 hypothetical protein CLORAM 02328 rClostridium ramosum DSM 359 ZP 07367500.1 exodeoxyribonuclease V alpha subunit rPediococcus acidilactici DSM 360 YP 004027603.1 helicase, recd/traa family [Caldicellulosiruptor kristianssonii 177R1B1 361 ZP 02420394. 1 hypothetical protein ANACAC 03011 rAnaerostives caccae DSM 146621 362 ZP 08707741.1 helicase, RecD/TraA family [Veillonella sp. oral taxon 780 str. F04221 363 ZP 08532372.1 helicase, RecD/TraA family [Caldalkalibacillus thermarum TA2.All 364 YP 003119362.1 helicase, RecD/TraA family [Catenulispora acidiphila DSM 449281 365 YPl 001821497.1 RecD/TraA family helicase ropitutus terrae PB90-11 >gblACB77897.11 366 YP 003427313.1 ATP-dependent exoDNAse V [Bacillus pseudofirmus OF41 367 ZP 07036639 1 helicase, RecD/TraA family [Peptoniphilus sp. oral taxon 386 str. F01311 368 YP 004091069.1 helicase, RecD/TraA family [Ethanoligenens harbinense YUAN-31 369 ZP 06197663.1 RecD/TraA family helicase [Pediococcus acidilactici 7 41 370 ZP 06409626. 1 helicase, RecD/TraA family [Clostridium hathewayi DSM 134791 371 ZP 08339411 1 RecD/TraA family helicase [Lachnospiraceae bacterium 2 1 46FAA] 372 YP 004883288.1 putative nuclease [Oscillibacter valericigenes Sim18-201 373 YP 004396914.1 RecD/TraA family helicase [Clostridium botulinum BKTO159251 374 YP 002950456.1 helicase, RecD/TraA family [Geobacillus so. WCH701 >gblACS25190.11 375 YP 387402,1 UvrD/REP helicase [Desulfovibrio alaskensis G201 >gblABB37707.11 376 YP 002771377.1 hypothetical protein BBR47 18960 rBrevibacillus brevis NBRC 1005991 377 ZP 01173819.1 YrrC [Bacillus so. NRRL B-149111 >gblEAR63466.11 YrrC [Bacillus so. 378 ZP 02074502. 1 hypothetical protein CLOL250 01272 [Clostridium sp. L2-501 379 ZP 02951515.1 helicase, RecD/TraA family [Clostridium butyricum 55211 380 ZP 07326697.1 helicase, RecD/TraA family [Acetivibrio cellulolyticus CD21 381 ZP 08662208. I helicase, RecD/TraA family [Streptococcus so. oral taxon 056 str. F04181 382 ZP 07709630. I helicase, RecD/TraA family protein rBacillus so. m3-131 383 ZP 08151018.1 RecD/TraA family helicase [Lachnospiraceae bacterium 4 1 37FAA] 384 ZP 05855961. 1 helicase, RecD/TraA family [Blautia hansenii DSM 205831 385 ZP 08333468.1 RecD/TraA family helicase [Lachnospiraceae bacterium 6 1 63FAAl 386 ZP 01967327.1 hypothetical protein RUMTOR 00874 rRuminococcus torques ATCC 387 ZP 07843612.1 helicase, RecD/TraA family [Staphylococcus hominis subso. hominis 388 ZP 08335292.1 RecD/TraA family helicase [Lachnospiraceae bacterium 9 1 43BFAA] 389 YP 002425519.1 helicase, RecD/TraA family [Acidithiobacillus ferrooxidans ATCC 390 ZP 08074741.1 Exodeoxyribonuclease V [Methylocystis so. ATCC 492421 391 ZP 03288021.1 hypothetical protein CLONEX 00200 [Clostridium nexile DSM 17871 WO 2013/098562 PCT/GB2012/053274 30 392 NP 349457.1 ATP-dependent exoDNAse (exonuclease V), alpha subunit, RecD 393 YP 535621.1 exodeoxyribonuclease V alpha chain [Lactobacillus salivarius UCC 1181 394 YP 001514053.1 RecD/TraA family helicase rAlkaliphilus oremlandii OhILAs] 395 ZP 06059658.1 RecD/TraA family helicase rStreptococcus sp. 2 1 36FAAl 396 ZP 04059935. 1 helicase, RecD/TraA family [Stavhvlococcus hominis SKI 191 397 AEN87514.1 Exodeoxyribonuclease V-like protein [Bacillus megaterium WSH-0021 398 YP 003988429.1 helicase, RecD/TraA family [Geobacillus so. Y4.1MC11 399 NP 781026.1 exodeoxyribonuclease V alpha chain [Clostridium tetani E881 400 YP 004707292.1 hypothetical protein CXIVA 02230 [Clostridium so. SY85191 401 YP 003590771.1 helicase, RecD/TraA family [Bacillus tusciae DSM 29121 402 ZP 03708405.1 hypothetical protein CLOSTMETH 03166 [Clostridium methylnentosum 403 ZP 07904646.1 RecD/TraA family helicase rEubacterium saburreum DSM 39861 404 ZP 08463854.1 exodeoxyribonuclease V alpha subunit [Desmospora sp. 84371 405 YP 003339261.1 exodeoxyribonuclease V [Streptosporangium roseum DSM 430211 406 ZP 07356412.1 helicase, RecD/TraA family [Desulfovibrio sp. 3 1 syn31 407 YP 004310482.1 helicase, RecD/TraA family [Clostridium lentocellum DSM 54271 408 YP 003565054.1 helicase, RecD/TraA family [Bacillus megaterium QM B15511 409 EGM50608.1 helicase, RecD/TraA family [Lactobacillus salivarius GJ-241 410 ZP 07454758.1 RecD/TraA family helicase [Eubacterium vurii subsp. margaretiae ATCC 411 CBL 17987.1 helicase, putative, RecD/TraA family [Ruminococcus sp. 18P131 412 ZP 03917092. 1 possible exodeoxyribonuclease V alpha subunit [Anaerococcus 413 ZP 08757131.1 helicase, RecD/TraA family [Parvimonas sp. oral taxon 393 str. F04401 414 EGL99465.1 recD-like DNA helicase YrrC [Lactobacillus salivarius NIAS8401 415 ZP 01725995.1 hypothetical protein BB14905 09550 [Bacillus sp. B149051 416 YP 003590260.1 helicase, RecD/TraA family [Bacillus tusciae DSM 29121 417 ZP 07206301.1 helicase, RecD/TraA family [Lactobacillus salivarius ACS-116-V-Col5al 418 YP 001680910.1 exodeoxyribonuclease V, alpha chain, RecD [Heliobacterium 419 YP 003821341.1 helicase, RecD/TraA family [Clostridium saccharolyticum WM 1] 420 ZP 08005791.1 YrrC protein [Bacillus so. 2 A 57 CT21 >gblEFV77442.11 YrrC protein 421 YP 002560662.1 exodeoxyribonuclease V alpha subunit [Macrococcus caseolyticus 422 ZP 05028653.1 hypothetical protein MC7420 1174 [Microcoleus chthonoplastes PCC 423 CCC58043.1 RecD-like DNA helicase YrrC [Caloramator australicus RC31 424 YPl 001699482.1 exodeoxyribonuclease V-like protein [Lysinibacillus sphaericus C3-411 425 ZP 02616886. 1 helicase, RecD/TraA family [Clostridium botulinum Bfl 426 XP 001420006.1 predicted protein [Ostreococcus lucimarinus CCE99011 427 YP 001779796.1 RecD/TraA family helicase [Clostridium botulinum BI str. Okral 428 YP 001389532.1 RecD/TraA family helicase [Clostridium botulinum F str. Langelandl 429 ZP 02993715.1 hypothetical protein CLOSPO 00789 [Clostridium sporogenes ATCC 430 ZP 01964108. 1 hypothetical protein RUMOBE 01832 [Ruminococcus obeum ATCC 431 ZP1 03227028.1 ATP-dependent exonuclease V [Bacillus coahuilensis m4-41 432 YP 001252714.1 helicase, RecD/TraA family [Clostridium botulinum A str. ATCC 35021 433 YP 001307573.1 RecD/TraA family helicase [Clostridium beiierinckii NCIMB 80521 434 ZP 08091201.1 hypothetical protein HMPREF9474 02952 [Clostridium symbiosum 435 CBZ01987.1 recd-like DNA helicase YrrC [Clostridium botulinum H04402 0651 436 ZP 06620580. 1 helicase, RecD/TraA family [Turicibacter sanguinis PC9091 437 ZP 02612165.1 helicase, RecD/TraA family [Clostridium botulinum NCTC 29161 438 ZP 03464124.1 hypothetical protein BACPEC 03225 [Bacteroides pectinonhilus ATCC 439 ZP 05427870.1 helicase, RecD/TraA family [Eubacterium saphenum ATCC 499891 440 ZP 04819493.1 exodeoxyribonuclease V alpha subunit [Staphylococcus evidermidis 441 CBL16176.1 helicase, putative, RecD/TraA family [Ruminococcus bromii L2-631 442 CBK73489.1 helicase, putative, RecD/TraA family [Butyrivibrio fibrisolvens 16/41 443 XP 003081706.1 Dehydrogenase kinase (ISS) [Ostreococcus tauri] >emblCAL56230.1| 444 ZP 06425429. 1 helicase, RecD/TraA family [Pentostrentococcus anaerobius 653-L1 445 ZP 08539226. 1 helicase, RecD/TraA family [Oribacterium sp. oral taxon 108 str. F04251 446 ZP 04008608.1 exodeoxyribonuclease V alpha chain [Lactobacillus salivarius ATCC 447 ZP 08525848.1 helicase, RecD/TraA family [Streptococcus anginosus SK521 448 ZP 08245897.1 helicase, RecD/TraA family [Streptococcus varauberis NCFD 20201 449 ZP 06290581.1 helicase, RecD/TraA family [Peptoninhilus lacrimalis 315-B1 450 ZP 08680798.1 RecD/TraA family helicase [Sporosarcina newyorkensis 26811 WO 2013/098562 PCT/GB2012/053274 31 451 NP 002802482.1 helicase, RecD/TraA family [Clostridium botulinum A2 str. Kyotol 452 YP 001449573.1 RecD/TraA family helicase [Streotococcus 2ordonii str. Challis substr. 453 ZP 01862085.1 hvothetical protein BSG1 18450 [Bacillus so. SG-11 >ablEDL62855.11 454 YP 001785497.1 RecD/TraA family helicase [Clostridium botulinum A3 str. Loch Mareel 455 EFV89168.1 exodeoxvribonuclease V aloha chain [Stahvlococcus epidermidis 456 ZP 07956105.1 RecD/TraA family helicase [Lachnospiraceae bacterium 5 1 63FAA] 457 ZP 03055915.1 helicase, RecD/TraA family [Bacillus pumilus ATCC 70611 458 ZP 04797338.1 exodeoxvribonuclease V aloha subunit [Staphylococcus evidermidis 459 EGS77340.1 helicase, RecD/TraA family [Staphylococcus epidermidis VCU1051 460 YP 004478259.1 hvothetical protein STP 0139 [Streptococcus parauberis KCTC 115371 461 ZP 08605488.1 RecD/TraA family helicase [Lachnospiraceae bacterium 462 ZP 08643227.1 hvothetical protein BRLA c44940 [Brevibacillus laterosporus LMG 463 ZP 06875615.1 putative exonuclease with DNA/RNA helicase motif [Bacillus subtilis 464 ZP 04678546.1 helicase, RecD/TraA family [Staphvlococcus warneri L376031 465 ZP 06613101.1 conserved hvoothetical protein [Staphylococcus evidermidis 466 ZP1 02441706.1 hypothetical protein ANACOL 00987 [Anaerotruncus colihominis DSM 467 YP 188759.1 RecD/TraA family helicase [Staphylococcus evidermidis RP62A1 468 ZP 02440294.1 hvothetical protein CLOSS21 02797 [Clostridium so. SS2/11 469 YP 001919909.1 helicase, RecD/TraA family [Clostridium botulinum E3 str. Alaska E431 470 YP 001884722.1 helicase, RecD/TraA family [Clostridium botulinum B str. Eklund 17B1 471 ZP 02039032. 1 hvothetical Drotein BACCAP 04681 [Bacteroides capillosus ATCC 472 ZP 07093704.1 helicase, RecD/TraA family [Petoniphilus so. oral taxon 836 str. F01411 473 YP 001487615.1 exodeoxyribonuclease V aloha subunit [Bacillus pumilus SAFR-0321 474 NP 764857.1 deoxvribonuclease [Staphylococcus evidermidis ATCC 122281 475 YP 804665.1 ATP-dependent RecD/TraA family DNA helicase rPediococcus 476 NP 942288.1 exodeoxyribonuclease V aloha chain [Synechocvstis so. PCC 68031 477 ZP 07054718.1 RecD/TraA family helicase [Listeria Qravi DSM 206011 >gblEFI83599.1| 478 EHA31059.1 hvothetical protein BSSC8 15020 [Bacillus subtilis subsp. subtilis str. 479 CBL39055.1 helicase. putative. RecD/TraA family [butvrate-producing bacterium 480 EGF05416.1 exodeoxyribonuclease V aloha subunit [Streotococcus sanguinis SK10571 481 YP 003974155.1 putative exonuclease [Bacillus atrophaeus 19421 >gblADP33224.1| 482 Z P 07822731.1 helicase, RecD/TraA family [Petoniphilus harei ACS-146-V-Sch2b] 483 ZP 06348052.1 helicase, RecD/TraA family [Clostridium so. M62/11 >gblEFE10725.11 484 ZP 05394746.1 helicase, RecD/TraA family [Clostridium carboxidivorans P71 485 YP 004204562.1 putative exonuclease [Bacillus subtilis BSn51 >dbilBAI86231.11 486 EGG96535.1 helicase, RecD/TraA family [Staohylococcus epidermidis VCU1211 487 YP 003471518.1 Exodeoxyribonuclease V subunit alpha [Staphylococcus luadunensis 488 ZP 04820712.1 helicase, RecD/TraA family [Clostridium botulinum El str. 'BoNT E 489 EGF05816.1 exodeoxyribonuclease V aloha subunit [Streotococcus sanguinis SKI] 490 YP 002634323.1 hvothetical protein Sea 1231 [Staphylococcus camosus subso. carnosus 491 YP 301232.1 ATP-dependent exonuclease V alpha subunit [Staphvlococcus 492 ZP 07841151.1 helicase, RecD/TraA family [Staphylococcus caprae C871 493 NP 846841.1 helicase [Bacillus anthracis str. Ames] >reflYP 021271.21 helicase 494 NP 390625.1 exonuclease with DNA/RNA helicase motif [Bacillus subtilis subsp. 495 ZP 07910981.1 RecD/TraA family helicase [Staohvlococcus lugdunensis M235901 496 YP 001727916.1 exonuclease V subunit alpha [Leuconostoc citreum KM201 497 YP 030537.1 helicase [Bacillus anthracis str. Sterne] >reflZP 00394720.11 COG0507: 498 ZP 04291295.1 Helicase, RecD/TraA [Bacillus cereus R3098031 >gblEEK76998.1| 499 YP 002751754.1 putative helicase [Bacillus cereus 03BB1021 >gblACO31219.11 putative 500 ZP 08091585.1 hvothetical protein HMPREF9474 03336 [Clostridium symbiosum The RecD helicase is more preferably one of the helicases shown in Table 5 below or a variant thereof. The RecD helicase more preferably comprises the sequence of one of the 5 helicases shown in Table 5, i.e. one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 421 and 44, or a variant thereof.
WO 2013/098562 PCT/GB2012/053274 32 Table 5 - More preferred RecD helicases SEQ % RecD-like ID Name Source NCBI ref Identity RecD motif I motif V NO to RecD2 (SEQ ID NO:) (SEQ ID Dra NO:) RecD Acaryochl NCBI Reference WAVTIH 18 2 oris 001521445. 29.8 GGPGTGKT (19) KSQG Ama marna 1 (20) NCBI Reference GGPGTGKS (22) YALTVH 21 RecD Deinococc Sequence: 12.3 RAQG 2 Dde us deserti YP_002786343. (23) 1 Deinococc NCBI Reference GGPGTGKS (22) YALTVH 24 RecD us Sequence: 79 RAQG 2 Dge geotherma YP604297.1 (23) hs - Haliangiu NCBI Reference GGPGVGKT (26) YAISVH 25 RecD m Sequence: 30 KSQG 2 Hoc ochraceum YP_003270118. (27) DSM 1 Natranaero NCBI Reference GGPGTGKT (19) YCISVH 28 RecD bius Sequence: 28.6 KSQG 2 Nth thermophil YP_001918465. (29) us 1 RecD Octadecab NCBI Reference GGPGVGKT (26) YAATIH 30 2 Oan acter Sequence: 31 KSQG antarcticus ZP_05054956.1 (31) NCBI Reference GGPGCGKS (33) YAMTIH 32 RecD Salinispor Sequence: 31.7 RSQG 2 Str a tropica YP_001157093. (34) 1 Desulfonat NCBI Reference GGPGTGKS (22) YAVSIH 35 RecD ronospira Sequence: 27.6 KSQG 2 Dth thiodismut ZP 07015918.1 (36) ans Nitrosococ NCBI Reference GGPGVGKT (26) YATSVH 37 RecD cus Sequence: 29.8 KSQG 2 Nha halophilus YP 10031528424. (38) Desulfohal NCBI Reference GGPGTGKT (19) YAVSVH 39 RecD obium Sequence: 32 KSQG 2 Dre retbaense -003197384. (40) Deinococc NCBI Reference GGPGTGKS (22) YALTVH 41 RecD us Sequence: - RAQG 2 Dra radiiduran NP295625.1 (23) S RecD Chlorobiu NCBI Reference GGPGVGKT (26) YATSIHK 42 2 Cch m Sequence: 30.8 SQG (43) chlorochro YP 379155.1 WO 2013/098562 PCT/GB2012/053274 33 matii RecD Deinococc NCBI Reference GGPGTGKS (22) YALTVH 44 2 us Sequence: 67 RGQG Dma maricopen YP_004170918. (45) sis 1 All sequences in the above Table comprise a RecD-like motif V (as shown). Only SEQ ID NOs: 18, 25, 28, 30, 35, 37, 39 and 42 comprise a RecD motif V (as shown). The RecD helicase is preferably a Tral helicase or a TraI subgroup helicase. Tral 5 helicases and Tral subgroup helicases may contain two RecD helicase domains, a relaxase domain and a C-terminal domain. The Tral subgroup helicase is preferably a TrwC helicase. The Tral helicase or Tral subgroup helicase is preferably one of the helicases shown in Table 6 below or a variant thereof. The TraI helicase or a TraI subgroup helicase typically comprises a RecD-like motif I as 10 defined above (SEQ ID NO: 8) and/or a RecD-like motif V as defined above (SEQ ID NO: 16). The Tral helicase or a Tral subgroup helicase preferably comprises both a RecDlike motif 1 (SEQ ID NO: 8) and a RecD-like motif V (SEQ ID NO: 16). The Tral helicase or a Tral subgroup helicase typically further comprises one of the following two motifs: - The amino acid motif H-(X)-X2-R-(X3) 1 A-H-X4-H (hereinafter called the MobF 15 motif III; SEQ ID NOs: 46 to 53 show all possible MobF motifs I1I (including all possible numbers of X3)), wherein XI and X3 are any amino acid and X2 and X4 are independently selected from any amino acid except D, E, K and R. (X1)2 is of course Xla-Xlb. Xia and Xlb can be the same of different amino acid. Xla is preferably D or E. Xlb is preferably T or ). (X1) is preferably DT or ED. (XI) 2 is most preferably DT. The 5 to 12 amino acids in (X3)su 2 20 can be the same or different, X2 and X4 are independently selected from G, P A, V, L, I, M, C, F, Y, W, H Q, N, S and T. X2 and X4 are preferably not charged. X2 and X4 are preferably not H-. X2 is more preferably N, S or A. X2 is most preferably N. X4 is most preferably F or T.
(X
