# Patent

## Title
Enzyme method

## Abstract

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national stage filing of International Application PCT/GB2012/052579, filed Oct. 18, 2012, which claims priority to U.S. Provisional Patent Application 61/549,998 filed Oct. 21, 2011 and U.S. Provisional Patent Application 61/599,244 filed on Feb. 15, 2012 in the United States. The contents of the aforementioned applications are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Hel308 helicase or a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor 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) 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 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 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
The inventors have demonstrated that a Hel308 helicase can control the movement of a polynucleotide through a pore especially when a potential, such as a voltage, is applied. The helicase is capable of moving a target polynucleotide in a controlled and stepwise fashion against or with the field resulting from the applied voltage. Surprisingly, the helicase is capable of functioning at a high salt concentration which is advantageous for characterising the 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:
(a) contacting the target polynucleotide with a transmembrane pore and a Hel308 helicase such that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and
(b) measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby 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 Hel308 helicase and thereby forming a sensor for characterising the target polynucleotide;
        use of a Hel308 helicase to control the movement of a target polynucleotide through a pore;
        a kit for characterising a target polynucleotide comprising (a) a pore and (b) a Hel308 helicase; and
        an analysis apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of a Hel308 helicase.
    
    


The inventors have also demonstrated that a molecular motor which is capable of binding to a target polynucleotide at an internal nucleotide can control the movement of the polynucleotide through a pore especially when a potential, such as a voltage, is applied. The motor is capable of moving the target polynucleotide in a controlled and stepwise fashion against or with the field resulting from the applied voltage. Surprisingly, when the motor is used in the method of the invention it is possible to control the movement of an entire strand of target polynucleotide through a nanopore. This is advantageous for characterising the polynucleotide and, in particular, for determining its sequence using Strand Sequencing.
Hence, the invention also provides a method of characterising a target polynucleotide, comprising:
(a) contacting the target polynucleotide with a transmembrane pore and a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide such that the molecular motor controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and
(b) measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby characterising the target polynucleotide.