3 ) i2 is preferably 6 or 10 residues in length (SEQ ID NOs: 47 and 51). Suitable embodimems of (X3)5. can be derived from SEQ ID Nfis: 61, 65, 69, 73, 74, 82, 86, 90, 94, 98, 25 102, 110 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168shown in Table 7 below (i.e. all but SEQ ID NOs: 78 and 106). Preferred embodiments of the MobF motif -111 are shown in Table 7 below - The amino acid motif G-XI-X2-X3-X4-X5-X6-X7-H-(X8)1u-H-X9 (hereinafter called the MobQ motif 111; SEQ ID N.s: 54 to 60 show all possible MobQ motifs 111I (including all 30 possible numbers of X8)), wherein X1, X2., X3, X5, X6, X7 and X9 are independently selected from any amino acid except D, E, K and R, X4 is D or E and X8 is any amino acid. X1,X2, X3, WO 2013/098562 PCT/GB2012/053274 34 X5, X6, X7 and X9 are independently selected from G,1 P, A, V, L, I, M, C, F, Y, W, -, Q, N, S and T. XL X2, X3, X5, X6, X7 and X9 are preferably not charged. XI, X2, X3, X5, X6, X7 and X9 are preferably not H-. The 6 to 12 amino acids in (X)SLu can be the same or different, Suitable embodiments of (X8)6- can be derived from SEQ ID NOs: 78 and 106 shown in Table 5 7 below. Preferred embodiments of the MobF rnotif III are shown in Table 7 below.
WO 2013/098562 PCT/GB2012/053274 35 Table 6 - Preferred Tral helicases and Tral subgroup helicases and their Accession Numbers 1 NP 061483.1 coniugal transfer nickase/helicase Tral [Plasmid F1 2 NP 862951.1 conjugal transfer nickase/helicase Tral [Escherichia colil 3 ZP 03047597. type IV secretion-like coniugative transfer relaxase protein Tral 4 YP 00203889 conjugal transfer nickase/helicase Tral [Salmonella enterica subsp. 5 YP 00173989 type IV secretion-like conjugative transfer relaxase protein Tral 6 YP 190115.1 conjugal transfer nickase/helicase Tral [Escherichia colil 7 ZP 08368984. coniugative transfer relaxase protein Tral rEscherichia coli TA2711 8 EFW76779.1 IncF plasmid coniugative transfer DNA-nicking and unwinding 9 YP 00382905 type IV secretion-like coniugative transfer relaxase protein 10 EGX 11991.1 coniugative transfer relaxase protein Tral [Escherichia coli 11 YP 00191916 type IV secretion-like coniugative transfer relaxase protein Tral 12 ZP 03051102. type IV secretion-like conjugative transfer relaxase protein Tral 13 EGH36328. 1 IncF plasmid coniugative transfer DNA-nicking and unwinding 14 ZP 03030171. type IV secretion-like coniugative transfer relaxase protein Tral 15 EGB88794. 1 coniugative transfer relaxase protein Tral rEscherichia coli MS 117-31 16 YP 00382916 nickase/helicase rEscherichia colil >gblADL14054.11 Tral 17 EFUT55615.1 coniugative transfer relaxase protein Tral rEscherichia coli MS 16-31 18 EGB69775 1 coniugative transfer relaxase TraI [Escherichia coli TW105091 19 YP 00303405 conjugal transfer nickase/helicase TraI [Escherichia coli Vir681 20 YP 443956.1 conjugal transfer nickase/helicase Tral [Escherichia colil 21 YP 00196541 oriT-specific relaxase; helicase rEscherichia colil >gblABG29544.11 22 AAQ98619.1 DNA helicase I [Escherichia colil 23 YP 00240109 conjugal transfer nickase/helicase TraI [Escherichia coli S881 24 YP 00233218 conjugal transfer nickase/helicase TraI [Escherichia coli 0127:H6 str. 25 CBG27820.1 DNA helicase I [Escherichia colil 26 YP 00171193 conjugal transfer nickase/helicase Tral [Escherichia colil 27 EGI88721.1 coniugative transfer relaxase protein Tral rShigella dysenteriae 155 28 ZP 06661276. coniugative transfer relaxase TraI [Escherichia coli B0881 29 YP 00148121 coniugal transfer nickase/helicase TraI [Escherichia coli APEC 01] 30 ZP 03070008. type IV secretion-like coniugative transfer relaxase protein Tral 31 YP 00191934 type IV secretion-like conjugative transfer relaxase protein Tral 32 YP 00129475 conjugal transfer nickase/helicase Tral [Escherichia colil 33 YP 00323254 conjugal transfer protein Tral [Escherichia coli 026:H1 1 str. 113681 34 YP 00323781 nickase [Escherichia coli O111:H- str. 111281 >dbilBAI39380.11 35 ADR29948.1 coniugative transfer relaxase protein Tral rEscherichia coli 083:H1 36 YP 00181654 conjugal transfer nickase/helicase TraI [Escherichia coli 15201 37 ZP 07104698. coniugative transfer relaxase protein Tral rEscherichia coli MS 119-71 38 ZP 06988741. conjugal transfer nickase/helicase TraI [Escherichia coli FVEC 13021 39 YP 00322510 putative Tral protein [Escherichia coli 0103:H2 str. 120091 40 AEE59988.1 IncF transfer nickase/helicase protein Tral rEscherichia coli 41 EFZ76933.1 coniugative transfer relaxase protein Tral rEscherichia coli RN587/11 42 YP 00487008 protein TraI [Escherichia colil >gblAEP03777.1| TraI [Escherichia 43 ZP 08376536, coniugative transfer relaxase protein Tral [Escherichia coli H5911 44 YP 538737.1 DNA helicase I [Escherichia coli UT1891 >reflZP 03035119.1 type 45 NP 052981.1 conjugal transfer nickase/helicase TraI [Plasmid RiO0 46 YP 00329403 conjugal transfer nickase/helicase [Escherichia coli ETEC H104071 47 YP 00240597 conjugal transfer protein Tral [Escherichia coli UMNO261 48 ZP 08344394. coniugative transfer relaxase protein Tral rEscherichia coli H7361 49 ZP 06648569. conjugal transfer nickase/helicase TraI [Escherichia coli FVEC14121 50 CBJ04377.1 DNA helicase I (TraI) (EC 3.6.1.-) [Escherichia coli ETEC H104071 51 YP 00332919 Tral rKlebsiella pneumoniael >gblACK98846.1| Tral rKlebsiella 52 ADN74088.1 conjugal transfer nickase/helicase TraI [Escherichia coli UM1461 53 YP 00351762 Tral rKlebsiella pneumoniael >gblADD6358 1.1 Tral rKlebsiella 54 YP 788091 1 conjugal transfer nickase/helicase Tral [Escherichia colil 55 P22706.1 RecName: Full=Multifunctional coniugation protein Tral Includes: 56 EGC0976 1.1 coniugative transfer relaxase TraI [Escherichia coli E 11671 57 EGX 11419. 1 conjugative transfer relaxase protein Tral rEscherichia coli 58 YP 00393764 protein TraI (DNA helicase I) [Escherichia colil >emblCBX35963.1| WO 2013/098562 PCT/GB2012/053274 36 59 EGW83369.1 conjugative transfer relaxase protein Tral rEscherichia coli 60 EGB59895.1 conjugative transfer relaxase Tral rEscherichia coli M8631 61 EFZ69441.1 conjugative transfer relaxase protein Tral rEscherichia coli 62 FU144479. I conjugative transfer relaxase protein Tral [Escherichia coli MS 63 YP 406350.1 oriT nicking and unwinding protein, fragment [Shigella bovdii 64 NP 085415.1 oriT nicking and unwinding protein, fragment [Shigella flexneri 65 EGW99614.1 conjugative transfer relaxase protein Tral [Escherichia coli 66 EGK37046.1 conjugative transfer relaxase protein Tral [Shigella flexneri K 67 EFW604341 IncF plasmid conjugative transfer DNA-nicking and unwinding 68 ZP 07678376.1 conjugative transfer relaxase protein Tral [Shigella dysenteriae 69 EFW49146. I IncF plasmid conjugative transfer DNA-nicking and unwinding 70 AEG39580.1 IncF plasmid conjugative transfer DNA-nicking and unwinding 71 NP 490592.1 conjugal transfer nickase/helicase Tral [Salmonella typhimurium 72 EFW56310.1 IncF plasmid conjugative transfer DNA-nicking and unwinding 73 YP 271768.1 conjugal transfer nickase/helicase Tral [Salmonella enterical 74 EGP2 1913.1 Protein trail [Escherichia coli PCN0331 75 EGR7091 1.1 conjugal transfer nickase/helicase Tral [Escherichia coli 76 EGB842 17.1 conjugative transfer relaxase protein Tral rEscherichia coli MS 77 ZP 07119795.1 conjugative transfer relaxase protein Tral rEscherichia coli MS 78 YP 313447.1 oriT nicking and unwinding protein, fragment [Shigella sonnei 79 EFU49447.1 conjugative transfer relaxase protein Tral rEscherichia coli MS 80 ZP 07197893.1 conjugative transfer relaxase protein Tral [Escherichia coli MS 81 ZP 08386420.1 conjugative transfer relaxase protein Tral rEscherichia coli 82 ZP 07246816.1 conjugative transfer relaxase protein Tral [Escherichia coli MS 83 YP 406123.1 oriT nicking and unwinding protein, fragment [Shigella 84 EFZ55097.1 conjugative transfer relaxase protein Tral [Shigella sonnei 53G] 85 YP 002213911. 1 conjugative transfer relaxase protein TraI [Salmonella enterica 86 YP 001716148.1 conjugative transfer oriT nicking-unwinding protein [Salmonella 87 EGE32684. 1 conjugative transfer oriT nicking-unwinding protein [Salmonella 88 EFZ60917.1 conjugative transfer relaxase protein Tral rEscherichia coli LT 89 EG340000.1 conjugative transfer relaxase Tral [Escherichia coli H1201 90 CA-164717 .1 putative DNA helicase I [uncultured bacterium] 91 ZP 08351681.1 conjugative transfer relaxase protein Tral rEscherichia coli 92 ZP 08351622.1 conjugative transfer relaxase protein Tral rEscherichia coli 93 YP 001338645.1 conjugal transfer nickase/helicase Tral [Klebsiella pneumoniae 94 EGK29I 111 conjugative transfer relaxase protein Tral [Shigella flexneri K 95 NP 858382.1 oriT nicking and unwinding protein [Shigella flexneri 2a str. 96 EGT71209.1 hypothetical protein C22711 5245 [Escherichia coli 0104:H4 97 YP 003560496.1 oriT nicking-unwinding rKlebsiella pneumoniael 98 YP 003517517.1 TraI [Klebsiella pneumoniael >reflYP 004249929.1 IncF 99 ZP 06015312.1 conjugal transfer nickase/helicase Tral FKlebsiella pneumoniae 100 EGJ9235 1.1 conjugative transfer relaxase protein Tral [Shigella flexneri K 101 YP 003754133.1 conjugal transfer nickase/helicase Tral [Klebsiella pneumoniael 102 ADA76996.1 OriT nicking and unwinding protein [Shigella flexneri 20020171 103 YP 001154759.1 conjugal transfer nickase/helicase Tral [Yersinia pestis Pestoides 104 YP 093987.1 conjugal transfer nickase/helicase Tral rYersinia pestis] 105 EGB74535. I conjugative transfer relaxase protein Tral [Escherichia coli MS 106 EGXI 5096.1 protein trail domain protein [Escherichia coli TX19991 107 ZP 07778521.1 tral domain protein [Escherichia coli 2362-751 >gblEFR1 8955.11 108 EGB44795.1 DNA helicase Tral [Escherichia coli H252] 109 ZP 07192950.1 putative conjugative transfer relaxase protein Tral [Escherichia 110 ZP 07692602.1 putative conjugative transfer relaxase protein Tral [Escherichia 111 ZP 07212721.1 putative conjugative transfer relaxase protein Tral [Escherichia 112 ZP 07122964.1 putative conjugative transfer relaxase protein Tral [Escherichia 113 ZP 06641688.1 conjugal transfer nickase/helicase Tral [Serratia odorifera DSM 114 ZP 07213112.1 putative coniugative transfer relaxase protein Tral [Escherichia 115 ZP 07125330.1 putative conjugative transfer relaxase protein Tral [Escherichia 116 AAA98086.1 helicase I [Plasmid F1 >gblAAC44187.11 TraI* [Escherichia 117 ZP 07692570 1 DNA helicase Tral [Escherichia coli MS 145-71 WO 2013/098562 PCT/GB2012/053274 37 118 EGB44794.1 conjugative relaxase domain-containing protein [Escherichia coli 119 CBA76609. 1 conjugal transfer nickase/helicase [Arsenophonus nasoniael 120 YP 004831100.1 conjugal transfer nickase/helicase TraI [Serratia marcescensi 121 AEJ60155.1 conjugal transfer protein Tral [Escherichia coli UMNF181 122 EGX24402.1 conjugative transfer relaxase protein Tral [Escherichia coli 123 AAM90727. I Tral [Salmonella enterica subsp. enterica serovar Typhi] 124 ZP 08374850.1 conjugative transfer relaxase protein Tral [Escherichia coli 125 CBY99022.1 conjugal transfer nickase/helicase TraI [Salmonella enterica 126 ZP 02347591 1 conjugative transfer relaxase protein TraI [Salmonella enterica 127 YP 001144265.1 Tral protein [Aeromonas salmonicida subsp. salmonicida A4491 128 YP 001144345.1 Tral protein [Aeromonas salmonicida subsp. salmonicida A4491 129 YP 003717559.1 putative Tral DNA helicase I [Escherichia coli ETEC 1392/751 130 ZP 08351623.1 protein Tral (DNA helicase I) [Escherichia coli M6051 131 ZP 06658914.1 conjugative transfer relaxase TraI [Escherichia coli B 1851 132 YP 002291232.1 Tral protein [Escherichia coli SE111 >dbilBAG80410.11 Tral 133 EFZ47384.1 protein tral domain protein rEscherichia coli E1280101 134 EGX19478.1 protein tral domain protein [Escherichia coli STEC S11911 135 YP 002527579 1 hypothetical protein p0103 123 [Escherichia colil 136 EGX15095.1 protein tral domain protein rEscherichia coli TX19991 137 EFZ47387.1 protein tral domain protein [Escherichia coli E1280101 138 ZP 06193648.1 protein Tral [Serratia odorifera 4Rx131 >gblEFA13747.11 protein 139 EGB39999.1 conjugative relaxase domain-containing protein [Escherichia coli 140 YP 003739388.1 conjugal transfer nickase/helicase [Erwinia billingiae Eb6611 141 YP 002539341.1 Tral [Escherichia colil >gb ACM18376.11 Tral [Escherichia colil 142 BAA31818.1 helicase I [Escherichia coli 0157:H7 str. Sakai] 143 ADA76995.1 OriT nicking and unwinding protein [Shigella flexneri 20020171 144 EFZ45075.1 protein tral domain protein [Escherichia coli E1280101 145 ZP 05940744.1 conjugal transfer protein Tral [Escherichia coli 0157:H7 str. 146 NP 858381.1 oriT nicking and unwinding protein [Shigella flexneri 2a str. 3011 147 Y1 325658.1 DNA helicase rEscherichia coli 0157:H7 EDL9331 148 EFZ04572.1 Tral protein [Salmonella enterica subsp. enterica serovar 149 YP 209287.1 Tral protein [Salmonella enterica subsp. enterica serovar 150 YP 001598090.1 hypothetical protein pOU7519 37 [Salmonella enterica subsp. 151 EGY27955.1 DNA helicase [Candidatus Regiella insecticola R5.151 152 ZP 02775703.2 protein Tral [Escherichia coli 0157:H7 str. EC41131 153 ZP 02802536.2 protein Tral (DNA helicase I) [Escherichia coli 0157:H7 str. 154 ZP 07779988.1 tral domain protein [Escherichia coli 2362-751 >gblEFR17488.11 155 ZP 08386421.1 protein Tral (DNA helicase I) [Escherichia coli H2991 156 1P4D A Chain A, F Factor Trai Relaxase Domain >pdb|1P4DIB Chain B, 157 2A01 A Chain A, F Factor Trai Relaxase Domain Bound To F Orit 158 EFZ47389 1 protein tral domain protein [Escherichia coli E1280101 159 YP 004118615.1 conjugative transfer relaxase protein Tral [Pantoea sp. At-9b] 160 YP 004119632.1 conjugative transfer relaxase protein Tral [Pantoea sp. At-9b] 161 EGW99739.1 protein tral domain protein [Escherichia coli G58-11 162 YP 004821631.1 conjugative transfer relaxase protein Tral [Enterobacter asburiae 163 YP 001165588.1 exonuclease V subunit alpha rEnterobacter sp. 6381 164 YP 003602677.1 conjugative transfer relaxase protein Tral [Enterobacter cloacae 165 YP 311531.1 hypothetical protein SSON 2674 [Shigella sonnei Ss0461 166 NP 073254.1 hypothetical protein pKDSC50 p30 [Salmonella enterica subs]. 167 2Q7T A Chain A, Crystal Structure Of The F Plasmid Trai Relaxase 168 EGB84260.1 conjugative relaxase domain protein [Escherichia coli MS 60-11 169 ZP 07119794. 1 conjugative relaxase domain protein [Escherichia coli MS 198-11 170 EGB74274.1 conjugative relaxase domain protein [Escherichia coli MS 57-21 171 ZP 04533197. 1 helicase I [Escherichia sp. 3 2 53FAAl >gblEEH89372.11 172 EFU49424.1 conjugative relaxase domain protein [Escherichia coli MS 153-11 173 EFZ60914.1 protein tral domain protein [Escherichia coli LT-681 174 CB1K86956.1 conjugative relaxase domain, TrwC/TraI family rEnterobacter 175 YP 313450.1 oriT nicking and unwinding protein, fragment [Shigella sonnei 176 EGP22056.1 hypothetical protein PPECC33 45560 [Escherichia coli PCN0331 WO 2013/098562 PCT/GB2012/053274 38 177 ZP 04533202.1 Tral protein rEscherichia sp. 3 2 53FAA] >gblEEH89366.1| Tral 178 ZP 07248320.1 DNA helicase Tral rEscherichia coli MS 146-11 >gblEFK88152.1| 179 EFZ45103.1 protein tral domain protein rEscherichia coli E1280101 180 YP 003502675.1 ATP-dependent exoDNAse (exonuclease V), alpha subunit 181 ZP 04533172.1 predicted protein rEscherichia sp. 3 2 53FAA] >gblEEH89397.1| 182 YP 406124.1 putative DNA helicase I, fragment [Shigella dysenteriae Sd1971 183 ZP 04533171.1 conserved hypothetical protein rEscherichia sp. 3 2 53FAA] 184 ZP 07192951.1 DNA helicase Tral [Escherichia coli MS 196-11 >gblEFI85454.1| 185 YP 001853797.1 putative conjugative transfer protein TraI [Vibrio tapetis] 186 YP 002261511.1 protein Tral (DNA helicase I) rAliivibrio salmonicida LF112381 187 NP 762615.1 conjugative transfer relaxase protein Tral [Vibrio vulnificus 188 YP 001393155.1 putative conjugative transfer protein Tral [Vibrio vulnificusi 189 EGB74536.1 DNA helicase Tral [Escherichia coli MS 57-21 190 NP 932226.1 putative conjugative transfer protein Tral [Vibrio vulnificus 191 YP 001557030.1 conjugative transfer relaxase protein Tral [Shewanella baltica 192 ADT96679.1 conjugative transfer relaxase protein Tral [Shewanella baltica 193 YP 001911094.1 Tral protein [Erwinia tasmaniensis Etl/991 >emb CA094972. 11 194 YP 002360275.1 conjugative transfer relaxase protein Tral [Shewanella baltica 195 AEG13610.1 conjugative transfer relaxase protein Tral [Shewanella baltica 196 EFZ04571.1 Tral protein [Salmonella enterica subsp. enterica serovar 197 ZP 01813760.1 putative conjugative transfer protein Tral [Vibrionales bacterium 198 YP 0 02 3 60 3 3 3.1 conjugative transfer relaxase protein Tral [Shewanella baltica 199 YP 001557007.1 conjugative transfer relaxase protein Tral [Shewanella baltica 200 YP 002364244.1 conjugative transfer relaxase protein Tral [Shewanella baltica 201 YP 209286.1 Tral protein [Salmonella enterica subsp. enterica serovar 202 YP 015476.1 DNA helicase Tral [Photobacterium profundum SS91 203 YP 001355447.1 conjugative transfer relaxase protein Tral [Shewanella baltica 204 EHC04201.1 conjugative transfer relaxase protein Tral [Shewanella baltica 205 ZP 06188936.1 conjugative transfer relaxase protein Tral [Legionella 206 ZP 06157867.1 IncF plasmid conjugative transfer DNA-nicking and unwinding 207 ZP 06157920.1 IncF plasmid conjugative transfer DNA-nicking and unwinding 208 ZP 08351743.1 protein Tral (DNA helicase I) [Escherichia coli M6051 209 ZP 08738328. 1 putative conjugative transfer protein Tral [Vibrio tubiashii ATCC 210 YP 003993727.1 incf plasmid conjugative transfer DNA-nicking and unwinding 211 ZP 06157811.1 IncF plasmid conjugative transfer DNA-nicking and unwinding 212 YP 003915110.1 putative conjugative transfer protein Tral [Legionella 213 YP 122194 1 hypothetical protein plpp0039 [Legionella pneumophila str. Parisl 214 ZP 07197892.1 conjugative relaxase domain protein [Escherichia coli MS 185-11 215 ZP 05884791.1 putative conjugative transfer protein Tral [Vibrio coralliilyticus 216 ZP 07222592.1 type-F conjugative transfer system pilin acetylase TraX 217 3 FLD A Chain A. Crystal Structure Of The Trai C-Terminal Domain 218 EGT71207.1 hypothetical protein C22711 5243 [Escherichia coli 0104:H4 str. 219 ZP 05440093. 1 conjugal transfer nickase/helicase TraI [Escherichia sp. 4 1 40B1 220 EGX2445 1.1 protein tral domain protein [Escherichia coli TX19991 221 EFZ55098.1 tral domain protein [Shigella sonnei 53G] 222 YP 003933505.1 DNA methylase [Pantoea vagans C9-11 >gblADO08159.11 223 AAA83930.1 tral [Plasmid F1 224 ADQ53972 1 putative conjugative transfer protein [Vibrio harveyil 225 ZP 07778522.1 tral domain protein [Escherichia coli 2362-751 >gblEFR18956.ll 226 ZP 07192561.1 conserved domain protein rEscherichia coli MS 196-11 227 AMV64824.1 oriT nicking and unwinding protein [Shigella flexneril 228 NP 085414.1 oriT nicking and unwinding protein, fragment [Shigella flexneri 229 ADA76994.1 OriT nicking and unwinding protein [Shigella flexneri 20020171 230 ZP 07197891.1 conjugative relaxase domain protein [Escherichia coli MS 185-11 231 EFU452 0.1 conjugative relaxase domain protein [Escherichia coli MS 110-31 232 ZP 07222591.1 conjugative relaxase domain protein [Escherichia coli MS 78-11 233 YP 406349 1 oriT nicking and unwinding protein, fragment [Shigella boydii 234 YP 406122.1 oriT nicking and unwinding protein, fragment [Shigella 235 EFW49145.1 conjugal transfer nickase/helicase TraI [Shigella dysenteriae CDC WO 2013/098562 PCT/GB2012/053274 39 236 ZP 07246817.1 conjugative relaxase domain protein [Escherichia coli MS 146-11 237 YP 004250852.1 putative protein tral (DNA helicase I) rVibrio nigripulchritudol 238 EFZ60915.1 protein tral domain protein rEscherichia coli LT-681 239 YP 617529,1 TrwC protein [Sohingooyxis alaskensis RB22561 240 ZP 01813650.1 ATP-dependent exoDNAse, alpha subunit [Vibrionales bacterium 241 ZP 01813651.1 ATP-dependent exoDNAse, alpha subunit [Vibrionales bacterium 242 N1 052850.1 hypothetical protein QpDV p09 [Coxiella burnetii] 243 CAA75825.1 hypothetical protein [Coxiella burnetiil 244 YP 002302593.1 DNA helicase rCoxiella burnetii CbuK 01541 >gblACJ21266.11 245 YP 003502676.1 Tral [Escherichia coli 055:H7 str. CB96151 >gblADD59692.1| 246 YP 001649308.1 putative protein trail rCoxiella burnetii 'MSU Goat Q177] 247 YP 001423428.2 DNA helicase rCoxiella burnetii Dugway 5J108-1 111 248 YP 001595803.1 putative protein trail rCoxiella burnetii RSA 3311 249 NP 052342.1 hypothetical protein QpH1 p10 rCoxiella burnetiil 250 ZP 01863208.1 hvothetical protein ED21 17597 FErythrobacter sp. SD-211 251 ZP 08645753. 1 conjugal transfer protein TraA [Acetobacter tropicalis NBRC 252 YP 497456.1 TrwC protein FNovosphingobium aromaticivorans DSM 124441 253 AAL78346.1 DNA helicase I [Escherichia colil 254 EFW6043 5.1 conjugal transfer nickase/helicase Tral [Shigella flexneri CDC 255 YP 001235537.1 exonuclease V subunit alpha rAcidiphilium cryptum JF-51 256 ZP 05038212.1 hypothetical protein S7335 4654 [Synechococcus sp. PCC 73351 257 ZP 08897263.1 exonuclease V subunit alpha rGluconacetobacter oboediens 258 N1 049139.1 DNA helicase [Novosphingobium aromaticivoransl 259 YP 004390567.1 coniugative relaxase domain-containing protein [Alicycliphilus 260 ZP 07678069.1 TrwC protein [Ralstonia sp. 5 7 47FAAl >reflZP 08896172.1 261 YP 974028.1 TrwC protein [Acidovorax so. JS421 >gblABM44293.11 TrwC 262 YP 001869867.1 mobilization protein Tral-like protein [Nostoc punctiforme PCC 263 YP 718086.1 DNA helicase rSphingomonas so. KAll >dbilBAF03374.1| DNA 264 YP 004534199.1 TrwC protein [Novosphingobium sp. PP1Y1 >emblCCA92381.1| 265 ZP 08701842.1 TrwC protein [Citromicrobium sp. JLT13631 266 YP 003602886.1 hypothetical protein ECL B 116 rEnterobacter cloacae subsp. 267 ZP 06861556.1 TrwC protein [Citromicrobium bathyomarinum JL3541 268 NP 542915.1 putative TraC protein [Pseudomonas putidal >emb|CAC86855.1| 269 YP 457045.1 TrwC protein [Erythrobacter litoralis HTCC25941 270 YP 122325.1 hypothetical protein P1100032 [Legionella pneumophila str. Lens] 271 YP 004030608.1 DNA helicase I Tral [Burkholderia rhizoxinica HKI 4541 272 YP 457732 1 TrwC protein [Erythrobacter litoralis HTCC25941 273 ZP 01039301.1 TrwC protein [Erythrobacter sp. NA-P11 >gblEAQ29772.11 TrwC 274 YP 737083.1 TrwC protein [Shewanella so. MR-71 >gblABI42026.1| TrwC 275 ZP 05040239.1 hypothetical protein S7335 1207 [Synechococcus sp. PCC 73351 276 A AP57243.1 putative TraC protein [Pseudomonas putidal 277 NP 942625.1 TrwC rXanthomonas citril >reflZP 06705283.11 Tral protein 278 1OMH A Chain A, Conjugative Relaxase Trwc In Complex With Orit Dna. 279 ZP 06485934.1 TrwC protein [Xanthomonas campestris pv. vasculorum 280 1()SB A Chain A, Conjugative Relaxase Trwc In Complex With Orit Dna. 281 ZP 08207479.1 coniugative relaxase region-like protein [Novosphingobium 282 2CDM A Chain A, The Structure Of Trwc Complexed With A 27-Mer Dna 283 ZP 01731779.1 hypothetical protein CY0110 01035 [Cyanothece sp. CCYO1101 284 NP 644759.1 TrwC protein [Xanthomonas axonopodis pv. citri str. 3061 285 ZP 06732867.1 Tral protein rXanthomonas fuscans subsp. aurantifolii str. ICPB 286 YP 361538.1 putative Tral protein rXanthomonas campestris pv. vesicatoria str. 287 CBJ36 129.1 putative traC, type IV secretion system rRalstonia solanacearum 288 YP 001260099.1 conjugative relaxase region-like protein rSphingomonas wittichii 289 YP 001451611.1 putative type IV conjugative transfer system coupling protein 290 ZP 08208941.1 coniugative relaxase region-like protein [Novosphingobium 291 AAO84912.1 DNA helicase I [Escherichia colil 292 Y1 002515847.1 DNA relaxase/coniugal transfer nickase-helicase TrwC 293 YP 001260037.1 coniugative relaxase region-like protein rSphingomonas wittichii 294 ZP 04629294. 1 hypothetical protein yberc0001 36240 rYersinia bercovieri ATCC WO 2013/098562 PCT/GB2012/053274 40 295 CAZ15897.1 probable conjugal transfer protein [Xanthomonas albilineans] 296 YP 315578.1 TrwC protein [Thiobacillus denitrificans ATCC 252591 297 ZP 01770026.1 TrwC protein [Burkholderia pseudomallei 3051 298 YP 001869963.1 exonuclease V subunit alpha rNostoc punctiforme PCC 731021 299 ZP 08210392.1 TrwC protein [Novosphingobium nitrogenifigens DSM 193701 300 YP 001692976.1 mobilization protein Tral [Yersinia enterocolitical 301 YP 003455306.1 conjugative transfer protein Tral [Legionella longbeachae 302 YP 745335,1 tral protein (DNA helicase I) rGranulibacter bethesdensis 303 YP 001911166.1 TrwC rSalmonella enterica subsp. enterica serovar Dublin 304 YPl 001874877.1 mobilisation protein rProvidencia rettgeril >emblCAQ48354.11 305 YP 001552064.1 trwC protein [Salmonella enterica subsp. enterica serovar 306 CAA44853.2 TrwC rEscherichia coli K-121 307 YP 096090.1 hypothetical protein lg2077 rLegionella pneumophila subsp. 308 YP 534815.1 putative plasmid transfer protein TraC rPseudomonas putidal 309 FAA00039.1 TPA: TrwC protein [Escherichia colil 310 NP 863125.1 putative TraC protein [Pseudomonas putidal 311 ZP 04868849. 1 conserved hypothetical protein rStaphvlococcus aureus subsp. 312 ZP 01304707.1 TrwC protein [Sphingomonas sp. SKA581 >gblEAT07464.11 313 ZP 05040124.1 TrwC relaxase family rSynechococcus sp. PCC 73351 314 YP 001798665.1 putative TrwC/TraI protein [Cyanothece sp. ATCC 511421 315 YP 002235496.1 putative coniugative transfer protein [Burkholderia 316 CAZ15872.1 probable mobilization protein train [Xanthomonas albilineans] 317 YP 840564.1 TrwC protein [Burkholderia cenocepacia H24241 318 ZP 08207332.1 TrwC protein [Novosphingobium nitrogenifigens DSM 193701 319 YPl 001966297.1 Tral [Pseudomonas so. CT141 >gblABA25997.11 Tral 320 3L6T A Chain A, Crystal Structure Of An N-Terminal Mutant Of The 321 YP 001736290.1 DNA helicase, TrwC and Tral like protein [Synechococcus so. 322 3L57 A Chain A, Crystal Structure Of The Plasmid Pcul Trai Relaxase 323 ZP 04532999.1 F pilin acetylation protein rEscherichia sp. 3 2 53FAAl 324 CAA40677.1 DNA helicase I [Escherichia colil 325 NP 478459.1 hypothetical protein alr8034 [Nostoc sp. PCC 71201 326 YP 001893556.1 coniugative relaxase domain protein [Burkholderia 327 YP 001033863.1 hypothetical protein RSP 3904 [Rhodobacter sphaeroides 328 ZP 06064648.1 TrwC protein [Acinetobacter iohnsonii SH0461 329 YP 003829308.1 nickase/helicase [Escherichia colil >gblADL14202.1| Tral 330 YP 002332893.1 conjugal transfer protein [Klebsiella pneumoniael 331 YP 002286896.1 Tral [Klebsiella pneumoniael >gblACI63157.11 Tral 332 YP 003813077.1 Tral [Klebsiella pneumoniael >gblADG84846.11 Tral 333 YP 002286953.1 TraI [Klebsiella pneumoniael >reflYP 003675776.11 Tral 334 YP 001096334.1 hypothetical protein pLEW517 p09 rEscherichia colil 335 YP 724504.1 hypothetical protein pMUR050 047 [Escherichia colil 336 NP 511201.1 hvothetical protein R46 023 rIncN plasmid R461 337 AD1H130046.1 conjugal transfer protein [Escherichia coli 025b:H4-ST131 str. 338 YP 002913254.1 TrwC protein [Burkholderia glumae BGR11 >gblACR32934.11 339 YP 004362462.1 TrwC protein [Burkholderia gladioli BSR31 >gblAEA65432.1| 340 ZP 02468056.1 TrwC protein [Burkholderia thailandensis MSMB431 341 YP 001840913.1 TrwC protein [Acinetobacter baumannii ACICUl 342 ZP 07239267.1 TrwC protein [Acinetobacter baumannii AB0591 343 YP 003853339.1 TrwC protein [Parvularcula bermudensis HTCC25031 344 ZP 07237891. 1 TrwC protein [Acinetobacter baumannii AB0581 345 YP 002491522.1 conjugative relaxase domain-containing protein 346 YP 002907678.1 TrwC protein [Burkholderia glumae BGR11 >gblACR32827.11 347 YT 004350971.1 TrwC protein [Burkholderia gladioli BSR31 >gblAEA65648.11 348 ZP 02834825.2 protein TraD rSalmonella enterica subsp. enterica serovar 349 ADX05370.1 TrwC protein [Acinetobacter baumannii 1656-21 350 YPl 003552078.1 TrwC protein [Candidatus Puniceispirillum marinum 351 EGB59894.1 tral protein [Escherichia coli M8631 352 YP 001522461.1 hypothetical protein AMI F0157 [Acaryochloris marina 353 ZP 05738733.1 protein Tral [Silicibacter sp. TrichCH4B1 >gblEEW61008.1| WO 2013/098562 PCT/GB2012/053274 41 354 AEM77047.1 putative conjugative relaxase [Escherichia colil 355 EHC7 1302.1 IncW plasmid conjugative relaxase protein TrwC [Salmonella 356 YP 004765041.1 Tral [Escherichia colil >gblAEK64833.11 Tral [Escherichia colil 357 YP 004553102.1 conjugative relaxase domain-containing protein [Sphingobium 358 NP 073253.1 hypothetical protein pKDSC50 p29 rSalmonella enterica subsp. 359 YP 004535774.1 DNA relaxase/coniugal transfer nickase-helicase TrwC 360 AEA76430.1 VirD2 [Klebsiella pneumoniael 361 YP 001806422.1 putative TrwC/TraI protein [Cyanothece sp. ATCC 511421 362 ZP 08138981.1 TrwC protein [Pseudomonas sp. TJI-511 >gblEGB99721.1| TrwC 363 YP 394134.1 exonuclease V subunit alpha [Sulfurimonas denitrificans DSM 364 EG061142.1 conjugative relaxase domain protein [Acidithiobacillus sp. GGI 365 ZP 06732944.1 Tral protein rXanthomonas fuscans subsp. aurantifolii str. ICPB 366 YP 004218965.1 conjugative relaxase domain protein [Acidobacterium sp. 367 YP 001941994.1 relaxase [Burkholderia multivorans ATCC 176161 368 EDZ39520. 1 Protein of unknown function rLeptospirillum sp. Group II'5-way 369 YP 004415459.1 TrwC protein [Pusillimonas sp. T7-71 >gb AEC18835.11 TrwC 370 YP 003545248.1 traI/trwC-like protein [Sohingobium iaponicum UT26S] 371 YP 004184501.1 conjugative relaxase domain-containing protein [Terriglobus 372 EGD06685.1 relaxase [Burkholderia sp. TJ1491 373 ADQ53945.1 putative conjugative transfer protein [Vibrio harveyil 374 YP 004089509.1 conjugative relaxase domain protein [Asticcacaulis excentricus 375 YP 004183694.1 conjugative relaxase domain-containing protein [Terriglobus 376 YP 003900289.1 conjugative relaxase domain-containing protein rCvanothece sp. 377 EDZ40407.1 Putative mobilization protein TraA rLeptospirillum sp. Group II 378 YP 004534918.1 TrwC protein [Novosphingobium sp. PP1Yl >emblCCA93 100.1 379 YP 004210530.1 conjugative relaxase domain protein [Acidobacterium sp. 380 YP 003642130.1 conjugative relaxase domain protein [Thiomonas intermedia K121 381 YP 004277247.1 putative relaxase TrwC [Acidiphilium multivorum AIU3011 382 NP 857772.1 DNA helicase I [Yersinia pestis KIM1 >gblAAC62598.1| DNA 383 EGB74534.1 hypothetical protein HIMPREF9532 05052 [Escherichia coli MS 384 ZP 08138968.1 putative TraC protein [Pseudomonas sp. TJI-511 385 YP 002756187.1 conjugative relaxase domain protein [Acidobacterium capsulatum 386 ZP 07392869 1 conjugative relaxase domain protein [Shewanella baltica OS1831 387 YP 004210680.1 conjugative relaxase domain protein [Acidobacterium sp. 388 EAY56629.1 probable TrwC protein rLeptospirillum rubaruml 389 YP 068423.1 hypothetical protein pYVOOO [Yersinia pseudotuberculosis IP 390 NP 995413.1 hvothetical protein YP pCD97 [Yersinia pestis biovar Microtus 391 ZP 08634947.1 Coniugative relaxase domain protein rAcidiphilium sp. PM] 392 ZP 01301850.1 hypothetical protein SKA58 02210 rSphingomonas sp. SKA581 393 YP 002754293.1 conjugative relaxase domain protein [Acidobacterium capsulatum 394 YP 001818827.1 conjugative relaxase domain-containing protein [Opitutus terrae 395 EGT71208.1 hypothetical protein C22711 5244 rEscherichia coli 0104:H4 str. 396 YP 001522273.1 hypothetical protein AMI E0190 [Acaryochloris marina 397 YP 003891048.1 conjugative relaxase domain protein [Cyanothece sp. PCC 7822] 398 YP 001521867.1 hypothetical protein AMI D0057 rAcaryochloris marina 399 YP 004748378.1 TraI protein rAcidithiobacillus caldus SM-I] >gblAEK57678.11 400 YP 002380579.1 relaxase [Cyanothece sp. PCC 74241 >gb ACK74122.11 401 ZP 08634902.1 Coniugative relaxase domain protein rAcidiphilium sp. PM] 402 ZP 05738878.1 Tral [Silicibacter sp. TrichCH4B] >gblEEW61153.1| Tral 403 YP 001821352.1 conjugative relaxase domain-containing protein ropitutus terrae 404 ZP 02733385.1 TrwC protein [Gemmata obscuriglobus UQM 22461 405 YP 004183160.1 conjugative relaxase domain-containing protein [Terriglobus 406 YP 001522155.1 TrwC protein, putative rAcarvochloris marina MBIC 110171 407 YP 002478348.1 conjugative relaxase domain protein [Cyanothece sp. PCC 74251 408 YP 002756241.1 conjugative relaxase domain protein [Acidobacterium capsulatum 409 YP 001521036.1 hypothetical protein AMI A0387 rAcaryochloris marina 410 YP 004416953.1 TrwC protein [Pusillimonas sp. T7-71 >gblAEC20329.1| TrwC 411 YP 001521806.1 hypothetical protein AMi C0379 rAcaryochloris marina 412 YP 001357151.1 hvothetical protein NIS 1688 rNitratiruptor so. SB155-21 WO 2013/098562 PCT/GB2012/053274 42 413 ZP 07030639. 