DESCRIPTION OF THE FIGURES
FIG. 1. a) Example schematic of use of a helicase to control DNA movement through a nanopore. 1) A ssDNA substrate (a) with an annealed primer (b) containing a cholesterol-tag (c) is added to the cis side of the bilayer (d). The cholesterol tag binds to the bilayer, enriching the substrate at the bilayer surface. 2) Helicase (e) 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. 3) Under an applied voltage, the DNA substrate is captured by the nanopore (f) via the leader section on the DNA. 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 the pore, preventing further uncontrolled DNA translocation. During this process dsDNA sections (such as the primer) are removed. The helicase movement along the DNA in a 3′ to 5′ direction pulls the threaded DNA out of the pore against the applied field. 4) The helicase pulls the DNA out of the nanopore, feeding it back to the cis compartment. The last section of DNA to pass through the nanopore is the 5′-leader. 5) When the helicase moves the DNA out of the nanopore it is lost back to the cis compartment. b) The DNA substrate design used in the Example (a=400 mer strand of DNA with a 50T leader (b), c=primer, d=cholesterol-tag).
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. Example helicase-DNA events (indicated by small arrows in the top section) (180 mV. 400 mM KCl, Hepes pH 8.0, 0.15 nM 400 mer DNA, 100 nM Hel308 Mbu, 1 mM DTT, 1 mM ATP, 1 mM MgCl2). Top) Section of current (y-axis, pA) vs. time α-axis, s) acquisition of Hel308 400 mer DNA events. The open-pore current is  ̃180 pA (labelled A). DNA is captured by the nanopore under the force of the applied potential (+180 mV). DNA with enzyme attached results in a long block (at  ̃60 pA in this condition) that shows stepwise changes in current as the enzyme moves the DNA through the pore. Middle) The middle section is an enlargement of one of the DNA events (l), showing DNA-enzyme capture, stepwise current changes as the DNA is pulled through the pore, and ending in a characteristic long polyT level before exiting the nanopore. Bottom) enlargement of the stepwise changes in current as DNA is moved through the nanopore.
FIG. 3. Helicase controlled DNA movement resulting in a consistent pattern of current transitions as DNA is passed through the nanopore (y-axis=current (pA), x-axis=time (s) for FIGS. 3a and 3b). Examples of the last  ̃80 current transitions from four typical DNA events that end in the polyT level. The four examples (two in 3a and two in 3b) illustrate that a consistent pattern of current transitions are observed.
FIG. 4. Increased salt concentration increases pore current and gives a larger DNA discrimination range (range=minimum current to maximum current across the DNA current transitions). Example helicase-DNA events (y-axis=current (pA), x-axis=time (s) for FIGS. 4a-c, 180 mV, Hepes pH 8.0, 0.15 nM 400 mer DNA SEQ ID NOs: 59 and 60, 100 nM Hel308 Mbu, 1 mM DTT, 1 mM ATP, 1 mM MgCl2) at 400 mM, 1 M, and 2 M KCl are shown in FIGS. 4 and 4c. Top traces show a full event that ends in the polyT level (with I-open indicated by A), and lower traces show a zoom section of the last 10 seconds of each event with a constant y-axis current scale of 150 pA. Increasing the salt concentration from 400 mM KCl to 2M KCl leads to a  ̃350% increase in the open-pore current (1-open from  ̃180 pA to  ̃850 pA), and a  ̃200% increase in discrimination range ( ̃25 pA to  ̃75 pA). FIG. 4d is a plot of DNA discrimination range as a function of salt concentration (y-axis=range (pA), x-axis=salt (mM)).
FIG. 5. The helicase (a) can control the movement of DNA (b) in at least two modes of operation. The helicase moves along the DNA in the 3′-5′ direction, but the orientation of the DNA in the nanopore (c) (dependent on which end of the DNA is captured) means that the enzyme can be used to either move the DNA out of the nanopore against the applied field (FIG. 5b), or move the DNA into the nanopore with the applied field (FIG. 5a). FIG. 5b) When the 5′ end of the DNA is captured the helicase works against the direction of the field applied by the voltage, pulling the threaded DNA out of the nanopore until the DNA is ejected back to the cis chamber. On the right is an example DNA-helicase event from Hel308 running 5′ down against the applied field (y-axis=current (pA), x-axis=time (s)). FIG. 5a) When the DNA is captured 3′-down in the nanopore, the enzyme moves the DNA into the nanopore in the direction of the field until it is fully translocated through the pore and lost on the trans side of the bilayer. On the right is an example DNA-helicase event from Hel308 running 3′-down with the applied field (y-axis=current (pA), x-axis=time (s)). Current traces vary between the 5′ down and 3′ down orientations of DNA.
FIG. 6. Fluorescence assay for testing enzyme activity. a) A custom fluorescent substrate was used to assay the ability of the helicase (a) to displace hybridised dsDNA. 1) The fluorescent substrate strand (100 nM final) has a 3′ ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strand (b) has a carboxyfluorescein base (c) at the 5′ end, and the hybridised complement (d) has a black-hole quencher (BHQ-1) base (e) at the 3′ end. When hybridised the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 μM of a capture strand (f) that is complementary to the shorter strand of the fluorescent substrate (d) is included in the assay. 2) In the presence of ATP (1 mM) and MgCl2 (5 mM), helicase (100 nM) added to the substrate binds to the 3′ tail of the fluorescent 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 activity (y-axis, relative activity) in buffer solutions (10 mM Hepes pH 8.0, 1 mM ATP, 5 mM MgCl2, 100 nM fluorescent substrate DNA, 1 μM capture DNA) containing different concentrations of KCl (x-axis, mM) from 400 mM to 2 M.
FIG. 7 shows examples of helicase controlled DNA events using different Hel308 helicases (y-axis=current (pA), x-axis=time (min) for FIGS. 7a-c, 180 mV, Hepes pH 8.0, 0.15 nM 400 mer DNA SEQ ID NOs: 59 and 60, 100 nM Hel308, 1 mM DTT, 1 mM ATP, 1 mM MgCl2): Hel308 Mhu (a), Hel308 Mok (b) and Hel308 Mma (c). These represent typical examples of DNA controlled movement through MspA nanopores that ended at the polyT level.
FIG. 8. Fluorescence assay for testing helicase internal binding activity. A) Custom fluorescent substrates were used to assay the ability of the helicases to bind to DNA lacking native 3′ ends, allowing them to subsequently displace hybridised dsDNA. The fluorescent substrate strand (50 nM final) has a 3′ ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strands (a) are modified with four consecutive non-DNA-derived triethylene glycol spacers (referred to as “spacer 9” groups, labelled b), either at the 3′ end, or internally, at the junction between the overhang and the dsDNA (as a negative control). Furthermore, the major upper strand has a carboxyfluorescein base (c) at the 5′ end, and the hybridised complement (d) has a black-hole quencher (BHQ-1) base (e) at the 3′ end. When hybridised, the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. A capture strand (1 f), that is complementary to the shorter strand (d) of the fluorescent substrate, is included in the assay. B) In the presence of ATP (1 mM) and MgCl2 (1 mM), a Hel308 helicase homologue (20 nM, g), added to the substrate containing 3′-terminal “spacer 9” groups, can bind to the ssDNA overhang of the fluorescent substrate, move along the major strand, and displace the complementary strand. C) Once the complementary strand with BHQ-1 is fully displaced the fluorescein on the major strand fluoresces. D) An excess of capture strand preferentially anneals to the complementary DNA to prevent re-annealing of initial substrate and loss of fluorescence.
FIG. 9 shows the relative rates of Hel308-mediated dsDNA turnover comparing 3′-unmodified DNA and 3′-“spacer 9” DNA in 400 mM NaCl, 10 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl2, 50 nM fluorescent substrate DNA, 1 μM capture DNA (y-axis=relative “3′-Sp9” activity (% wrt native 3′), x-axis=a (Mbu), b (Csy), c (Tga), d (Mma), e (Mhu), f (Min), g (Mig), h (Mmaz), i (Mac), j (Mok), k (Mth), l (Mba), m (Mzh)).
FIG. 10. Schematic of the use of a helicase to control DNA movement through a nanopore which is employed in example 5. A) A DNA substrate (SEQ ID NOs 67 (labelled w) and 68 (labelled x)) with an annealed primer (SEQ ID NO 69 (labelled y)) with an attached cholesterol-tag (labelled z) is added to the cis side of the bilayer. The cholesterol tag binds to the bilayer, enriching the substrate at the bilayer surface. Helicase (labelled l) added to the cis compartment binds to the 4 bp leader of SEQ ID NO 67. B) Under an applied voltage, the DNA substrate is captured by the nanopore via the 5′ leader section on the DNA, which strips off SEQ ID NO 69. C) Under the force of the applied field the DNA is pulled into the pore until the bound helicase (l) contacts the top of the pore and prevents further uncontrolled translocation. In this process the antisense strand SEQ ID NO 68 is stripped from the DNA strand. D) In the presence of divalent metal ions and NTP substrate, the helicase (l) on top of the pore moves along the DNA and controls the translocation of the DNA through the pore. The helicase movement along the DNA in a 3′ to 5′ direction pulls the threaded DNA out of the pore against the applied field. The exposed single stranded DNA on the cis side (3′ in this case) is available for further helicases (2-4) to bind either at the terminal nucleotide or at an internal nucleotide. E) If the helicase at the pore (1) disengages from the DNA, the DNA is pulled into the pore by the field until the next helicase (2) on the DNA reaches the pore. The helicase at the pore pulls the DNA out of the nanopore, feeding it back to the cis compartment. The last section of DNA to pass through the nanopore is the 5′-leader. F) When the helicase moves the DNA out of the nanopore it is lost back to the cis compartment. Arrows indicate the direction of DNA movement.
FIG. 11 shows data plots (y-axis=position in the 900 mer, x-axis=index) which indicate how the position of the region of DNA in the nanopore of the 900 mer (y-axis) varied as the Hel308 helicase homologue Mbu controlled the translocation of the DNA strand through the MspA pore (x-axis) during each helicase event. A-C show examples of typical translocation events of the entire DNA strand from approximately the beginning of the strand through to the end of the strand (exiting via polyT leader), whereas event D shows an example of incomplete DNA translocation, where enzyme detachment means the DNA never makes it to the end of the strand. The slips (eg. such as the large slips highlighted by dotted circles) indicate the sequence falling back to a previous point in the strand, and are the result of enzyme detachment. When an enzyme detaches the DNA will be pulled back under the force of the field into the nanopore until another enzyme further along the strand contacts the pore, then continuing helicase movement.
FIG. 12 shows data plots (y-axis=position in the 900 mer, x-axis=index) which indicate how the position of the 900 mer varied as the Hel308 helicase homologue Tga controlled the translocation of the DNA strand through the MspA pore. Events A-D show translocation of the entire DNA strand.
FIG. 13 shows a fluorescence assay used to compare the enzyme processivity of Hel308 Mbu helicase (SEQ ID NO: 10) to that of Hel 308 Mok helicase (SEQ ID NO: 29). A custom fluorescent substrate was used to assay the ability of the helicase to displace hybridised dsDNA. The fluorescent substrate (50 nM final) has a 3′ ssDNA overhang, and 80 (a1) and 33 base-pair (a2) sections of hybridised dsDNA (section A, SEQ ID NO: 70). The major bottom “template” strand (1) is hybridised to an 80 nt “blocker” strand (2, SEQ ID NO: 71), adjacent to its 3′ overhang, and a 33 nt fluorescent probe (3, SEQ ID NO: 72), labelled at its 5′ and 3′ ends with carboxyfluorescein (FAM) (4) and black-hole quencher (BHQ-1) (5) bases, respectively. When hybridised, the FAM is distant from the BHQ-1 and the substrate is essentially fluorescent. In the presence of ATP (1 mM) and MgCl2 (10 mM) (addition of ATP and MgCl2 is indicated by 7 and 8 respectively), the helicase (6, nM) binds to the substrate's 3′ overhang (SEQ ID NO: 70), moves along the lower strand, and begins to displace the 80 nt blocker strand (SEQ ID NO: 71), as shown in section B. If processive, the helicase displaces the fluorescent probe too (section C, SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end). The fluorescent probe is designed in such a way that its 5′ and 3′ ends are self-complementary and thus form a kinetically-stable hairpin once displaced, preventing the probe from re-annealing to the template strand (section D). Upon formation of the hairpin product, the FAM is brought into the vicinity of the BHQ-1 and its fluorescence is quenched. A processive enzyme, capable of displacing the 80 mer “blocker” (SEQ ID NO: 71) and fluorescent (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) strands will therefore lead to a decrease in fluorescence over time. However, if the enzyme has a processivity of less than 80 nt it would be unable to displace the fluorescent strand (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and, therefore, the “blocker” strand (SEQ ID NO: 71) would reanneal to the major bottom strand (section E).
FIG. 14 shows additional custom fluorescent substrates which were also used for control purposes. The substrate used as a negative control was identical to that of the one described in FIG. 3 but lacking the 3′ overhang (section A, (SEQ ID NOs: 71 (labelled 1), 72 (labelled 2) (labeled with a carboxyfluorescein (FAM) (3) at its 5′ end a black-hole quencher (BHQ-1) (4) at its 3′ end) and 73 (labelled 5, consisting of an 80 bp section (a1) and a 33 bp section (a2)))). A similar substrate to that described in FIG. 3 but lacking the 80 base pair section (SEQ ID NOs: 72 (labelled 2) (labeled with a carboxyfluorescein (FAM) (3) at its 5′ end a black-hole quencher (BHQ-1) (4) at its 3′ end) and 74 (labelled 6, consisting of a 28 bp section (a3))), was used as a positive control for active, but not necessarily processive, helicases (section B).
FIG. 15 shows a graph (y-axis=fluorescence (arbitrary units), x-axis=time (min)) of the time-dependent fluorescence changes upon testing Hel308 Mbu helicase (SEQ ID NO: 10, empty circles) and Hel 308 Mok helicase (SEQ ID NO: 29, black triangles) against the processivity substrate shown in FIG. 13 in buffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl2, 50 nM fluorescent substrate DNA (SEQ ID NOs: 70, 71 and 72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end). The decrease in fluorescence exhibited by Hel308 Mok denotes the increased processivity of these complexes as compared to Hel308 Mbu (SEQ ID NO: 10).
FIG. 16 shows a graph (y-axis=fluorescence (arbitrary units), x-axis=time (min)) of the time-dependent fluorescence changes upon testing Hel308 Mbu helicase (SEQ ID NO: 10, empty circles) and Hel 308 Mok helicase (SEQ ID NO: 29, black triangles) against the positive control processivity substrate (shown in FIG. 14 section B, SEQ ID NOs: 72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and 74) in buffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl2, 50 nM fluorescent substrate DNA (SEQ ID NOs: 72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and 74)). This positive control demonstrated that both helicases were indeed active, as denoted by a fluorescence decrease for both samples.