1 conjugative relaxase domain protein [Acidobacterium sp. 414 YP 530542.1 putative ATP-dependent exoDNAse (exonuclease V) subunit 415 NP 052442 1 hypothetical protein pYVe227 p65 [Yersinia enterocolitical 416 YP 004783051.1 conjugative relaxase domain-containing protein [Acidithiobacillus 417 YP 003262832.1 relaxase [Halothiobacillus neapolitanus c2] >gblACX95785.11 418 AD053973.1 putative coniugative transfer protein [Vibrio harveyil 419 EDZ37984.1 Coniugal transfer protein, TraA rLeptospirillum sp. Group II'5 420 YP 001522591.1 hypothetical protein AMI G0097 rAcarvochloris marina 421 EAY56417.1 putative conjugal transfer protein (TraA) rLeptospirillum 422 YP 459829 1 hypothetical protein ELI 14700 [Erythrobacter litoralis 423 YP 001818081.1 conjugative relaxase domain-containing protein [Opitutus terrae 424 ZP 07745472.1 conjugative relaxase domain protein [Mucilaginibacter paludis 425 CBA73 957 1 conjugal transfer nickase/helicase Tral [Arsenophonus nasoniael 426 YP 002248140.1 hypothetical protein THEYE A0292 rThermodesulfovibrio 427 ZP 06641691 1 conserved hypothetical protein rSerratia odorifera DSM 45821 428 ZIP 05056614.1 TrwC relaxase family rVerrucomicrobiae bacterium DG12351 429 ZP 03723740.1 conjugative relaxase domain protein [Opitutaceae bacterium 430 YP 004210579.1 conjugative relaxase domain protein [Acidobacterium sp. 431 YPl 001522671.1 hypothetical protein AMI H0004 [Acarvochloris marina 432 YP 001573657.1 conjugative relaxase domain-containing protein rBurkholderia 433 EDZ39038.1 Coniugal protein, TraA [Leptospirillum sp. Group II'5-way CG'l 434 YP 001632380.1 conjugal transfer protein [Bordetella petrii DSM 128041 435 ZP 06242489.1 conjugative relaxase domain protein [Victivallis vadensis ATCC 436 EDZ37956.1 Coniugal protein, TraA [Leptospirillum sp. Group II'5-way CG'l 437 YP 004488214.1 conjugative relaxase domain-containing protein [Delftia sp. Csl 438 ZP 00208504.1 COG0507: ATP-dependent exoDNAse (exonuclease V), alpha 439 ACJ47794.1 Tral [Klebsiella pneumoniael 440 ZP 02730551.1 TrwC protein [Gemmata obscuriglobus UQM 22461 441 ZP 06244759.1 TrwC relaxase [Victivallis vadensis ATCC BAA-5481 442 YP 003022160.1 relaxase [Geobacter sp. M211 >gblACT18402.11 coniugative 443 YP 001521304.1 hypothetical protein AMI B0272 rAcaryochloris marina 444 ZP 01091846.1 hypothetical protein DSM3645 02833 [Blastopirellula marina 445 CAZ88117.1 putative ATP-dependent exoDNAse (exonuclease V), alpha 446 YP 004718365.1 conjugative relaxase domain-containing protein [Sulfobacillus 447 YP 002553030.1 conjugative relaxase domain-containing protein [Acidovorax 448 YP 003386820.1 conjugative relaxase domain-containing protein rSvirosoma 449 YP 001119893.1 exonuclease V subunit alpha [Burkholderia vietnamiensis G41 450 YP 315444.1 putative ATP-dependent exoDNAse (exonuclease V) subunit 451 YP 003071370.1 hypothetical protein p2METDI0024 rMethylobacterium 452 YP 003125939.1 conjugative relaxase [Chitinophaga pinensis DSM 25881 453 ZP 08495729 1 TrwC relaxase [Microcoleus vaginatus FGP-21 >gblEGK83455.1 454 EFZ53417.1 tral domain protein [Shigella sonnei 53G1 455 EGR709O10 I conjugal transfer nickase/helicase Tral [Escherichia coli 0104:H4 456 YP 002912178.1 ATP-dependent exoDNAse (exonuclease V) subunit alpha 457 ZP 01089566.1 hypothetical protein DSM3645 27912 [Blastopirellula marina 458 YP 002753784.1 DNA helicase domain protein rAcidobacterium capsulatum ATCC 459 EFZ60916.1 protein tral domain protein [Escherichia coli LT-681 460 ZP 08262009.1 protein tral [Asticcacaulis biprosthecum C191 >gblEGF93811.11 461 AE111045.1 TrwC relaxase [[Cellvibriol gilvus ATCC 131271 462 ZP 05040209.1 hypothetical protein S7335 1177 [Synechococcus sp. PCC 73351 463 YP 001840830.1 ATP-dependent exoDNAse (exonuclease V) [Mycobacterium 464 YP 001700713.1 TraA/ATP-dependent exoDNAse/relaxase [Mycobacterium 465 CAC86586.1 conjugal transfer protein [Agrobacterium tumefaciensl 466 NP 355808.2 conjugation protein [Agrobacterium tumefaciens str. C581 467 YP 001120496.1 hypothetical protein Bcepl1808 2669 [Burkholderia vietnamiensis 468 EGW76304.1 protein tral domain protein rEscherichia coli STEC B2F11 469 YP 002979543.1 Ti-type conjugative transfer relaxase TraA [Rhizobium 470 EB-144041.1 TrwC relaxase [Mycobacterium rhodesiae JS601 471 YP 002984810.1 Ti-type conjugative transfer relaxase TraA [Rhizobium WO 2013/098562 PCT/GB2012/053274 43 472 ZP 068418350.1 ATP-dependent exoDNAse (exonuclease V) rMvcobacterium 473 YP 001972793.1 putative coniugal transfer protein TraA [Stenotroohomonas 474 ZP 06760230. 1 utative coniugative relaxase domain protein [Veillonella sp. 475 YP 001840914.1 TrwC protein rAcinetobacter baumannii ACICU1 476 YP 003311407.1 TrwC relaxase rVeillonella oarvula DSM 20081 477 ZP 08208016.1 TrwC protein rNovosphingobium nitrogenifigens DSM 193701 478 XP 003342708.1 hvothetical protein SMAC 10304 rSordaria macrospora k-hell] 479 YP 004074482.1 TrwC relaxase rMycobacterium so. Spvr11 >blADU02001.11 480 YP 935511 1 exonuclease V subunit alpha rMycobacterium so. KMS1 481 YP 004100308.1 TrwC relaxase rntrasporangium calvum DSM 430431 482 YP 003326911.1 TrwC relaxase rXvlanimonas cellulosilvtica DSM 158941 483 YP 001136860.1 exonuclease V subunit alpha rMycobacterium silvum PYR 484 YP 001776789.1 coniugative relaxase domain-containing protein 485 NP 862296.1 transfer protein homolog TraA rCorvnebacterium glutamicum] 486 YP 001851874.1 ATP-dependent exoDNAse (exonuclease V) rMycobacterium 487 AAS20144.1 TraA-like protein [Arthrobacter aurescens] 488 YP 949993.1 putative TraA-like protein rArthrobacter aurescens TC1] 489 YP 004271377.1 TrwC relaxase rPlanctomvces brasiliensis DSM 53051 490 YP 001243088.1 putative ATP-dependent exoDNAse rBradvrhizobium so. 491 YP 7713091 putative conjugal transfer protein TraA [Rhizobium 492 ZP 02730298.1 TrwC protein rGemmata obscuriglobus UOM 22461 493 EG061143.1 coniugative relaxase domain protein rAcidithiobacillus sp. GGI 494 YP 002978744.1 Ti-tve coniusative transfer relaxase TraA rRhizobium 495 YP 002973152.1 Ti-tve coniugative transfer relaxase TraA [Rhizobium 496 ZP 06846967.1 ATP-dependent exoDNAse (exonuclease V) rMycobacterium 497 YP 003377696.1 TraA rCorvnebacterium alutamicum] >dbi BA16603 1. 1 TraA, 498 ZP 06846356.1 Ti-tve coniugative transfer relaxase TraA rBurkholderia so. 499 YP 001136826.1 exonuclease V subunit alpha rMvcobacterium silvum PYR 500 YP 949954.1 putative TraA-like coniugal transfer protein [Arthrobacter The Tral helicase or Tral subgroup helicase is more preferably one of the helicases shown in Table 7 below or a variant thereof The Tral helicase or Tral subgroup helicase more 5 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 thereof. Table 7 - More preferred Tral helicase and Tral subgroup helicases RecD- RecD- Mob F SEQ Name % Identity like like or Q ID Strain NCBI ref to TraI Eco motif I motif V motif III NO (SEQ ID (SEQ ID (SEQ ID NO:) NO:) NO:) NCBI GYAGV YAITA HDTSR Reference GKT HGAQG DQEPQ 61 Tral Escherichi Sequence: (62) (63) LHTH Eco a coli NP_061483.1 (64) Genbank AAQ98619.1 Citromicro NCBI GIAGA YALNV HDTNR 65 TrwC bium Reference 15% GKS HMAQG NQEPN Cba bathyomar Sequence: (66) (67) LHFH inum ZP_06861556. (68) WO 2013/098562 PCT/GB2012/053274 44 JL354 1 Halothiob NCBI GAAGA YCITIH HEDAR TrwC acillus Reference GKT RSQG TVDDI 69 Hne neapolitan Sequence: 11.5% (70) (71) ADPQL us c2 YP_00326283 HTH 2.1 (72) Erythroba NCBI GIAGA YALNA I-DTNR TrwC eter Reference GKS HMAQG NQEPN 73 Eli litoralis Sequence: 16% (66) (67) LFH HTCC259 YP_457045.1 (68) 4 GFAGT YATTV HETSRE 74 TrwC E.coli CAA44853.2 11.998% GKS HSSQG RDPQL Eco (75) (76) HTH (77) Agrobacte GRAGA YATTIH GMVAD TraA rium GKT KSQG WVYH 78 Atu tumefacie AAC17212.1 12.68% (79) (80) DNPGN ns C58 PHIH (81) Sulfobacill GAAGT YASTA HSTSR 82 TrwC us YP_00471836 9.487% GKT HKSQG AQDPH Sac acidophilu 5.1 (83) (84) LHSH s TPY (85) Acidithiob GHAGA YAGTT HASSR 86 TrwC acillus YP_00478305 10.247% GKT HRNQG EQDPQI Afe ferrivorans 1.1 (87) (88) HSH SS3 (89) GLAGT YAVTS HDTAR TrwC Terriglobu YP00418450 GKT HSSQG PVNGY 90 Tsa s saanensis 1 14.689% (91) (92) AAPQL SP1PR4 HTH (93) Microluna GPAGA YAITA HYDSR 94 TrwC tus YP_00457419 11.467% GKT HRAQG AGDPQ Mph phosphovo 6.1 (95) (96) LHTH rus NM-1 (97) Thermode GWAG YAVTA HLCGR TrwC sulfovibrio YP_00224814 VGKT DHMQG LDPQIH 98 Tye yellowston 0.1 8.487% (99) (100) NH ii DSM (101) 11347 Rhodother GVAGA YALTID HMTSG TrwC mus YP_00482654 GKT SAQG DGSPH 102 Rma mannus 2.1 11.909% (103) (104) LHVH SGO.5JP1 (105) 7-172 Oceanicau GYAGT YAATI GMIAD 106 TraA lis ZP_00953568. 12.099% GKS HKAQG LVNVH Oma alexandrii 1 (107) (108) WDIGE HTCC263 DGKAK WO 2013/098562 PCT/GB2012/053274 45 3 PHAH (109) Citromicro GIAGA YALNA HDTNR 110 TrwC bium sp. ZP_08701842. 12.371% GKS HMAQG NQEPN Cjlt JLT1363 1 (66) (67) LHFH (111) Erythroba GIAGA YALNA HDTNR 112 TrwC eter sp. ZP_01863208. 12.907% GKS HMAQG NQEPN Esd SD-21 1 (66) (67) LHFH (111) Erythroba GIAGA YALNA HDTNR 113 TrwC eter sp. ZP_01039301. 12.969% GKS HMAQG NQEPN Enap NAP1 1 (66) (67) LHFH (111) Novosphin GVAGA YALNA HDTNR TrwC gobium ZP09190449 GKS HMAQG NQEPN 114 Npe pentaroma - 12.765% (115) (67) ATFH tivorans (116) US6-1 Novosphin GGAGV YAINV HDVSR TrwC gobium ZP_08210392. GKS HIAQG NNDPQ 117 Nni nitrogenif 11.82% (118) (119) LHVH gens DSM (120) 19370 Sphingom GIAGA YALNM HDTSR 121 TrwC onas YP_00126009 13.945% GKS HMAQG ALDPQ Swi wittichii 9.1 (66) (122) GHIH RW1 (123) Sphingom GVAGA YALNA HDTSR 124 TrwC onas sp. YP718086.1 14.119% GKS HMAQG ALDPQ Ska KA1 (115) (67) GHIH (123) Candidatu GRAGT FASTA HEASR s GKT HGAQG NLDPQ TrwC Puniceispi YP00355207 (126) (127) LHSH 125 Pma rillum 8.1 12.91% (128) mannum IMCC132 2 Parvularcu GYAGT YAMTS HDISRD la GKT HAAQG KDPQL 129 TrwC bermudens YP_00385333 13.141% (130) (131) HTH Pbe is 9.1 (132) HTCC250 3 GLAGT YAQTV HNTSR 133 TrwC Acidovora YP_974028.1 12.52% GKT HASQG DLDPQ Ajs x sp. JS42 (91) (134) THTH (135) 136 TrwC Caulobact YP_00251584 13.137% GFAGT YVQTA HETSR Ccr er 7.1 AKT FAAQG AQDPQ WO 2013/098562 PCT/GB2012/053274 46 crescentus (137) (138) LHTH NA1OOO (139) Sphingopy GYAGT YVDTA HGTSR 140 TrwC xis YP617529.1 14.193% AKT FAAQG AQDPQ Sal alaskensis (141) (142) LHTH RB2256 (143) Acetobact GYAGT YASTA HGTSR TrwC er ZP_08645753 AKT FAAQG ALDPQ 144 Atr tropicahs - 13.171% (141 (145) LHSH NBRC (146) 101654 Acidobact GSAGS YAVTS HDTAR TrwC erium YP_00275624 GKT YSAQG PVGGY 147 Aca capsulatu 1.1 11.338% (148) (149) AAPQL HTH m (150) GLAGT YAVTS HDTAR TrwC Granulicel YP_00421896 GKT HSSQG PVNGY 151 Gtu la 5.1 14.12% (91) (92) AAPQL tundricola HTH (93) Burkholde GEAGT YAHTS HETNR TrwC ria YP 00194199 GKT YKEQG ENEPQ 152 Bmu multivoran 4.1 13.347% (153) (154) LHNH s ATCC (155) 17616 Legionella GYAGV YVLTN QPSSRA 156 TrwC longbeach YP_00345530 11.612% AKT YKVQG NDPAL Llo ae 6.1 (157) (158) HTH NSW150 (159) Asticcacau GSAGT YSLTA HSMSR 160 TrwC lis YP_00408950 11.86% GKT NRAQG AGDPE Aex excentricu 9.1 (161) (162) MHNH s CB 48 (163) Methyloba AGAGT YAGTV HYTTR TrwC eterium YP00177678 GKT YAAQG EGDPNI 164 Mra radiotolera 11.565% (165) (166) HTH ns JCM (167) 2831 Mycobact APAGA YAVTV HETSR erium GKT HAAQG AGDPH 168 TrwC parascrofu ZP06848350 11.394% (169) (170) LHTH Mpa laceum (171) ATCC BAA-614 SEQ ID NOs: 78 and 106 comprise a MobQ motif III, whereas the other sequences in Table 7 comprise a MobF motif III.
WO 2013/098562 PCT/GB2012/053274 47 The Tral helicase preferably comprises the sequence shown in SEQ ID NO: 61 or a variant thereof. A variant of a RecD helicase is an enzyme that has an amino acid sequence which varies from that of the wild-type helicase and which retains polynucleotide binding activity. In 5 particular, a variant of any one 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 is an enzyme that has an amino acid sequence which varies from that of any one 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, 10 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 and which retains polynucleotide binding activity. A variant of SEQ ID NO: 18 or 61 is an enzyme that has an amino acid sequence which varies from that of SEQ ID NO: 18 or 61 and which retains polynucleotide binding activity. The variant retains helicase activity. Methods for measuring helicase activity are known in the art. Helicase activity can also be measured as described in the 15 Examples. The variant must work in at least one of the two modes discussed below. Preferably, the variant works in both modes. The variant may include modifications that facilitate handling of the polynucleotide encoding the helicase and/or facilitate its activity at high salt concentrations and/or room temperature. Variants typically differ from the wild-type helicase in regions outside of the motifs discussed above. However, variants may include modifications 20 within these motif(s). Over the entire length of the amino acid sequence of any one 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 as SEQ ID NO: 18 or 61, a variant will preferably be at least 10% homologous to that 25 sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 65, 30 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 as SEQ ID NO: 18 or 61, over the entire sequence. There may be at least 70%, for example at least 80%, at least 85%, at least 90% or at least 95%, amino acid identity over a stretch of 150 or more, for example 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more, contiguous amino acids ("hard homology").