DESCRIPTION OF THE SEQUENCE LISTING
SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the MS-B1 mutant MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.
SEQ ID NO: 2 shows the amino acid sequence of the mature form of the MS-B1 mutant of the MspA monomer. This mutant lacks the signal sequence and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K.
SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit of α-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19): 7702-7707).
SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HL-NN.
SEQ ID NOs: 5 to 7 shows the amino acid sequences of MspB, C and D.
SEQ ID NO: 8 shows the amino acid sequence of the Hel308 motif
SEQ ID NO: 9 shows the amino acid sequence of the extended Hel308 motif
SEQ ID NOs: 10 to 58 show the amino acid sequences of the Hel308 helicases and motifs in Table 5.
SEQ ID NOs: 59 to 74 show the sequences used in the Examples.
SEQ ID NO: 75 shows the sequence of Hel308 Dth in the alignmenton on page 57 onwards.
SEQ ID NO: 76 shows the sequence of Hel308 Mmar in the alignment on on page 57 onwards.
SEQ ID NO: 77 shows the sequence of Hel308 Nth in the alignment on page 57 onwards.
SEQ ID NO: 78 shows the consensus sequence in the alignment on page 57 onwards.
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 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 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.
Hel308 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 Hel308 helicase such that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore. One or more characteristics of the target polynucleotide are then measured using standard methods known in the art. Steps (a) and (b) are preferably carried out with a potential applied across the pore. As discussed in more detail below, the applied potential typically results in the formation of a complex between the pore and the helicase. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example of this is using a salt gradient across the lipid membrane. A salt gradient is disclosed in Holden et al., J Am Chem. Soc. 2007 Jul. 11; 129(27):8650-5.
In some instances, the current passing through the pore during one or more interactions 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 Hel308 helicases have a surprisingly high salt tolerance and so the method of the invention may be 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 passes through the pore. Since the measurement signal is dependent on the concentration of the salt, it is advantageous to use high salt concentrations to increase the magnitude of the acquired signal. 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. For Strand Sequencing, salt concentrations in excess of 100 mM are ideal and salt concentrations of 1 M and above are preferred. The inventors have surprisingly shown that Hel308 helicases will function effectively at salt concentrations as high as, for example, 2 M.
Second, when a voltage is applied, Hel308 helicases can surprisingly move the target polynucleotide in two directions, namely with or against the field resulting from the applied 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 through the pore, ie in the direction of or against the field. This is discussed in more detail below.
Third, Hel308 helicases typically move the target polynucleotide through the pore one nucleotide at a time. Hel308 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, Hel308 helicases are capable of controlling the movement of single stranded polynucleotides and double stranded polynucleotides. This means that a variety of different target polynucleotides can be characterised in accordance with the invention.
Fifth, Hel308 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. 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, Hel308 helicases are easy to produce and easy to handle. Their use therefore 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 polynucleotide can be oxidized or methylated. One or more nucleotides in the target 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. 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 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 monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, 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 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), 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 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 expected.
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 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 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 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 typically stored prior to assay, preferably below −70° C.
A transmembrane pore is a structure that permits hydrated ions driven by an applied potential to flow from one side of the membrane to the other side of the membrane.
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 hydrophilic and lipophilic properties. The amphiphilic layer may be a monolayer or a bilayer.
The membrane is preferably a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as bio sensors 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 Application No. PCT/GB08/000,563 (published as WO 2008/102121), International Application No. PCT/GB08/004,127 (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 (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 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/GB08/004,127 (published as WO 2009/077734).
In another preferred embodiment, the membrane is a solid state layer. A solid-state layer is not of biological origin. In other words, a solid state layer is not derived from or isolated from a biological environment such as an organism or cell, or a synthetically 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 Si3N4, Al2O3, and SiO, organic and inorganic polymers such as polyamide, plastics such 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 bilayer comprising a pore, (ii) an isolated, naturally-occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is preferably carried out using an artificial lipid bilayer. The bilayer 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.
The polynucleotide may be coupled to the membrane. This may be done using any known method. If the membrane is an amphiphilic layer, such as a lipid bilayer (as discussed in detail above), the polynucleotide is preferably 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.
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 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 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 polynucleotide may be transiently coupled to an amphiphilic layer or lipid bilayer using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from 6 to 30 carbon atoms, such as hexadecanoic acid, may be used.
In preferred embodiments, polynucleotide is coupled to a lipid bilayer. Coupling of polynucleotides to synthetic lipid bilayers has been carried out previously with 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




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 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 either end of DNA has been reported previously. A thiol group can be added to the 5′ of ssDNA using polynucleotide kinase and ATPγS (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 (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 (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 USA 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. 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.
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 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 polypeptide or a collection of polypeptides that permits hydrated ions, such as analyte, to flow from one side of a membrane to the other side of the membrane. In the present invention, the transmembrane protein pore is capable of forming a pore that permits hydrated ions driven by an applied potential to flow from one side of the membrane to the other. The transmembrane protein pore preferably permits analyte such as nucleotides to flow from one side of the membrane, such as a lipid bilayer, to the other. 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 or 8 subunits. The pore is more preferably a heptameric or octameric 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 β barrel or channel or a transmembrane α-helix bundle or channel.
The barrel or channel of the transmembrane protein pore typically comprises amino acids 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, polynucleotides or nucleic acids.
Transmembrane protein pores for use in accordance with the invention can be derived from β-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-strands. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, 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). α-helix bundle pores comprise a barrel or channel that is formed from α-helices. Suitable α-helix bundle pores include, but are not limited to, inner membrane proteins and a outer membrane proteins, such as WZA and ClyA toxin. The transmembrane pore may be derived from Msp or from α-hemolysin (α-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 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 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 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 membranes, such as lipid bilayers. 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 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 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 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 et at (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et at (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/).
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 variant may comprise one or more of the following mutations present in MspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, 149H, 168V, D91G, A96Q, N102D, S103T, V104I, 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 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 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 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


















Ala
aliphatic, hydrophobic,
Met
hydrophobic, neutral



neutral




Cys
polar, hydrophobic, neutral
Asn
polar, hydrophilic, neutral


Asp
polar, hydrophilic,
Pro
hydrophobic, neutral



charged (−)




Glu
polar, hydrophilic,
Gln
polar, hydrophilic, neutral



charged (−)




Phe
aromatic, hydrophobic,
Arg
polar, hydrophilic,



neutral

charged (+)


Gly
aliphatic, neutral
Ser
polar, hydrophilic, neutral


His
aromatic, polar, hydrophilic,
Thr
polar, hydrophilic, neutral



charged (+)




Ile
aliphatic, hydrophobic,
Val
aliphatic, hydrophobic,



neutral

neutral


Lys
polar, hydrophilic,
Trp
aromatic, hydrophobic,



charged (+)

neutral


Leu
aliphatic, hydrophobic,
Tyr
aromatic, polar,



neutral

hydrophobic
















TABLE 3







Hydropathy scale










Side Chain
Hydropathy













Ile
4.5



Val
4.2



Leu
3.8



Phe
2.8



Cys
2.5



Met
1.9



Ala
1.8



Gly
−0.4



Thr
−0.7



Ser
−0.8



Trp
−0.9



Tyr
−1.3



Pro
−1.6



His
−3.2



Glu
−3.5



Gln
−3.5



Asp
−3.5



Asn
−3.5



Lys
−3.9



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.
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: 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 extension may be quite short, for example from 1 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.
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 β-barrel, is provided by β-sheets in each subunit. A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form β-sheets. One or more modifications can be made to the regions of SEQ ID NO: 2 that form β-sheets 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 α-helices and/or loop regions.
The monomers derived from Msp may be modified to assist their identification or 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 engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 July; 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. 125I, 35S, enzymes, antibodies, 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 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 amino acids by reaction with an aldehyde followed by reduction with NaBH4, 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 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/GB09/001,690 (published as WO 2010/004273), PCT/GB09/001,679 (published as WO 2010/004265) or PCT/GB10/000,133 (published as WO 2010/086603). Methods for inserting pores into membranes are discussed.
The transmembrane protein pore is also preferably derived from α-hemolysin (α-HL). The wild type α-HL pore is formed of seven identical monomers or subunits (i.e. it is heptameric). The sequence of one monomer or subunit of α-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 to 51, 63 to 66, 72, 92 to 97, 104 to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217, 218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290 and 294 of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4 form part of a constriction of the barrel or channel of α-HL.
In such embodiments, a pore comprising seven proteins or monomers each comprising 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 form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into 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 membranes, such as lipid bilayers. Suitable methods are discussed above.
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 position 8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 with cysteine (A8C, T9C, N17C, K237C, S239C or E287C). The variant is preferably any one of the variants described in International Application No. PCT/GB09/001,690 (published as WO 2010/004273), PCT/GB09/001,679 (published as WO 2010/004265) or PCT/GB10/000,133 (published as WO 2010/086603).
The variant may also include modifications that facilitate any interaction with 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 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 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. 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 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 the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. The extension may be quite short, for example from 1 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 sequence which varies from that of SEQ ID NO: 4 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 4 that are responsible for pore formation. The pore forming ability of α-HL, which contains a β-barrel, is provided by β-strands in each subunit. A variant of SEQ ID NO: 4 typically comprises the regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQ ID NO: 4 that form β-strands are discussed above. One or more modifications can be made to the regions of SEQ ID NO: 4 that form β-strands as long as the resulting variant retains its ability to form a pore. Specific modifications that can be made to the β-strand regions of SEQ ID NO: 4 are discussed above.
A variant of SEQ ID NO: 4 preferably includes one or more modifications, such as substitutions, additions or deletions, within its α-helices and/or loop regions. Amino acids that form α-helices and loops are discussed above.
The variant may be modified to assist its identification or purification as discussed above.
Pores derived from α-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 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 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.
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.
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/GB09/001,690 (published as WO 2010/004273), PCT/GB09/001,679 (published as WO 2010/004265) or PCT/GB10/000,133 (published as WO 2010/086603).
Any Hel308 helicase may be used in accordance with the invention. Hel308 helicases are also known as ski2-like helicases and the two terms can be used interchangeably.
The Hel308 helicase typically comprises the amino acid motif Q-X1-X2-G-R-A-G-R (hereinafter called the Hel308 motif; SEQ ID NO: 8). The Hel308 motif is typically part of the helicase motif VI (Tuteja and Tuteja, air. J. Biochem 271, 1849-1863 (2004)). X1 may be C, M or L. X1 is preferably C. X2 may be any amino acid residue. X2 is typically a hydrophobic or neutral residue. X2 may be A. M, C, V, S, T, P or R. X2 is preferably A, F, M, C, V, L, I, S, T or P. X2 is more preferably A, M or L. X2 is most preferably A or M.
The Hel308 helicase preferably comprises the motif Q-X1-X2-G-R-A-G-R-P (hereinafter called the extended Hel308 motif; SEQ ID NO: 9) wherein X1 and X2 are as described above.
The most preferred Hel308 motifs and extended Hel308 motifs are shown in Table 5 below. The Hel308 helicase may comprise any of these preferred motifs.
The Hel308 helicase is preferably one of the helicases shown in Table 4 below or a variant thereof.