WO 2013/098562 PCT/GB2012/053274 48 Homology is determined as described above. The variant may differ from the wild-type sequence in any of the ways discussed above with reference to SEQ ID NOs: 2 and 4. 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, 5 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168. Such fragments retain polynucleotide binding activity. Fragments may 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 800, at least about 900 or at least about 1000 amino acids in length. The length of the fragment will depend on the length of the wild-type sequence. As discussed in 10 more detail below, fragments preferably comprise the RecD-like motif I and/or the RecD-like motif V of the relevant wild-type sequence. As discussed above, Tral helicases and Tral subgroup helicases comprise a relaxase domain. The relaxase domain comprises the MobF motif III or the the MobQ motif III and is typically found at the amino (N) terminus of the Tral helicase or Tral subgroup helicase. 15 Preferred fragments of Tral helicases and Tral subgroup helicases, such as preferred fragments 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, lack the N terminal domain of the wild-type sequence. The N-terminal domain typically corresponds to the about the N terminal third of the protein. In SEQ ID NO: 61 (which is 1756 amino acids in 20 length), the N-terminal domain is typically from about 500 to about 700 amino acids in length, such as from about 550 to about 600 amino acids in length. In SEQ ID NOs: 65, 69 and 73 (which are 970, 943 and 960 amino acids in length respectively), the N-terminal domain is typically from about 300 to about 350 amino acids in length, such as from about 320 to about 340 amino acids in length. 25 Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 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. The substitutions are preferably conservative substitutions as discussed above. One or more substitutions may be 30 made at amino acid positions K555, R554, T644, R647, P666, M667, H646, N604, N596, Y598, V470, G391, H409, T407, R410 and Y414 of SEQ ID NO: 41. In SEQ ID NOs: 18, 21, 24,25, 28, 30, 32, 35, 37, 39, 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, substitutions may be made at one or more amino acid positions which correspond to amino acid 35 positions K555, R554, T644, R647, P666, M667, H646, N604, N596, Y598, V470, G391, H409, WO 2013/098562 PCT/GB2012/053274 49 T407, R410 and Y414 of SEQ ID NO: 41. It is straightforward to determine corresponding amino acid positions in different protein sequences. For instance, the proteins may be aligned based on their homology. Homology may be determined as discussed above. A variant, such as a fragment, of any one of SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 5 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 the RecD-like motif I (or RecD motif I) and/or RecD-like motif V (or RecD motif V) of the relevant wild-type sequence. A variant, such as a fragment, of any one 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, 10 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises the RecD-like motif I (or RecD motif I) and the RecD-like motif V (or RecD motif V) of the relevant wild-type sequence. For instance, a variant of SEQ ID NO: 18 preferably comprises the RecD motif I GGPGTGKT (SEQ ID NO: 19) and the RecD motif V WAVTIHKSQG (SEQ ID NO: 20). The RecD-like motifs I and V (or RecD motifs I 15 and V) of 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, 102, 106, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 are shown in Tables 5 and 7. However, a variant of any one 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, 20 151, 152, 156, 160, 164 and 168 may comprise the RecD-like motif I (or RecD motif I) and/or RecD-like motif V (or RecD motif V) from a different wild-type sequence. For instance, a variant of SEQ ID NO: 28 or SEQ ID NO: 35 may comprise the RecD motif I and RecD-like motif V of SEQ ID NO: 21 (GGPGTGKS and YALTVHRAQG respectively; SEQ ID NOs: 22 and 23). A variant of any one SEQ ID NOs: 18, 21, 24, 25, 28, 30, 32, 35, 37, 39, 41, 42, 44, 61, 25 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 may comprise any one of the preferred motifs shown in Tables 5 and 7. Variants of any one 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 may also 30 include modifications within the RecD-like motifs I and V of the relevant wild-type sequence. Suitable modifications are discussed above when defining the two motifs. The discussion in the paragraph equally applies to the MobF motif III in SEQ ID NOs: 61, 65, 69, 73, 74, 82, 86, 90, 94, 98, 102, 110, 112, 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 and MobQ motif III in SEQ ID NOs: 78 and 106. In particular, a variant, such 35 as a fragment, of any one of SEQ ID NOs: 61, 65, 69, 73, 74, 82, 86, 90, 94, 98, 102, 110, 112, WO 2013/098562 PCT/GB2012/053274 50 113, 114, 117, 121, 124, 125, 129, 133, 136, 140, 144, 147, 151, 152, 156, 160, 164 and 168 preferably comprises the MobF motif III of the relevant wild-type sequence. A variant, such as a fragment, of SEQ ID NO: 78 or 106 preferably comprises the MobQ motif III of the relevant wild-type sequence. A variant, such as a fragment, of any one of SEQ ID NOs: 61, 65, 69, 73, 5 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 the RecD-like motif I (or RecD motif I), RecD-like motif V (or RecD motif V) and MobF or MobQ motif III of the relevant wild-type sequence. The helicase may be covalently attached to the pore. The helicase is preferably not 10 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. Any of the proteins described herein, i.e. the transmembrane protein pores or RecD helicases, may be modified to assist their identification or purification, for example by the 15 addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their 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 onto a native or engineered position on the pore or helicase. An example of this would be to react a gel-shift 20 reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 Jul;4(7):497-505). The pore and/or helicase may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125 , 35 S, enzymes, antibodies, antigens, 25 polynucleotides and ligands such as biotin. Proteins may be made synthetically or by recombinant means. For example, the pore and/or helicase may be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the pore and/or helicase may be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When a protein is produced by synthetic 30 means, such amino acids may be introduced during production. The pore and/or helicase may also be altered following either synthetic or recombinant production. The pore and/or helicase may also be produced using D-amino acids. For instance, the pore or helicase may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.
WO 2013/098562 PCT/GB2012/053274 51 The pore and/or helicase may also contain other non-specific modifications as long as they do not interfere with pore formation or helicase function. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an 5 aldehyde followed by reduction with NaBH 4 , amidination with methylacetimidate or acylation with acetic anhydride. The pore and helicase can be produced using standard methods known in the art. Polynucleotide sequences encoding a pore or helicase may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a pore or helicase may be 10 expressed in a bacterial host cell using standard techniques in the art. The pore and/or helicase may 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 the expression of the polypeptide. These methods are described in described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor 15 Laboratory Press, Cold Spring Harbor, NY. The pore and/or helicase may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system. 20 The method of the invention involves measuring one or more characteristics of the target polynucleotide. The method may involve measuring two, three, four or five or more characteristics of the target polynucleotide. The one or more characteristics are preferably selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of the 25 target polynucleotide and (v) whether or not the target polynucleotide is modified. Any combination of (i) to (v) may be measured in accordance with the invention. For (i), the length of the polynucleotide may be measured using the number of interactions between the target polynucleotide and the pore. For (ii), the identity of the polynucleotide may be measured in a number of ways. The 30 identity of the polynucleotide may be measured in conjunction with measurement of the sequence of the target polynucleotide or without measurement of the sequence of the target polynucleotide. The former is straightforward; the polynucleotide is sequenced and thereby identified. The latter may be done in several ways. For instance, the presence of a particular motif in the polynucleotide may be measured (without measuring the remaining sequence of the WO 2013/098562 PCT/GB2012/053274 52 polynucleotide). Alternatively, the measurement of a particular electrical and/or optical signal in the method may identify the target polynucleotide as coming from a particular source. For (iii), the sequence of the polynucleotide can be determined as described previously. Suitable sequencing methods, particularly those using electrical measurements, are described in 5 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. For (iv), the secondary structure may be measured in a variety of ways. For instance, if the method involves an electrical measurement, the secondary structure may be measured using a change in dwell time or a change in current flowing through the pore. This allows regions of 10 single-stranded and double-stranded polynucleotide to be distinguished. For (v), the presence or absence of any modification may be measured. The method preferably comprises determining whether or not the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers. Specific modifications will result in specific interactions with the pore which can be 15 measured using the methods described below. For instance, methylcyotsine may be distinguished from cytosine on the basis of the current flowing through the pore during its interation with each nucleotide. A variety of different types of measurements may be made. This includes without limitation: electrical measurements and optical measurements. Possible electrical measurements 20 include: current measurements, impedance measurements, tunnelling measurements (Ivanov AP et al., Nano Lett. 2011 Jan 12;1 1(1):279-85), and FET measurements (International Application WO 2005/124888). Optical measurements may be combined 10 with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan;81(1):014301). The measurement may be a transmembrane current measurement such as measurement of ionic current flowing through 25 the pore. Electrical measurements may be made using standard single channel recording equipment as describe 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. Alternatively, electrical measurements may be made using a multi-channel 30 system, for example as described in International Application WO-2009/077734 and International Application WO-2011/067559. 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 35 movement of the target polynucleotide through the pore; and WO 2013/098562 PCT/GB2012/053274 53 (b) measuring the current passing through the pore as the polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide. The methods may be carried out using any apparatus that is suitable for investigating a 5 membrane/pore system in which a pore is inserted into a membrane. The method may be carried out using any apparatus that is suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier has an aperture in which the membrane containing the pore is formed. 10 The methods may be carried out using the apparatus described in International Application No. PCT/GB08/000562 (WO 2008/102120). The methods may involve measuring the current passing through the pore as the polynucleotide moves with respect to the pore. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the 15 membrane and pore. The methods may be carried out using a patch clamp or a voltage clamp. The methods preferably involve the use of a voltage clamp. The methods of the invention may involve the measuring of a current passing through the pore as the polynucleotide moves with respect to the pore. Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the 20 Example. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically from +2 V to -2 V, typically -400 mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, + 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The 25 voltage used is more preferably in the range 100 mV to 240mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different nucleotides by a pore by using an increased applied potential. The methods are typically carried out in the presence of any charge carriers, such as metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali 30 metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazolium chloride. In the exemplary apparatus discussed above, the salt is present in the aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl) or caesium chloride (CsCl) is typically used. KCl is preferred. 35 The salt concentration may be at saturation. The salt concentration may be 3M or lower and is WO 2013/098562 PCT/GB2012/053274 54 typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from 150 mM to 1 M. As discussed above, RecD helicases surprisingly work under high salt concentrations. The method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at 5 least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations. The methods are typically carried out in the presence of a buffer. In the exemplary 10 apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The methods are typically carried out at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5. 15 The methods may be carried out at from 0 C to 100 'C, from 15 C to 95 'C, from 16 C to 90 'C, from 17 C to 85 'C, from 18 C to 80 0 C, 19 0 C to 70 0 C, or from 20 0 C to 60 0 C. The methods are typically carried out at room temperature. The methods are optionally carried out at a temperature that supports enzyme function, such as about 37 C. The method is typically carried out in the presence of free nucleotides or free nucleotide 20 analogues and an enzyme cofactor that facilitate the action of the helicase. The free nucleotides may be one or more of any of the individual nucleotides discussed above. The free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate 25 (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate 30 (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are 35 preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMIP or dCMP. The WO 2013/098562 PCT/GB2012/053274 55 free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows the helicase to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg 2 +, Mn 2 +, Ca2+ or Co2+. The enzyme cofactor is most preferably Mg2+ 5 The target polynucleotide may be contacted with the RecD helicase and the pore in any order. In is preferred that, when the target polynucleotide is contacted with the RecD helicase and the pore, the target polynucleotide firstly forms a complex with the helicase. When the voltage is applied across the pore, the target polynucleotide/helicase complex then forms a complex with the pore and controls the movement of the polynucleotide through the pore. 10 As discussed above, RecD helicases may work in two modes with respect to the pore. First, the method is preferably carried out using the RecD helicase such that it moves the target sequence through the pore with the field resulting from the applied voltage. In this mode the 5' end of the DNA is first captured in the pore, and the enzyme moves the DNA into the pore such that the target sequence is passed through the pore with the field until it finally translocates 15 through to the trans side of the bilayer. Alternatively, the method is preferably carried out such that the enzyme moves the target sequence through the pore against the field resulting from the applied voltage. In this mode the 3' end of the DNA is first captured in the pore, and the enzyme moves the DNA through the pore such that the target sequence is pulled out of the pore against the applied field until finally ejected back to the cis side of the bilayer. 20 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 may be used in combination. 25 Other methods The invention also provides a method of forming a sensor for characterising a target polynucleotide. The method comprises forming a complex between a pore and a RecD helicase. The complex may be formed by contacting the pore and the helicase in the presence of the target polynucleotide and then applying a potential across the pore. The applied potential may be a 30 chemical potential or a voltage potential as described above. Alternatively, the complex may be formed by covalently attaching the pore to the helicase. Methods for covalent attachment are known in the art and disclosed, for example, in International Application Nos. PCT/GB09/001679 (published as WO 2010/004265) and PCT/GB1O/000133 (published as WO 2010/086603). The complex is a sensor for characterising the target polynucleotide. The 35 method preferably comprises forming a complex between a pore derived from Msp and a RecD WO 2013/098562 PCT/GB2012/053274 56 helicase. Any of the embodiments discussed above with reference to the method of the invention equally apply to this method. Kits 5 The present invention also provides kits for characterising 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 equally apply to the kits. The kit may further comprise the components of a membrane, such as the phospholipids needed to form an amphiphilic layer, such as a lipid bilayer. 10 The kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to amplify and/or express polynucleotides, a membrane as defined above or 15 voltage or patch clamp apparatus. Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding which patients the method may be used for. The kit may, optionally, comprise nucleotides. 20 Apparatus The invention also provides an apparatus for characterising a target polynucleotide. The apparatus comprises a plurality of pores and a plurality of a RecD helicase. The apparatus preferably further comprises instructions for carrying out the method of the invention. The apparatus may be any conventional apparatus for polynucleotide analysis, such as an array or a 25 chip. Any of the embodiments discussed above with reference to the methods of the invention are equally applicable to the apparatus of the invention. The apparatus is preferably set up to carry out the method of the invention. The apparatus preferably comprises: a sensor device that is capable of supporting the membrane and plurality of pores and 30 being operable to perform polynucleotide characterising using the pores and helicases; at least one reservoir for holding material for performing the characterising; a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and a plurality of containers for receiving respective samples, the fluidics system being 35 configured to supply the samples selectively from the containers to the sensor device. The WO 2013/098562 PCT/GB2012/053274 57 apparatus may be any of those described in International Application No. No. PCT/GB08/004127 (published as WO 2009/077734), PCT/GB 10/000789 (published as WO 2010/122293), International Application No. PCT/GB1O/002206 (not yet published) or International Application No. PCT/US99/25679 (published as WO 00/28312). 5 Characterisation without a pore In some embodiments, the target polynucleotide is characterised, such as partially or completely sequenced, using a RecD helicase, but without using a pore. In particular, the invention also provides a method of characterising a target polynucleotide which comprises 10 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 polynucleoide is preferably not contacted with a pore, such as a transmembrane pore. The method involves taking one or more measurements as the RecD helicase controls the movement of the polynucleotide and thereby characterising the target polynucleotide. The measurements are indicative of one or 15 more characteristics of the target polynucleotide. Any such measurements may be taken 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 may be used in the method lacking a pore. For instance, any of the RecD helicases discussed above may be used. 20 The invention also provides an analysis apparatus comprising a RecD helicase. The invention also provides a kit a for characterising a target polynucleotide comprising (a) an analysis apparatus for characterising target polynucleotides and (b) a RecD helicase. These apparatus and kits preferably do not comprise a pore, such as a transmembrane pore. Suitable apparatus are discussed above. 25 The following Examples illustrate the invention. Example 1 This example illustrates the use of a Tral helicase (Tral Eco; SEQ ID NO: 61) to control 30 the movement of intact DNA strands through a nanopore. The general method and substrate employed throughout this example is shown in Fig. 1 and described in the figure caption Materials and Methods Primers were designed to amplify a ~400 bp fragment of PhiX174. Each of the 5'-ends 35 of these primers included a 50 nucleotide non-complimentary region, either a homopolymeric WO 2013/098562 PCT/GB2012/053274 58 stretch or repeating units of 10 nucleotide homopolymeric sections. These serve as identifiers for controlled translocation of the strand through a nanopore, as well as determining the directionality of translocation. In addition, the 5'-end of the forward primer was "capped" to include four 2'-0-Methyl-Uracil (mU) nucleotides and the 5'-end of the reverse primer was 5 chemically phosphorylated. These primer modifications then allow for the controlled digestion of predominantly only the antisense strand, using lambda exonuclease. The mU capping protects the sense strand from nuclease digestion whilst the P04 at the 5' of the antisense strand promotes it. Therefore after incubation with lambda exonuclease only the sense strand of the duplex remains intact, now as single stranded DNA (ssDNA). The generated ssDNA was then 10 PAGE purified as previously described. The DNA substrate design used in all the experiments described here is shown in Fig. 1B. The DNA substrate consists of a 400 base section of ssDNA from PhiX, with a 50T 5'-leader to aid capture by the nanopore (SEQ ID NO: 172). Annealed to this strand just after the 50T leader is a primer (SEQ ID NO: 173) containing a 3' cholesterol tag to enrich the DNA on the surface 15 of the bilayer, and thus improve capture efficiency. An additional primer (SEQ ID NO: 174) is used towards the 3' end of the strand to aid the capture of the strand by the 3' end. Buffered solution: 400 mM NaCl, 10 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl 2 , 1 mM DTT 20 Nanopore: E.coli MS(B2)8 MspA ONLP3476 MS-(L88N/D90N/D91N/D93N/ D118R/D134R/E139K)8 Enzyme: Tral Eco (SEQ ID NO: 61; ONLP3572, -4. 3 ptM) 23.3 il -> 100 nM final. Electrical measurements were acquired from single MspA nanopores inserted in 1,2 25 diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers. Bilayers were formed across ~100 pm diameter apertures in 20 tm thick PTFE films (in custom Delrin chambers) via the Montal-Mueller technique, separating two 1 mL buffered solutions. All experiments were carried out in the stated buffered solution. Single-channel currents were measured on Axopatch 200B amplifiers (Molecular Devices) equipped with 1440A digitizers. 30 Ag/AgC1 electrodes were connected to the buffered solutions so that the cis compartment (to which both nanopore and enzyme/DNA are added) is connected to the ground of the Axopatch headstage, and the trans compartment is connected to the active electrode of the headstage. After achieving a single pore in the bilayer, DNA polynucleotide and helicase were added to 50 piL of buffer and pre-incubated for 5 mins (DNA = 12.0 nM, Enzyme = 2 [tM). This pre-incubation 35 mix was added to 950 pL of buffer in the cis compartment of the electrophysiology chamber to WO 2013/098562 PCT/GB2012/053274 59 initiate capture of the helicase-DNA complexes in the MspA nanopore (to give final concentrations of DNA = 0.6 nM, Enzyme = 0.1 pM). Helicase ATPase activity was initiated as required by the addition of divalent metal (1 mM MgC1 2 ) and NTP (1 mM ATP) to the cis compartment. Experiments were carried out at a constant potential of +140 mV. 5 Results and Discussion The addition of Helicase-DNA substrate to MspA nanopores as shown in Fig. 1 produces characteristic current blocks as shown in Figs. 2 and 3. DNA without helicase bound interacts transiently with the nanopore producing short-lived blocks in current (<< 1 second). DNA with 10 helicase bound and active (ie. moving along the DNA strand under ATPase action) produces long characteristic block levels with stepwise changes in current as shown in Figs. 2 and 3. Different DNA motifs in the nanopore give rise to unique current block levels. For a given substrate, we observe a characteristic pattern of current transitions that reflects the DNA sequence (examples in Fig. 3). 15 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 strand. Salt tolerance Nanopore strand sequencing experiments of this type generally require ionic salts. The 20 ionic salts are necessary to create a conductive solution for applying a voltage offset to capture and translocate DNA, and to measure the resulting sequence dependent current changes as the DNA passes through the nanopore. Since the measurement signal is dependent on the concentration of the ions, it is advantageous to use high concentration ionic salts to increase the magnitude of the acquired signal. For nanopore sequencing salt concentrations in excess of 100 25 mM KCl are ideal, and salt concentrations of 400mM KCl and above are preferred. However, many enzymes (including some helicases and DNA motor proteins) do not tolerate high salt conditions. Under high salt conditions the enzymes either unfold 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 30 approximately 100 mM KCl/NaCl, and there are no reported helicases that show correct activity in conditions of 400 mM KCl and above. While potentially halophilic variants of similar enzymes from halotolerant species exist, they are extremely difficult to express and purify in standard expression systems (e.g. E. coli).
WO 2013/098562 PCT/GB2012/053274 60 We surprisingly show in this Example that Tral displays salt tolerance up to very high levels of salt. We find that the enzyme retains functionality in salt concentrations of 400 mM KCl through to 1 M KCl, either in fluorescence experiments or in nanopore experiments. 5 Forward and reverse modes of operation Most helicases move along single-stranded polynucleotide substrates in uni-directional manner, moving a specific number of bases for each NTPase turned over. Helicase movement can be exploited in different modes to feed DNA through the nanopore in a controlled fashion. Fig. 1 illustrates two basic 'forward' and 'reverse' modes of operation. In the forward mode, the 10 DNA is fed into the pore by the helicase in the same direction as the DNA would move under the force of the applied field. This direction is shown by the trans arrows. For Tral, which is a 5'-3' helicase, this requires capturing the 5' 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 from the applied potential, ie. moving from cis to trans. The reverse mode 15 requires capturing the 3' end of the DNA, after which the helicase proceeds to pull the threaded DNA back out of the nanopore against the field from the applied potential, ie. moving from trans to cis. Fig. 1 shows these two modes of operation using Tral Eco. Example 2 20 This example illustrates the salt tolerance of RecD helicases using a fluorescence assay for testing enzyme activity. A custom fluorescent substrate was used to assay the ability of the helicase to displace hybridised dsDNA (Fig. 4A). As shown in 1) of Fig. 4A, the fluorescent substrate strand (50 nM final) has a 5' ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper 25 strand has a carboxyfluorescein base at the 3' end, and the hybrised complement has a black-hole quencher (BHQ-1) base at the 5' end. When hybrised the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 pM of a capture strand that is complementary to the shorter strand of the fluorescent substrate is included in the assay. As shown in 2), in the presence of ATP (1 mM) and MgCl 2 (10 mM), helicase (100 nM) 30 added to the substrate binds to the 5' tail of the fluorescent substrate, moves along the major strand, and displaces the complementary strand as shown. As shown in 3), once the complementary strand with BHQ-1 is fully displaced the fluorescein on the major strand fluoresces. As shown in 4), an excess of capture strand preferentially anneals to the complementary DNA to prevent re-annealing of initial substrate and loss of fluorescence. 35 WO 2013/098562 PCT/GB2012/053274 61 Substrate DNA: SEQ ID NO: 175 with a carboxyfluorescein near the 3' end and SEQ ID NO: 176 with a Black Hole Quencher-I at the 5' end Capture DNA: SEQ ID NO: 177 5 The graph in Fig. 4B shows the initial rate of activity of two RecD helicases (RecD Nth and Dth, SEQ IDs 28 and 35) in buffer solutions (100 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgC 2 , 50 nM fluorescent substrate DNA, 1 pM capture DNA) containing different concentrations of KCl from 100 mM to 1 M. The helicase works at 1 M. 10 Example 3 In this Example, a different Tral helicase was used, namely TrwC Cba (SEQ ID NO: 65). All experiments were carried out as previously described in Example 1 under the same experimental conditions (pore = MspA B2, DNA = 400mer SEQ ID NO: 172, 173 and 174, buffer = 400mM KCl, 10mM Hepes pH 8.0, 1mM DTT, 1mM ATP, 1mM MgCl 2 ). Fig. 5 shows 15 two typical examples of helicase controlled DNA events using this enzyme. Example 4 In this Example a number of different TrwC helicases (TrwC (Atr) (SEQ ID NO: 144), TrwC (Sal) (SEQ ID NO: 140), TrwC (Ccr) (SEQ ID NO: 136) and TrwC (Eco) (SEQ ID NO: 20 74)) were investigated for their ability to control the movement of DNA (SEQ ID NOs: 178, 179 (with /iSp18//iSp18//iSp18//iSp18//iSp18//iSpl8/TT/3CholTEG/ at the 3' end) and 180) through an MspA nanopore (MS (G75S/G77S/L88N/D90N/D91N/D93N/D1 18R/Q126R/D134R/E139K)8, i.e. 8 x SEQ ID NO: 2 with G75S/G77S/L88N/Q126R. 25 Materials and Methods Buffered solution: 625mM KCl, 75mM K Ferrocyanide, 25mM K Ferricyanide, 100mM Hepes at pH 8.0 for TrwC (Atr),TrwC (Eco) and TrwC (CcR), and at pH 9.0 for TrwC (Sal). Enzyme: TrwC (Atr) (100 nM) or TrwC (Sal) (100 nM) or TrwC (Ccr) (100 nM) or TrwC (Eco) 30 (100 nM) all at a final concentration of 100 nM Electrical measurements were acquired from single MspA nanopores inserted in 1,2 diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers as described in Example 1, except platinum electrodes were used instead of Ag/AgCl. After achieving a single 35 pore in the bilayer, MgCl 2 (10 mM) and dTTP (5 mM, for TrwC (Atr), TrwC (Ccr) and TrwC WO 2013/098562 PCT/GB2012/053274 62 (Eco)) or ATP (1 mM for TrwC (Sal)) were added to the cis chamber and a control experiment was run for 5 mins at an applied potential of +120 mV. DNA polynucleotide (SEQ ID NO: 178 hybridized to 179 and 180, 0.1 nM) was added to the cis chamber and another control experiment was run for 5 mins at an applied potential of +120 mV. Finally, the appropriate 5 helicase (TrwC (Atr), TrwC (Sal), TrwC (Ccr) or TrwC (Eco) all added at a final concentration of 100 nM) was added to the cis compartment of the electrophysiology chamber to initiate capture of the helicase-DNA complexes in the MspA nanopore. Experiments were carried out at a constant potential of +120 mV. 10 Results and Discussion Helicase controlled DNA movement was observed for each of the helicases investigated. Example traces are shown in Figures 6-9 respectively. 15 Example 5 In this example, a number of different TrwC helicases (TrwC (Oma) (SEQ ID NO: 106), TrwC (Afe) (SEQ ID NO: 86), and TrwC (Mph) (SEQ ID NO: 94)) were investigated for their 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 3' end) for TrwC (Afe) 20 and TrwC (Mph)) through an MspA nanopore (MS (G75S/G77S/L88N/D90N/D91N/D93N/D1 18R/Q126R/D134R/E139K)8 i.e. 8 x SEQ ID NO: 2 with G75S/G77S/L88N/Q126R. Buffered solution: 625mM KCl, 75mM K Ferrocyanide, 25mM K Ferricyanide, 100mM Hepes. 25 pH8.0 Enzyme: TrwC (Oma), TrwC (Afe), and TrwC (Mph) all at a final concentration of 100 nM Electrical measurements were acquired from single MspA nanopores inserted in 1,2 diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers as described in 30 Example 1, except platinum electrodes were used instead of Ag/AgCl. After achieving a single pore in the bilayer, MgCl 2 (10 mM) were added to the cis chamber and a control experiment was run for 5 mins at an applied potential of 120 mV. 0. 15nM final of DNA polynucleotide (SEQ ID NOs: 172 to 174 (as in Example 1) for TrwC (Oma), or SEQ ID NO: 181 hybridized to 182 for TrwC (Afe) and TrwC (Mph)) and 100nM final of the appropriate helicase (TrwC (Oma), TrwC 35 (Afe), and TrwC (Mph) were added to the cis chamber and another control experiment was run WO 2013/098562 PCT/GB2012/053274 63 for 10 mins at an applied potential of +120 mV. Finally, helicase ATPase activity was initiated by the addition of ATP (1 mM) to the cis compartment of the electrophysiology chamber. Experiments were carried out at a constant potential of +120 mV. 5 Results and Discussion Helicase controlled DNA movement was observed for each of the helicases investigated. Example traces are shown in Figures 10-12 respectively.