TABLE 4







Preferred Hel308 helicases








Accession
Description





NP_578406.1
ski2-like helicase [Pyrococcus furiosus DSM 3638] >sp|O73946.1|HELS_PYRFU



RecName: Full = Putative ski2-type helicase >pdb|2ZJ2|A Chain A, Archaeal Dna



Helicase Hjm Apo State In Form 1 >pdb|2ZJ5|A Chain A, Archaeal Dna Helicase



Hjm Complexed With Adp In Form 1 >pdb|2ZJ8|A Chain A, Archaeal Dna



Helicase Hjm Apo State In Form 2 >pdb|2ZJA|A Chain A, Archaeal Dna Helicase



Hjm Complexed With Amppcp In Form 2 >dbj|BAA32016.1| helicase



[Pyrococcus furiosus] >gb|AAL80801.1| helicase [Pyrococcus furiosus DSM



3638]


NP_126564.1
ski2-like helicase [Pyrococcus abyssi GE5] >sp|Q9V0A9.1|HELS_PYRAB



RecName: Full = Putative ski2-type helicase >emb|CAB49795.1| DNA helicase



[Pyrococcus abyssi GE5]


NP_143168.1
ski2-like helicase [Pyrococcus horikoshii OT3] >sp|O59025.1|HELS_PYRHO



RecName: Full = Putative ski2-type helicase >dbj|BAA30383.1| 715aa long



hypothetical protein [Pyrococcus horikoshii OT3]


YP_004424773.1
ski2-like helicase [Pyrococcus sp.NA2] >gb|AEC52769.1| ski2-like helicase



[Pyrococcus sp. NA2]


YP_004623750.1
ski2-like helicase [Pyrococcus yayanosii CH1] >gb|AEH24478.1| ski2-like



helicase [Pyrococcus yayanosii CH1]


YP_002307730.1
ski2-like helicase [Thermococcus onnurineus NA1] >gb|ACJ16833.1| DNA



helicase [Thermococcus onnurineus NA1]


YP_004763427.1
ski2-like helicase [Thermococcus sp. 4557] >gb|AEK73750.1| ski2-like helicase



[Thermococcus sp. 4557]


YP_002959236.1
ski2-like helicase [Thermococcus gammatolerans EJ3] >gb|ACS33372.1| ski2-



type helicase, putative [Thermococcus gammatolerans EJ3]


YP_004071709.1
ski2-type helicase [Thermococcus barophilus MP] >gb|ADT84486.1| putative



ski2-type helicase [Thermococcus barophilus MP]


YP_002994328.1
Putative ski2-type helicase [Thermococcus sibiricus MM 739] >gb|ACS89979.1|



Putative ski2-type helicase [Thermococcus sibiricus MM 739]


ZP_04875329.1
Type III restriction enzyme, res subunit family [Aciduliprofundum boonei



T469] >gb|EDY35111.1| Type III restriction enzyme, res subunit family



[Aciduliprofundum boonei T469]


YP_003436565.1
DEAD/DEAH box helicase [Ferroglobus placidus DSM 10642] >gb|ADC66290.1|



DEAD/DEAH box helicase domain protein [Ferroglobus placidus DSM 10642]


YP_004485304.1
ski2-type helicase [Methanotorris igneus Kol 5] >gb|AEF97239.1| ski2-type



helicase [Methanotorris igneus Kol 5]


YP_004616424.1
DEAD/DEAH box helicase domain-containing protein [Methanosalsum zhilinae



DSM 4017] >gb|AEH61205.1| DEAD/DEAH box helicase domain protein



[Methanosalsum zhilinae DSM 4017]


ZP_04873370.1
Type III restriction enzyme, res subunit family [Aciduliprofundum boonei



T469] >ref|YP_003482774.1| DEAD/DEAH box helicase domain protein



[Aciduliprofundum boonei T469] >gb|EDY36687.1| Type III restriction enzyme,



res subunit family [Aciduliprofundum boonei T469] >gb|ADD08212.1|



DEAD/DEAH box helicase domain protein [Aciduliprofundum boonei T469]


YP_004342552.1
ski2-type helicase [Archaeoglobus veneficus SNP6] >gb|AEA47837.1| ski2-type



helicase [Archaeoglobus veneficus SNP6]


NP_071282.1
SKI2-family helicase [Archaeoglobus fulgidus DSM 4304]


2P6R_A
Chain A, Crystal Structure Of Superfamily 2 Helicase Hel308 In Complex With



Unwound Dna >pdb|2P6U|A Chain A, Apo Structure Of The Hel308 Superfamily



2 Helicase


YP_685308.1
ski2-like helicase [uncultured methanogenic archaeon



RC-I] >sp|Q0W6L1.1|HELS_UNCMA RecName: Full = Putative ski2-type



helicase >emb|CAJ35982.1| putative ski2-type helicase [uncultured



methanogenic archaeon RC-I]


YP_001048404.1
ski2-like helicase [Methanoculleus marisnigri JR1] >gb|ABN58422.1|



DEAD/DEAH box helicase domain protein [Methanoculleus marisnigri JR1]


YP_919908.1
DEAD/DEAH box helicase domain-containing protein [Thermofilum pendens



Hrk 5] >gb|ABL77905.1| DEAD/DEAH box helicase domain protein



[Thermofilum pendens Hrk 5]


YP_843229.1
ski2-like helicase [Methanosaeta thermophila PT] >gb|ABK14589.1|



DEAD/DEAH box helicase domain protein [Methanosaeta thermophila PT]


ZP_08045937.1
ski2-like helicase [Haladaptatus paucihalophilus DX253] >gb|EFW90585.1| ski2-



like helicase [Haladaptatus paucihalophilus DX253]


NP_280985.1
ski2-like helicase [Halobacterium sp.NRC-1] >ref|YP_001690117.1| ski2-like



helicase [Halobacterium salinarum R1] >sp|Q9HMV6.1|HELS_HALSA



RecName: Full = Putative ski2-type helicase >sp|B0R7Q2.1|HELS_HALS3



RecName: Full = Putative ski2-type helicase >gb|AAG20465.1| DNA repair



protein [Halobacterium sp. NRC-1] >emb|CAP14771.1| putative DNA helicase



[Halobacterium salinarum R1]


YP_003357840.1
Holliday junction migration helicase [Methanocella paludicola



SANAE] >dbj|BAI62857.1| Holliday junction migration helicase



[Methanocella paludicola SANAE]


YP_003457479.1
DEAD/DEAH box helicase domain protein [Methanocaldococcus sp.



FS406-22] >gb|ADC68743.1| DEAD/DEAH box helicase domain protein



[Methanocaldococcus sp. FS406-22]


YP_003127632.1
DEAD/DEAH box helicase domain protein [Methanocaldococcus fervens



AG86] >gb|ACV24132.1| DEAD/DEAH box helicase domain protein



[Methanocaldococcus fervens AG86]



ski2-like helicase [Halalkalicoccus jeotgali B3] >gb|ADJ13543.1| ski2-like


YP_003735335.1
helicase [Halalkalicoccus jeotgali B3]


YP_503885.1
ski2-1ike helicase [Methanospirillum hungatei JF-1] >gb|ABD42166.1|



DEAD/DEAH box helicase-like protein [Methanospirillum hungatei JF-1]


BAJ48115.1
helicase [Candidatus Caldiarchaeum subterraneum] >dbj|BAJ48144.1| helicase



[Candidatus Caldiarchaeum subterraneum] >dbj|BAJ50919.1| helicase



[Candidatus Caldiarchaeum subterraneum]


YP_001405615.1
ski2-like helicase [Candidatus Methanoregula boonei



6A8] >sp|A7IB61.1|HELS_METB6 RecName: Full = Putative ski2-type



helicase >gb|ABS56972.1| DEAD/DEAH box helicase domain protein



[Methanoregula boonei 6A8]


YP_306959.1
ski2-like helicase [Methanosarcina barkeri str.



Fusaro] >sp|Q465R3.1|HELS_METBF RecName: Full = Putative ski2-type



helicase >gb|AAZ72379.1| helicase [Methanosarcina barkeri str. Fusaro]


YP_001031179.1
ski2-like helicase [Methanocorpusculum labreanum Z] >gb|ABN07912.1|



DEAD/DEAH box helicase domain protein [Methanocorpusculum labreanum Z]


YP_003541733.1
DEAD/DEAH box helicase [Methanohalophilus mahii DSM



5219] >gb|ADE36088.1| DEAD/DEAH box helicase domain protein



[Methanohalophilus mahii DSM 5219]


YP_004384692.1
putative Ski2-type helicase [Methanosaeta concilii GP6] >gb|AEB68874.1|



putative Ski2-type helicase [Methanosaeta concilii GP6]


YP_003725904.1
DEAD/DEAH box helicase domain-containing protein [Methanohalobium



evestigatum Z-7303] >gb|AD173108.1| DEAD/DEAH box helicase domain



protein [Methanohalobium evestigatum Z-7303]


YP_003405271.1
DEAD/DEAH box helicase [Haloterrigena tarkmenica DSM



5511] >gb|ADB62598.1| DEAD/DEAH box helicase domain protein



[Haloterrigena turkmenica DSM 5511]


YP_004244914.1
DEAD/DEAH box helicase [Vulcanisaeta moutnovskia



768-28] >gb|ADY01412.1| DEAD/DEAH box helicase domain protein



[Vulcanisaeta moutnovskia 768-28]


YP_001540156.1
DEAD/DEAH box helicase domain-containing protein [Caldivirga



maquilingensis IC-167] >sp|A8MB76.1|HELS_CALMQ RecName: Full = Putative



ski2-type helicase >gb|ABW01166.1| DEAD/DEAH box helicase domain protein



[Caldivirga maquilingensis IC-167]