权利要求:
Claims (37)
[1] 1. A method of characterising 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) taking one or more measurements as the polynucleotide moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.
[2] 2. A method according to claim 1, wherein the one or more characteristics are selected from (i) the length of the target polynucleotide, (ii) the identity of the target polynucleotide, (iii) the sequence of the target polynucleotide, (iv) the secondary structure of of the target polynucleotide and (v) whether or not the target polynucleotide is modified.
[3] 3. A method according to claim 2, wherein the target polynucleotide is modified by methylation, by oxidation, by damage, with one or more proteins or with one or more labels, tags or spacers.
[4] 4. A method according to any one of claims I to 3, wherein the one or more characteristics of the target polynucleotide are measured by electrical measurement and/or optical measurement.
[5] 5. A method according to claim 4, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.
[6] 6. A method according to claim 1, wherein 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 wherein the current is indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide. WO 2013/098562 PCT/GB2012/053274 65
[7] 7. A method according to any one of the preceding claims, wherein step (b) involves taking one or more measurements as the polynucleotide moves through the pore.
[8] 8. A method according to any one of the preceding claims, wherein the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the helicase
[9] 9. A method according to any one of the preceding claims, wherein at least a portion of the polynucleotide is double stranded.
[10] 10. A method according to any one of the preceding claims, wherein the pore is a transmembrane protein pore or a solid state pore.
[11] 11. A method according to claim 10, wherein the transmembrane protein pore is selected from a hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA.
[12] 12. A method according to claim 11, wherein the transmembrane protein is (a) formed of eight identical subunits as shown in SEQ ID NO: 2 or (b) 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 over the entire sequence and retains pore activity.
[13] 13. A method according to claim 11, wherein the transmembrane protein is (a) a-hemolysin formed of seven identical subunits as shown in SEQ ID NO: 4 or (b) a variant thereof in which one or more of the seven subunits has at least 50% homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains pore activity.
[14] 14. A method according to any one of the preceding claims, wherein the RecD helicase comprises: - the amino acid motif XI-X2-X3-G-X4-X5-X6-X7 (SEQ ID NO: 8), wherein X1 is G, S or A, X2 is any amino acid, X3 is P, A, S or G, X4 is T, A, V, S or C, X5 is G or A, X6 is K or R and X7 is T or S; and/or WO 2013/098562 PCT/GB2012/053274 66 - the amino acid motifX1-X2~X3--X4-X5-(X6)-Q-X.7 (SEQ ID NOs: 9, 0 and 11), wherein X1 is Y, W or F. X2 is A, T, S, M, C or V., X3 is any amino acid, X4 is T or N, X5 is A, T, G, S, V or I, X6 is any amino acid and X7 is G or S, 1 z' - A method according to claim 14, wherein the RecD helicase comprises the following motifs: (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); (c) 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); (f) GGPGCGKS (SEQ ID NO: 33) and/or YAMTTFIRSQG (SEQ ID NO: 34); (g) 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); (i) GGPGTGKT (SEQ ID NO: 19) and/or YAVSVHKSQG (SEQ ID NO: 40); (j) GGPGVGKT (SEQ ID NO: 26) and/or YATSIHKSQG (SEQ ID NO: 43); or (k) GGPGTGKS (SEQ ID NO: 22) and/or YALTVHRGQG (SEQ ID NO: 45).
[15] 16. A method according to any one of the preceding claims, wherein the RecD helicase is one of the helicases shown in Table 4 or 5 or a variant thereof.
[16] 17. A method according to claim 16, wherein the RecD helicase comprises: (a) the sequence shown in any one of 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 amino acid identity over the entire sequence and which retains helicase activity.
[17] 18. A method according to any one of claims I to 14, wherein the RecD helicase is a Tral helicase or Tral subgroup helicase.
[18] 19. A method according to claim 18, wherein the Tral helicase or TraIl subgroup helicase further comprises: - the amino acid motif H.(X1) 2 X2~R.(X3)sr2H-X44- (SEQ ID NOs: 46 to 53), wherein X1 and X3 are any amino acid and X2 and X4 are independently selected from any amino acid except 1D, E, K and R; or WO 2013/098562 PCT/GB2012/053274 67 - the amino acid motif G-XI -X2-X3-X4-X5-X6-X7-1-(S 2 -IX9 (SEQ I) NOs: 54 to 60), wherein Xl, X2, X3, X5, X6, X7 and X9 are independently selected from any amino acid except D, E, K and R, X4 is D or E and X8 is any amino acid. 20, A method according to claim 14, wherein the Tral helicase or Tral subgroup helicase comprises the following motifs: (a) GYAGVGKT (SEQ ID NO: 62), YAITAHGAQG (SEQ ID NO: 63) and HDTSRDQEPQLHTH (SEQ ID NO: 64); (b) GIAGAGKS (SEQ ID NO: 66), YALNVM4AQG (SEQ ID NO: 67) and HIDTNRNQEPNLHFH (SEQ ID NO: 68), (c) GAAGAGK T (SEQ ID NO: 70), YCITIHRSQG (SEQ ID NO: 71) and HEDARTVDDIADPQLHTH (SEQ ID NO: 72); (d) GFAGTGKS (SEQ I D NO: 75), YATTVHSSQG (SEQ ID NO: 76) and HIETSRERDPQLHTH (SEQ ID NO: 77); (e) GRACAGKT (SEQ ID NO: 79), YAT' TIHIKSQG (SEQ I) NO: 80) and GMVADWVYHDNPGN-PHIH (SEQ ID NO: 81); (f) GAAGTGKT (SEQ I) NO: 83), YASTAIIKSQG (SEQ 11) NO: 84) and HSTSRAQDPILHSH (SEQ ID NO: 85); (g) GHAGAGKT (SEQ ID NO: 87), YAGTTHRNQG (SEQ ID NO: 88) and HASSREQDPQIHSH (SEQ ID NO: 89); (h) GLAGTGKT (SEQ ID NO: 91), YAVTSIISSQG (SEQ ID NO. 92) and HDTARPVNGYAAPQLHTH (SEQ ID NO: 93); (i) GPAGAGKT (SEQ ID NO: 95), YAITAHRAQG (SEQ ID NO: 96) and H YDSRAGDPQLIATH (SEQ ID NO. 97): (j) GWAGVGKT (SEQ ID NO: 99), YAVTADMJQG (SEQ ID NO: 100) and H1L CGRLDPQIHJlNH (SEQ ID NO: 101), (k) GVAGAGKT (SEQ ID NO: 103), YALTIDSAQG (SEQ ID NO: 104) and HMTSGDGSPHLHVH (SEQ ID NO: 105); 0) GYAGTGKS (SEQ ID NO: 107), YAATIHKAQG (SEQ ID NO: 108) and GMIADLVNVIHWDIGEDGKAKPIAH (SEQ ID NO: 109): (rn) GIAGA GKS (SEQ ID NO: 66), YALNAHMAQG (SEQ ID NO: 67) and HDTNRNQEPNLIFH (SEQ ID NO: 111); (n) GVAGAG-KS (SEQ ) NO: I15), YALNAHMAQG (SEQ ID NO: 67) and HDTNRNQEPNAHFH (SEQ ID NO: 116); WO 2013/098562 PCT/GB2012/053274 68 (a) GGAGVGKS (SEQ [D NO: 118), YAINVFHAQG (SEQ I) NO: 119) and HDVSRNNDPQLHVH (SEQ ID NO: 120); (p) GTAGAGKS (SEQ ID NO: 66), YANMHMAQG (SEQ ID NO: 122) and H1-DTSRALDPQGHIH (SEQ ID NO: 123); (q) GVAGAGK S (SEQ ID NO: 115), YALNAHMAQG (SEQ ID NO: 67) and HDTSRALDPQGHIH (SEQ ID NO: 123); (r) GRAGTGKT (SEQ ID NO: 126), FASTAHGAQG (SEQ ID NO: 127) and HEASRNLDPQLHSH (SEQ 1D NO: 128); (S) GYAGTGKT (SEQ ID NO: 130), YAMTSHAAQG (SEQ ID NO: 131) and HDISRDKDPQLHT (SEQ I) NO: 132); (t) GLAGTGKT (SEQ ID NO: 91), YAQTVHASQG (SEQ ID NO: 134) and INTSRDLDPQTHTH (SEQ ID NO: 135); (u) GFAGTAKT SEQ ID NO: 137), YVQTAFAAQG (SEQ ID NO: 138) and IETSRAQDPQLHTH (SEQ IDNO: 139); (v) GYAGTAKT (SEQ ID NO: 141), YVDTAF AAQG (SEQ )ID NO: 142) and HGTSRAQDPQLHTH (SEQ ID NO: 143); (w) GYAGTAKT (SEQ ID NO: 141), YASTAFAAQG (SEQ ID NO: 145) and HGTSRALDPQLHSH (SEQ ID NO: 146); (x) GSAGSGKT (SEQ ID NO: 148), YAVTSYSAQG (SEQ ID NO: 149) and HDTARPVGGYAAPQLH'TH (SEQ ID NO: 150), (y) GEAGTGKT (SEQ ID NO: 153), YAHTSYKEQG (SEQ ID NO: 154) and HETNRENEPQLHNH (SEQ ID NO: 155); (z) GYAGVAKT (SEQ ID NO: 157), YVITNYKVQG (SEQ ID NO: 158) and QPSSRANDPALHITH (SEQ I) NO. 159): (aa) GSAGTGKT (SEQ ID NO: 161), YSLTANRAQG (SEQ ID NO: 162) and IISMSRAGDPEMHNIH (SEQ ID NO: 163); (bb) AGAGTGKT (SEQ ID NO: 165), YAGTVYAAQG (SEQ ID NO: 166) and HYTTREGDPNIHTH (SEQ ID NO: 167); or (cc) APAGAGKT (SEQ ID NO: 169), YAVTVHAAQG (SEQ ID NO: 170) and HETSRAGDPHLHITH (SEQ ID NO: 171).
[19] 21. A method according to any one of claims 18 to 20, wherein the Tral helicase or Tral subgroup helicase is one of the helicases shown in Table 6 or 7 or a variant thereof. WO 2013/098562 PCT/GB2012/053274 69
[20] 22. A method according to claim 21, wherein the Tral helicase or Tral subgroup helicase comprises (a) the sequence shown in any 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 (b) a variant thereof having at least 10% homology to the relevant sequence based on amino acid identity over the entire sequence and retains helicase activity.
[21] 23. A method according to any one of the preceding claims, wherein the method is carried out using a salt concentration of at least 0.3 M and the salt is optionally KCl.
[22] 24. A method according to claim 23, wherein the salt concentration is at least 1.0 M.
[23] 25. A method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore and a RecD helicase and thereby forming a sensor for characterising the target polynucleotide.
[24] 26. A method according to claim 25, wherein the complex is formed by (a) contacting the pore and the helicase in the presence of the target polynucleotide and (a) applying a potential across the pore.
[25] 27. A method according to claim 26, wherein the potential is a voltage potential or a chemical potential.
[26] 28. A method according to claim 26, wherein the complex is formed by covalently attaching the pore to the helicase.
[27] 29. Use of a RecD helicase to control the movement of a target polynucleotide through a pore.
[28] 30. A kit for characterising a target polynucleotide comprising (a) a pore and (b) a RecD helicase.
[29] 31. A kit according to claim 29, wherein the kit further comprises a chip comprising an amphiphilic layer. WO 2013/098562 PCT/GB2012/053274 70
[30] 32. An analysis apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of a RecD helicase.
[31] 33. An analysis apparatus according to claim 32, wherein the analysis apparatus comprises: a sensor device that is capable of supporting the plurality of pores and being operable to perform polynucleotide characterisation using the pores and helicases; at least one reservoir for holding material for performing the characterisation; a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and a plurality of containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the containers to the sensor device.
[32] 34. A method of characterising a target polynucleotide, comprising: (a) contacting the target polynucleotide with a RecD helicase such that the RecD helicase controls the movement of the target polynucleotide; and (b) taking one or more measurements as the RecD helicase controls the movement of the polynucleotide wherein the measurements are indicative of one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.
[33] 35. A method according to claim 34, wherein: (a) the one or more characteristics are as defined in claim 2; (b) the target polynucleotide is as defined in claim 3 or 9; (c) the one or more characteristics are measured as defined in claim 4 or 5; (d) the RecD is as defined in any one of claims 14 to 22; or (e) the method is carried out as defined in any claim 23 or 24.
[34] 36. Use of a RecD helicase to control the movement of a target polynucleotide during characterisation of the polynucleotide.
[35] 37. Use of a RecD helicase to control the movement of a target polynucleotide during sequencing of part or all of the polynucleotide.
[36] 38. An analysis apparatus for characterising target polynucleotides in a sample, characterised in that it comprises a RecD helicase. WO 2013/098562 PCT/GB2012/053274 71
[37] 39. A kit for characterising a target polynucleotide comprising (a) an analysis apparatus for characterising target polynucleotides and (b) a RecD helicase.