NP_618094.1
ski2-like helicase [Methanosarcina acetivorans



C2A] >sp|Q8TL39.1|HELS_METAC RecName: Full = Putative ski2-type



helicase >gb|AAM06574.1| helicase [Methanosarcina acetivorans C2A]


YP_003900980.1
DEAD/DEAH box helicase domain-containing protein [Vulcanisaeta distributa



DSM 14429] >gb|ADN49929.1| DEAD/DEAH box helicase domain protein



[Vulcanisaeta distributa DSM 14429]


YP_003896003.1
DEAD/DEAH box helicase domain-containing protein [Methanoplanus



petrolearius DSM 11571] >gb|ADN37565.1| DEAD/DEAH box helicase domain



protein [Methanoplanus petrolearius DSM 11571]


YP_003615773.1
DEAD/DEAH box helicase domain protein [Methanocaldococcus infernus



ME] >gb|ADG12809.1| DEAD/DEAH box helicase domain protein



[Methanocaldococcus infernus ME]


YP_183745.1
RNA helicase Ski2-like protein [Thermococcus kodakarensis



KOD1] >sp|Q5JGV6.1|HELS_PYRKO RecName: Full = Putative ski2-type



helicase; Contains: RecName: Full = Endonuclease PI-PkoHel; AltName:



Full = Pko Hel intein >dbj|BAD85521.1| RNA helicase Ski2 homolog



[Thermococcus kodakarensis KOD1]


YP_001322557.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus vannielii



SB] >sp|A6UN73.1|HELS_METVS RecName: Full = Putative ski2-type



helicase >gb|ABR53945.1| DEAD/DEAH box helicase domain protein



[Methanococcus vannielii SB]


YP_002467772.1
ski2-like helicase [Methanosphaerula palustris E1-9c] >gb|ACL18049.1|



DEAD/DEAH box helicase domain protein [Methanosphaerula palustris E1-9c]


YP_003480097.1
DEAD/DEAH box helicase [Natrialba magadii ATCC 43099] >gb|ADD05535.1|



DEAD/DEAH box helicase domain protein [Natrialba magadii ATCC 43099]


YP_004577043.1
ski2-type helicase [Methanothermococcus okinawensis IH1] >gb|AEH07265.1|



ski2-type helicase [Methanothermococcus okinawensis IH1]


YP_004742641.1
superfamily II helicase [Methanococcus maripaludis XI] >gb|AEK19898.1|



superfamily II helicase [Methanococcus maripaludis X1]


NP_632449.1
ski2-like helicase [Methanosarcina mazei Go1] >sp|Q8PZR7.1|HELS_METMA



RecName: Full = Putative ski2-type helicase >gb|AAM30121.1| helicase



[Methanosarcina mazei Go1]


YP_001097223.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis C5] >gb|ABO35008.1| DEAD/DEAH box helicase domain protein



[Methanococcus maripaludis C5]


YP_004742247.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis XI] >gb|AEK19504.1| DEAD/DEAH box helicase domain-containing



protein [Methanococcus maripaludis X1]


YP_004794766.1
ski2-like helicase [Haloarcula hispanica ATCC 33960] >gb|AEM55778.1| ski2-



like helicase [Haloarcula hispanica ATCC 33960]


NP_988010.1
superfamily II helicase [Methanococcus maripaludis S2] >emb|CAF30446.1|



superfamily II helicase [Methanococcus maripaludis S2]


YP_565780.1
ski2-like helicase [Methanococcoides burtonii DSM



6242] >sp|Q12WZ6.1|HELS_METBU RecName: Full = Putative ski2-type



helicase >gb|ABE52030.1| DEAD/DEAH box helicase-like protein



[Methanococcoides burtonii DSM 6242]


YP_001549808.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis C6] >gb|ABX02576.1| DEAD/DEAH box helicase domain protein



[Methanococcus maripaludis C6]


YP_001548609.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis C6] >gb|ABX01377.1| DEAD/DEAH box helicase domain protein



[Methanococcus maripaludis C6]


YP_001329359.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis C7] >gb|ABR65208.1| DEAD/DEAH box helicase domain protein



[Methanococcus maripaludis C7]


YP_004595982.1
ski2-type helicase [Halopiger xanaduensis SH-6] >gb|AEH36103.1|



ski2-type helicase [Halopiger xanaduensis SH-6]


YP_656795.1
ski2-like helicase [Haloquadratum walsbyi DSM 16790] >emb|CAJ51138.1|



ATP-dependent DNA helicase [Haloquadratum walsbyi DSM 16790]


CCC38992.1
ATP-dependent DNA helicase Hel308 [Haloquadratum walsbyi C23]


YP_004035272.1
superfamily ii helicase [Halogeometricum borinquense DSM



11551] >gb|ADQ65833.1| superfamily II helicase



[Halogeometricum borinquense DSM 11551]


YP_137330.1
ski2-like helicase [Haloarcula marismortui ATCC



43049] >sp|Q5UYM9.1|HELS_HALMA RecName: Full = Putative ski2-type



helicase >gb|AAV47624.1| putative ski2-type helicase



[Haloarcula marismortui ATCC 43049]


YP_001581577.1
DEAD/DEAH box helicase domain-containing protein [Nitrosopumilus



maritimus SCM1] >gb|ABX12139.1| DEAD/DEAH box helicase domain protein



[Nitrosopumilus maritimus SCM1]


EET90255.1
DEAD/DEAH box helicase domain protein [Candidatus Micrarchaeum



acidiphilum ARMAN-2]


NP_376477.1
helicase [Sulfolobus tokodaii str. 7] >sp|Q974S1.1|HELS_SULTO RecName:



Full = Putative ski2-type helicase >dbj|BAK54341.1| Holliday junction migration



helicase [Sulfolobus tokodaii str. 7]


YP_001097792.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis C5] >gb|ABO35578.1| DEAD/DEAH box helicase domain protein



[Methanococcus maripaludis C5]


ZP_08667240.1
DEAD/DEAH box helicase domain protein [Nitrosopumilus sp.



MY1] >gb|EGP92972.1| DEAD/DEAH box helicase domain protein [Nitrosopumilus



sp. MY1]


YP_254972.1
DNA helicase [Sulfolobus acidocaldarius DSM



639] >sp|Q4JC00.1|HELS_SULAC RecName: Full = Putative ski2-type



helicase >gb|AAY79679.1| DNA helicase [Sulfolobus acidocaldarius



DSM 639]


EFD92533.1
DEAD/DEAH box helicase domain protein [Candidatus Parvarchaeum



acidophilus ARMAN-5]


YP_003176527.1
ski2-like helicase [Halomicrobium mukohataei DSM 12286] >gb|ACV46820.1|



DEAD/DEAH box helicase domain protein [Halomicrobium mukohataei DSM12286]


EGD71904.1
DEAD/DEAH box helicase domain protein [Candidatus Parvarchaeum



acidophilus ARMAN-5_‘5-way FS’]


YP_001040230.1
DEAD/DEAH box helicase domain-containing protein [Staphylothermus marinus



F1] >gb|ABN69322.1| DEAD/DEAH box helicase domain protein



[Staphylothermus marinus F1]


ABZ07376.1
putative DEAD/DEAH box helicase [uncultured marine crenarchaeote



HF4000_ANIW133M9]


YP_001097458.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus



maripaludis C5] >gb|ABO35243.1| DEAD/DEAH box helicase domain protein



[Methanococcus maripaludis C5]


ABZ08606.1
putative DEAD/DEAH box helicase [uncultured marine crenarchaeote



HF4000_APKG3H9]


YP_325906.1
ski2-like helicase [Natronomonas pharaonis DSM



2160] >sp|Q31U46.1|HELS_NATPD RecName: Full = Putative ski2-type



helicase >emb|CAI48337.1| ATP-dependent DNA helicase 1



[Natronomonas pharaonis DSM 2160]


YP_930665.1
DEAD/DEAH box helicase domain-containing protein [Pyrobaculum islandicum



DSM 4184] >gb|ABL88322.1| DEAD/DEAH box helicase domain protein



[Pyrobaculum islandicum DSM 4184]


YP_001435870.1
DEAD/DEAH box helicase [Ignicoccus hospitalis KIN4/1] >gb|ABU82463.1|



DEAD/DEAH box helicase domain, protein [Ignicoccus hospitalis KIN4/I]


YP_003668634.1
DEAD/DEAH box helicase domain-containing protein [Staphylothermus



hellenicus DSM 12710] >gb|ADI31735.1| DEAD/DEAH box helicase domain



protein [Staphylothermus hellenicus DSM 12710]


ZP_08558598.1
ski2-like helicase [Halorhabdus tiamatea SARL4B] >gb|EGM36528.1| ski2-like



helicase [Halorhabdus tiamatea SARL4B]


YP_002428409.1
DEAD/DEAH box helicase domain-containing protein [Desulfurococcus



kamchatkensis 1221n] >gb|ACL11042.1| DEAD/DEAH box helicase domain



protein [Desulfurococcus kamchatkensis 1221n]


YP_004336918.1
ATP-dependent, DNA binding helicase [Thermoproteus uzoniensis



768-20] >gb|AEA11606.1| ATP-dependent, DNA binding helicase



[Thermoproteus uzoniensis 768-20]


ZP_08257442.1
DEAD/DEAH box helicase domain-containing protein [Candidatus



Nitrosoarchaeum limnia SFB1] >gb|EGG41989.1| DEAD/DEAH box helicase



domain-containing protein [Candidatus Nitrosoarchaeum limnia SFB1]


YP_004459284.1
DEAD/DEAH box helicase domain-containing protein [Acidianus hospitalis



W1] >gb|AEE94986.1| DEAD/DEAH box helicase domain protein



[Acidianus hospitalis W1]


NP_558924.1
ATP-dependent, DNA binding helicase [Pyrobaculum aerophilum str.



IM2] >gb|AAL63106.1| ATP-dependent, DNA binding helicase



[Pyrobaculum aerophilum str. IM2]


YP_004409449.1
DEAD/DEAH box helicase domain-containing protein [Metallosphaera cuprina



Ar-4] >gb|AEB94965.1| DEAD/DEAH box helicase domain-containing protein



[Metallosphaera cuprina Ar-4]


YP_003649556.1
DEAD/DEAH box helicase domain-containing protein [Thermosphaera



aggregans DSM 11486] >gb|ADG90604.1| DEAD/DEAH box helicase domain



protein [Thermosphaera aggregans DSM 11486]


ZP_06387115.1
DEAD/DEAH box helicase domain protein [Sulfolobus solfataricus



98/2] >gb|ACX90562.1| DEAD/DEAH box helicase domain protein



[Sulfolobus solfataricus 98/2]


2VA8_A
Chain A, Dna Repair Helicase Hel308 >pdb|2VA8|B Chain B, Dna Repair



Helicase Hel308 >emb|CAO85626.1| DNA helicase [Sulfolobus solfataricus]


YP_004809267.1
ski2-type helicase [halophilic archaeon DL31] >gb|AEN06894.1| ski2-type



helicase [halophilic archaeon DL31]


ADX84345.1
DEAD/DEAH box helicase domain protein [Sulfolobus islandicus



REY15A] >gb|ADX81629.1| DEAD/DEAH box helicase domain protein



[Sulfolobus islandicus HVE10/4]


YP_002828439.1
DEAD/DEAH box helicase [Sulfolobus islandicus



M.14.25] >ref|YP_002842325.1| DEAD/DEAH box helicase domain protein [Sulfolobus



islandicus M.16.27] >gb|ACP37141.1| DEAD/DEAH box helicase domain protein



[Sulfolobus islandicus M.14.25] >gb|ACP54280.1| DEAD/DEAH box helicase



domain protein [Sulfolobus islandicus M.16.27]


YP_002913571.1
DEAD/DEAH box helicase domain protein [Sulfolobus islandicus



M.16.41 >gb|ACR40903.1| DEAD/DEAH box helicase domain protein



[Sulfolobus islandicus M.16.4]


Q97VY9.1
RecName: Full = Putative ski2-type helicase


YP_002841682.1
DEAD/DEAH box helicase domain protein [Sulfolobus islandicus



Y.N.15.51] >gb|ACP49760.1| DEAD/DEAH box helicase domain protein



[Sulfolobus islandicus Y.N.15.51]


YP_002831080.1
DEAD/DEAH box helicase domain protein [Sulfolobus islandicus



L.S.2.15] >ref|YP_003418425.1| DEAD/DEAH box helicase domain protein



[Sulfolobus islandicus L.D.8.5] >gb|ACP34435.1| DEAD/DEAH box helicase



domain protein [Sulfolobus islandicus L.S.2.15] >gb|ADB86055.1|



DEAD/DEAH box helicase domain protein [Sulfolobus islandicus L.D.8.5]


YP_001054984.1
DEAD/DEAH box helicase domain-containing protein [Pyrobaculum calidifontis



JCM 11548] >sp|A3MSA1.1|HELS_PYRCJ RecName: Full = Putative ski2-type



helicase >gb|ABO07518.1| DEAD/DEAH box helicase domain protein



[Pyrobaculum calidifontis JCM 11548]


NP_343811.1
DNA helicase related protein [Sulfolobus solfataricus P2] >ref|YP_002836469.1|



DEAD/DEAH box helicase [Sulfolobus islandicus Y.G.57.14] >gb|AAK42601.1|



DNA helicase related protein [Sulfolobus solfataricus P2] >gb|ACP44547.1|



DEAD/DEAH box helicase domain protein [Sulfolobus islandicus Y.G.57.14]


YP_001152379.1
DEAD/DEAH box helicase domain-containing protein [Pyrobaculum arsenaticum



DSM 135141 >gb|ABP49727.1| DEAD/DEAH box helicase domain protein



[Pyrobaculum arsenaticum DSM 13514]


YP_001191456.1
DEAD/DEAH box helicase domain-containing protein [Metallosphaera sedula



DSM 5348] >gb|ABP95532.1| DEAD/DEAH box helicase domain protein



[Metallosphaera sedula DSM 5348]


NP_147034.2
holliday junction migration helicase [Aeropyrum pernix



K1] >sp|Q9YFQ8.2|HELS_AERPE RecName: Full = Putative ski2-type



helicase >dbj|BAA79103.2| holliday junction migration helicase



[Aeropyrum pernix K1]


YP_024158.1
ski2-like helicase [Picrophilus torridus DSM 9790] >gb|AAT43965.1| helicase



involved in UV-protection [Picrophilus torridus DSM 9790]


YP_003816358.1
Putative ski2-type helicase [Acidilobus saccharovorans



345-15] >gb|ADL19327.1| Putative ski2-type helicase



[Acidilobus saccharovorans 345-15]


YP_003860265.1
DEAD/DEAH box helicase domain protein [Ignisphaera aggregans DSM



17230] >gb|ADM28385.1| DEAD/DEAH box helicase domain protein



[Ignisphaera aggregans DSM 17230]


NP_394295.1
ski2-like helicase [Thermoplasma acidophilum DSM



1728] >sp|Q9HJX7.1|HELS_THEAC RecName: Full = Putative ski2-type



helicase >emb|CAC11964.1| DNA helicase related protein



[Thermoplasma acidophilum]


YP_876638.1
superfamily II helicase [Cenarchaeum symbiosum A] >gb|ABK78334.1|



superfamily II helicase [Cenarchaeum symbiosum A]


ZP_05571398.1
ski2-like helicase [Ferroplasma acidarmanus fer1]


YP_004176252.1
DEAD/DEAH box helicase domain-containing protein [Desulfurococcus mucosus



DSM 2162] >gb|ADV64770.1| DEAD/DEAH box helicase domain protein



[Desulfurococcus mucosus DSM 2162]


YP_001737782.1
DEAD/DEAH box helicase domain-containing protein [Candidatus Korarchaeum



cryptofilum OPF8] >gb|ACB08099.1| DEAD/DEAH box helicase domain protein



[Candidatus Korarchaeum cryptofilum OPF8]


EGQ40435.1
superfamily II helicase [Candidatus Nanosalinarum sp. J07AB56]


YP_002567343.1
ski2-like helicase [Halorubrum lacusprofundi ATCC 49239] >gb|ACM58273.1|



DEAD/DEAH box helicase domain protein [Halorubrum lacusprofundi ATCC 49239]


YP_001793507.1
DEAD/DEAH box helicase domain-containing protein [Thermoproteus



neutrophilus V24Sta] >gb|ACB39061.1| DEAD/DEAH box helicase domain



protein [Thermoproteus neutrophilus V24Sta]


YP_003534088.1
ATP-dependent DNA helicase Hel308a [Haloferax volcanii



DS2] >gb|ADE04048.1| ATP-dependent DNA helicase Hel308a



[Haloferax volcanii DS2]


YP_004037165.1
superfamily ii helicase [Halogeometricum borinquense DSM



11551] >gb|ADQ67720.1| superfamily II helicase [Halogeometricum borinquense



DSM 11551]


NP_111333.1
ski2-like helicase [Thermoplasma volcanium



GSS1] >sp|Q97AI2.1|HELS_THEVO RecName: Full = Putative ski2-type



helicase >dbj|BAB59970.1| DNA helicase [Thermoplasma volcanium GSS1]


YP_002565871.1
DEAD/DEAH box helicase [Halorubrum lacusprofundi ATCC



49239] >gb|ACM56801.1| DEAD/DEAH box helicase domain protein.



[Halorubrum lacusprofundi ATCC 49239]


CCC39675.1
ATP-dependent DNA helicase Hel308 [Haloquadratum walsbyi C23]


YP_657401.1
ATP-dependent DNA helicase [Haloquadratum walsbyi DSM



16790] >emb|CAJ51759.1| ATP-dependent DNA helicase [Haloquadratum walsbyi



DSM 16790]


YP_003535028.1
ATP-dependent DNA helicase Hel308b [Haloferax volcanii



DS2] >gb|ADE02398.1| ATP-dependent DNA helicase Hel308b



[Haloferax volcanii DS2]


YP_003706863.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus voltae



A3] >gb|ADI35890.1| DEAD/DEAH box helicase domain protein



[Methanococcus voltae A3]


ABD17736.1
helicase [Methanococcus voltae PS]


NP_613398.1
superfamily II helicase [Methanopyrus kandleri AV19] >gb|AAM01328.1|



Predicted Superfamily II helicase [Methanopyrus kandleri AV19]


CBH38575.1
putative ski2-type helicase [uncultured archaeon]


EEZ93258.1
DEAD/DEAH box helicase domain protein [Candidatus Parvarchaeum



acidiphilum ARMAN-4]


EGQ40350.1
superfamily II helicase [Candidatus Nanosalinarum sp.J07AB56]


YP_004004246.1
dead/deah box helicase domain protein [Methanothermus fervidus DSM



2088] >gb|ADP77484.1| DEAD/DEAH box helicase domain protein



[Methanothermus fervidus DSM 2088]


YP_003850109.1
helicase [Methanothermobacter marburgensis str. Marburg] >gb|ADL58796.1|



predicted helicase [Methanothermobacter marburgensis str. Marburg]


YP_003424423.1
DEAD/DEAH box helicase domain-containing protein [Methanobrevibacter



ruminantium M1] >gb|ADC47531.1| DEAD/DEAH box helicase domain-



containing protein [Methanobrevibacter ruminantium M1]


YP_004291107.1
DEAD/DEAH box helicase domain-containing protein [Methanobacterium sp.