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GB201917060D0|2019-11-22|2020-01-08|Oxford Nanopore Tech Ltd|Method|

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WO2021111125A1|2019-12-02|2021-06-10|Oxford Nanopore Technologies Limited|Method of characterising a target polypeptide using a nanopore|

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GB201917742D0|2019-12-04|2020-01-15|Oxford Nanopore Tech Ltd|Method|

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GB202004944D0|2020-04-03|2020-05-20|King S College London|Method|

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WO2021255476A2|2020-06-18|2021-12-23|Oxford Nanopore Technologies Limited|Method|

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GB202009349D0|2020-06-18|2020-08-05|Oxford Nanopore Tech Ltd|Method|

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WO2021255475A1|2020-06-18|2021-12-23|Oxford Nanopore Technologies Limited|A method of selectively characterising a polynucleotide using a detector|

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WO2022020461A1|2020-07-22|2022-01-27|Oxford Nanopore Technologies Inc.|Solid state nanopore formation|

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GB202107192D0|2021-05-19|2021-06-30|Oxford Nanopore Tech Ltd|Method|

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GB202107354D0|2021-05-24|2021-07-07|Oxford Nanopore Tech Ltd|Method|

法律状态:
2018-12-20| FGA| Letters patent sealed or granted (standard patent)|

优先权:

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申请号 | 申请日 | 专利标题

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US201161581332P| true| 2011-12-29|2011-12-29||

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US61/581,332||2011-12-29||

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PCT/GB2012/053274|WO2013098562A2|2011-12-29|2012-12-28|Enzyme method|

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