AL-21] >gb|ADZ10135.1| DEAD/DEAH box helicase domain protein



[Methanobacterium sp.AL-21]


YP_447162.1
helicase [Methanosphaera stadtmanae DSM 3091] >gb|ABC56519.1| predicted



helicase [Methanosphaera stadtmanae DSM 3091]


YP_004519549.1
DEAD/DEAH box helicase domain-containing protein [Methanobacterium sp.



SWAN-1] >gb|AEG17748.1| DEAD/DEAH box helicase domain protein



[Methanobacterium sp. SWAN-1]


NP_275949.1
DNA helicase related protein [Methanothermobacter thermautotrophicus str.



Delta H] >sp|O26901.1|HELS_METTH RecName: Full = Putative ski2-type



helicase >gb|AAB85310.1| DNA helicase related protein [Methanothermobacter



thermautotrophicus str. Delta H]


ZP_05975717.2
putative Ski2-type helicase [Methanobrevibacter smithii DSM



2374] >gb|EFC93382.1| putative Ski2-type helicase



[Methanobrevibacter smithii DSM 2374]


ZP_03607647.1
hypothetical protein METSMIALI_00751 [Methanobrevibacter smithii DSM



2375] >gb|EEE41862.1| hypothetical protein METSMIALI_00751



[Methanobrevibacter smithii DSM 2375]


YP_001273412.1
ATP-dependent helicase [Methanobrevibacter smithii ATCC



35061] >gb|ABQ87044.1| ATP-dependent helicase [Methanobrevibacter smithii



ATCC 35061]


YP_003247505.1
DEAD/DEAH box helicase domain protein [Methanocaldococcus vulcanius



M7] >gb|ACX73023.1| DEAD/DEAH box helicase domain protein



[Methanocaldococcus vulcanius M7]


NP_248116.1
SKI2 family helicase [Methanocaldococcus jannaschii DSM



2661] >sp|Q58524.1| HELS_METJA RecName: Full = Putative ski2-type helicase;



Contains: RecName: Full = Endonuclease PI-MjaHel; AltName: Full = Mja Hel



intein; AltName: Full = Mja Pep3 intein >gb|AAB99126.1| putative SKI2-family



helicase [Methanocaldococcus jannaschii DSM 2661]


YP_001324295.1
DEAD/DEAH box helicase domain-containing protein [Methanococcus aeolicus



Nankai-3] >gb|ABR55683.1| DEAD/DEAH box helicase domain protein



[Methanococcus aeolicus Nankai-3]


YP_003536960.1
Pre-mRNA splicing helicase [Haloferax volcanii DS2] >gb|ADE02332.1| Pre-



mRNA splicing helicase [Haloferax volcanii DS2]


YP_003131029.1
DEAD/DEAH box helicase domain protein [Halorhabdus utahensis DSM



12940] >gb|ACV12296.1| DEAD/DEAH box helicase domain protein



[Halorhabdus utahensis DSM 12940]


YP_002567151.1
DEAD/DEAH box helicase [Halorubrum lacusprofundi ATCC



49239] >gb|ACM58081.1| DEAD/DEAH box helicase domain protein



[Halorubrum lacusprofundi ATCC 49239]


YP_004035351.1
superfamily ii helicase [Halogeometricum borinquense DSM



11551] >gb|ADQ65912.1| superfamily II helicase [Halogeometricum borinquense



DSM11551]


YP_004808851.1
DEAD/DEAH box helicase domain-containing protein [halophilic archaeon



DL31] >gb|AEN06478.1| DEAD/DEAH box helicase domain protein [halophilic



archaeon DL31]


XP_002716686.1
PREDICTED: DNA polymerase theta isoform 1 [Oryctolagus cuniculus]


YP_656834.1
ATP-dependent DNA helicase [Haloquadratum walsbyi DSM



16790] >emb|CAJ51176.1| ATP-dependent DNA helicase [Haloquadratum walsbyi



DSM 16790]


XP_003248103.1
PREDICTED: DNA polymerase theta-like isoform 1 [Acyrthosiphon pisum]


ABC72356.1
ATP-dependent DNA helicase [Haloquadratum walsbyi]


CCC39031.1
DEAD/DEAH box helicase [Haloquadratum walsbyi C23]


XP_001165150.2
PREDICTED: DNA polymerase theta isoform 1 [Pan troglodytes]


XP_003225852.1
PREDICTED: DNA polymerase theta-like [Anolis carolinensis]


XP_615375.3
PREDICTED: DNA polymerase theta [Bos taurus] >ref|XP_002684835.1|



PREDICTED: polymerase (DNA directed), theta-like



[Bos taurus] >gb|DAA33456.1| polymerase (DNA directed),



theta-like [Bos taurus]


XP_002813286.1
PREDICTED: LOW QUALITY PROTEIN: DNA polymerase theta-like [Pongo



abelii]


AAR08421.2
DNA polymerase theta [Homo sapiens]


EAW79510.1
polymerase (DNA directed), theta, isoform CRA_a [Homo sapiens]


NP_955452.3
DNA polymerase theta [Homo sapiens] >sp|O75417.2|DPOLQ_HUMAN



RecName: Full = DNA polymerase theta; AltName: Full = DNA polymerase



eta >gb|AAI72289.1| Polymerase (DNA directed), theta [synthetic polynucleotide]


NP_001099348.1
DNA polymerase theta [Rattus norvegicus] >gb|EDM11249.1| polymerase (DNA



directed), theta (predicted), isoform CRA_a [Rattus norvegicus]


XP_003341262.1
PREDICTED: LOW QUALITY PROTEIN: DNA polymerase theta-like



[Monodelphis domestica]


XP_001502374.3
PREDICTED: DNA polymerase theta [Equus caballus]


XP_545125.3
PREDICTED: LOW QUALITY PROTEIN: DNA polymerase theta [Canis lupus



familiaris]


XP_002928855.1
PREDICTED: LOW QUALITY PROTEIN: DNA polymerase theta-like



[Ailuropoda melanoleuca]


NP_084253.1
DNA polymerase theta isoform 1 [Mus musculus] >gb|AAL77225.1| DNA



polymerase theta [Mus musculus] >gb|EDK97951.1| polymerase (DNA directed),



theta, isoform CRA_a [Mus musculus] >gb|AAI38361.1| Polymerase (DNA



directed), theta [Mus musculus] >gb|AAI57901.1| Polymerase (DNA directed),



theta [Mus musculus]


AAK39635.1
DNA polymerase theta [Homo sapiens]


AAN39838.1
DNA polymerase Q [Mus musculus]


XP_003412882.1
PREDICTED: DNA polymerase theta [Loxodonta africana]


YP_003735206.1
DEAD/DEAH box helicase domain-containing protein [Halalkalicoccus jeotgali



B3] >gb|ADJ13414.1| DEAD/DEAH box helicase domain protein



[Halalkalicoccus jeotgali B3]


YP_004794841.1
pre-mRNA splicing helicase [Haloarcula hispanica ATCC



33960] >gb|AEM55853.1| pre-mRNA splicing helicase



[Haloarcula hispanica ATCC 33960]


XP_416549.2
PREDICTED: similar to DNA polymerase theta [Gallus gallus]


XP_003427319.1
PREDICTED: helicase POLQ-like isoform 2 [Nasonia vitripennis]


XP_003202748.1
PREDICTED: DNA polymerase theta-like [Meleagris gallopavo]


XP_969311.1
PREDICTED: similar to DNA polymerase theta



[Tribolium castaneum] >gb|EEZ97532.1| hypothetical protein



TcasGA2_TC011380 [Tribolium castaneum]


ZP_08046037.1
DEAD/DEAH box helicase domain protein [Haladaptatus paucihalophilus



DX253] >gb|EFW90685.1| DEAD/DEAH box helicase domain protein



[Haladaptatus paucihalophilus DX253]


YP 461714.1
helicase [Syntrophus aciditrophicus SB] >gb|ABC77546.1| helicase [Syntrophus



aciditrophicus SB]


YP 003176510.1
DEAD/DEAH box helicase [Halomicrobium mukohataei DSM



12286] >gb|ACV46803.1] DEAD/DEAH box helicase domain protein



[Halomicrobium mukohataei DSM 12286]


YP_137400.1
Pre-mRNA splicing helicase [Haloarcula marismortui ATCC



43049] >gb|AAV47694.1| Pre-mRNA splicing helicase [Haloarcula marismortui



ATCC 43049]


NP_001184156.1
polymerase (DNA directed), theta [Xenopus (Silurana) tropicalis]


NP_280861.1
pre-mRNA splicing helicase [Halobacterium sp. NRC-1] >ref|YP_001689987.1|



ATP-dependent DNA helicase [Halobacterium salinarum R1] >gb|AAG20341.1|



pre-mRNA splicing helicase [Halobacterium sp.NRC-1] >emb|CAP14641.1|



ATP-dependent DNA helicase [Halobacterium salinarum R1]


YP_004595640.1
DEAD/DEAH box helicase domain-containing protein [Halopiger xanaduensis



SH-6] >gb|AEH35761.1| DEAD/DEAH box helicase domain protein [Halopiger



xanaduensis SH-6]


XP_001521144.2
PREDICTED: DNA polymerase theta, partial [Ornithorhynchus anatinus]


XP_003261953.1
PREDICTED: DNA polymerase theta, partial [Nomascus leucogenys]


XP_001358456.2
GA19301 [Drosophila pseudoobscura pseudoobscura] >gb|EAL27595.2|



GA19301 [Drosophila pseudoobscura pseudoobscura]


ZP_08560003.1
DEAD/DEAH box helicase domain protein [Halorhabdus tiamatea



SARL4B] >gb|EGM34502.1| DEAD/DEAH box helicase domain protein



[Halorhabdus tiamatea SARL4B]


XP_002187783.1
PREDICTED: similar to polymerase (DNA directed), theta [Taeniopygia guttata]


XP_002112587.1
hypothetical protein TRIADDRAFT_25163 [Trichoplax adhaerens] >gb|EDV24697.1|



hypothetical protein TRIADDRAFT_25163 [Trichoplax adhaerens]


YP_003405139.1
DEAD/DEAH box helicase [Haloterrigena turkmenica DSM



5511] >gb|ADB62466.1| DEAD/DEAH box helicase domain protein



[Haloterrigena turkmenica DSM 5511]


EGV92665.1
DNA polymerase theta [Cricetulus griseus]


CBY24305.1
unnamed protein product [Oikopleura dioica]


YP_003130565.1
DEAD/DEAH box helicase domain protein [Halorhabdus utahensis DSM



12940] >gb|ACV11832.1| DEAD/DEAH box helicase domain protein.



[Halorhabdus utahensis DSM 12940]


YP_003479811.1
DEAD/DEAH box helicase [Natrialba magadii ATCC 43099] >gb|ADD05249.1|



DEAD/DEAH box helicase domain protein [Natrialba magadii ATCC 43099]


EFB22383.1
hypothetical protein PANDA_000253 [Ailuropoda melanoleuca]


YP_003357334.1
putative ATP-dependent helicase [Methanocella paludicola



SANAE] >dbj|BAI62351.1| putative ATP-dependent helicase [Methanocella paludicola



SANAE]


YP_325942.1
ATP-dependent DNA helicase 2 [Natronomonas pharaonis DSM



2160] >emb|CA148373.2| ATP-dependent DNA. helicase 2



[Natronomonas pharaonis DSM 2160]


XP_002912509.1
PREDICTED: LOW QUALITY PROTEIN: helicase POLQ-like [Ailuropoda



melanoleuca]


XP_002704678.1
PREDICTED: helicase, POLQ-like [Bos taurus]


CAE47762.2
novel protein similar to humna, DNA-directed polymerase theta (POLQ) [Danio



rerio]


XP_003205636.1
PREDICTED: helicase POLQ-like [Meleagris gallopavo]


XP_544959.2
PREDICTED: helicase, POLQ-like [Canis lupus familiaris]


EFX86757.1
hypothetical protein DAPPUDRAFT_312857 [Daphnia pulex]


YP_003389641.1
DEAD/DEAH box helicase [Spirosoma linguale DSM 74] >gb|ADB40842.1|



DEAD/DEAH box helicase domain protein [Spirosoma linguale DSM 74]


XP_002602932.1
hypothetical protein BRAFLDRAFT_251779



[Branchiostoma floridae] >gb|EEN58944.1| hypothetical protein



BRAFLDRAFT_251779 [Branchiostoma floridae]


YP_004144962.1
peptidase C14 caspase catalytic subunit p20 [Mesorhizobium ciceri biovar



biserrulae WSM1271] >rel|YP_004614892.1| DEAD/DEAH box helicase



domain-containing protein [Mesorhizobium opportunistum



WSM2075] >gb|ADV14912.1| peptidase C14 caspase catalytic subunit p20



[Mesorhizobium ciceri biovar biserrulae WSM1271] >gb|AEH90798.1|



DEAD/DEAH box helicase domain protein [Mesorhizobium opportunistum



WSM2075]


XP_002124758.1
PREDICTED: similar to DNA polymerase theta [Ciona intestinalis]


XP_694437.5
PREDICTED: DNA polymerase theta [Danio rerio]


XP_420565.1
PREDICTED: similar to DNA helicase HEL308 [Gallus gallus]


XP_003129397.1
PREDICTED: helicase POLQ-like [Sus scrofa]


EDL20278.1
mCG128467, isoform CRA_b [Mus musculus]


XP_001517710.2
PREDICTED: helicase POLQ, partial [Ornithorhynchus anatinus]


AAH82601.1
Helicase, mus308-like (Drosophila) [Mus musculus]


XP_003384429.1
PREDICTED: DNA polymerase theta-like [Amphimedon queenslandica]


XP_003221282.1
PREDICTED: helicase POLQ-like [Anolis carolinensis]


NP_524333.1
mutagen-sensitive 308 [Drosophila melanogaster] >gb|AAB67306.1| Mus308



[Drosophila melanogaster] >gb|AAF54S58.1| mutagen-sensitive 308 [Drosophila



melanogaster] >gb|ACH92234.1| FI03732p [Drosophila melanogaster]


AAX33507.1
LP14642p [Drosophila melanogaster]


NP_001074576.1
helicase POLQ-like [Mus musculus] >sp|Q2VPA6.2|HELQ_MOUSE RecName:



Full = Helicase POLQ-like; AltName: Full = Mus308-like helicase: AltName:



Full = POLQ-like helicase >gb|AAI09171.2| Helicase, mus308-like (Drosophila)



[Mus musculus]


YP_003523727.1
DEAD/DEAH box helicase domain protein [Sideroxydans lithotrophicus



ES-1] >gb|ADE11340.1| DEAD/DEAH box helicase domain protein



[Sideroxydans lithotrophicusES-1]


XP_002120889.1
PREDICTED: similar to DNA helicase HEL308 [Ciona intestinalis]


XP_001892566.1
Type III restriction enzyme, res subunit family protein



[Brugia malayi] >gb|EDP38603.1| Type III restriction enzyme, res subunit



family protein [Brugia malayi]


ABZ09232.1
putative helicase conserved C-terminal domain protein [uncultured marine



crenarchaeote HF4000_APKG7F11]


XP_002814981.1
PREDICTED: LOW QUALITY PROTEIN: helicase POLQ-like [Pongo abelii]


XP_002717082.1
PREDICTED: DNA helicase HEL308 [Oryctolagus cuniculusl


XP_001104832.1
PREDICTED: helicase, POLQ-like [Macaca mulatta]


AAL85274.1
DNA helicase HEL308 [Homo sapiens]


NP_598375.2
helicase POLQ-like [Homo sapiens] >gb|EAX05934.1| DNA helicase HEL308,



isoform CRA_a [Homo sapiens] >gb|AAI41525.1| Helicase, POLQ-like



[synthetic polynucleotide]


Q8TDG4.2
RecName: Full = Helicase POLQ-like; AltName: Full = Mus308-like helicase:



AltName: Full = POLQ-like helicase


XP_003265889.1
PREDICTED: helicase POLQ [Nomascus leucogenys]


XP_002745688.1
PREDICTED: helicase POLQ-like [Callithrix jacchus]


XP_003310356.1
PREDICTED: LOW QUALITY PROTEIN: helicase POLQ-like [Pan



troglodytes]


NP_001014156.2
helicase, POLQ-like [Rattus norvegicus] >rf|XP_001060858.1 PREDICTED:



helicase, POLQ-like [Rattus norvegicus] >gb|EDL99554.1| rCG37823, isoform



CRA_c [Rattus norvegicus]


XP_001850567.1
ATP-dependent DNA helicase MER3 [Culex quinquefasciatus] >gb|EDS32308.1|



ATP-dependent DNA helicase MER3 [Culex quinquefasciatus]


XP_003427318.1
PREDICTED: helicase POLQ-like isoform 1 [Nasonia vitripennis]


XP_003143912.1
hypothetical protein LOAG_08332 [Loa loa; >gb|EFO20157.1| hypothetical



protein LOAG_08332 [Loa loa]


CAG11187.1
unnamed protein product [Tetraodon nigroviridis]


XP 001111254.2
PREDICTED: DNA polymerase theta isoform 2 [Macaca mulatta]


XP 003414242.1
PREDICTED: helicase POLQ [Loxodonta africana]


XP_002681870.1
predicted protein [Naegleria gruberi] >gb|EFC49126.1| predicted protein



[Naegleria gruberi]


EAX05935.1
DNA helicase HEL308, isoform CRA_b [Homo sapiens]


AAH59917.1
Ascc3 protein [Mus musculus]


ZP_07082808.1
DEAD/DEAH box helicase domain protein [Sphingobacterium spiritivorum



ATCC 33861] >gb|EFK55937.1| DEAD/DEAH box helicase domain protein



[Sphingobacterium spiritivorum ATCC 33861]


XP_001494572.3
PREDICTED: LOW QUALITY PROTEIN: helicase POLQ-like [Equus Caballus]


XP_002714920.1
PREDICTED: activating signal cointegrator 1 complex subunit 3 [Oryctolagus



cuniculus]


XP_002598278.1
hypothetical protein BRAFLDRAFT_204526



[Branchiostoma floridae] >gb|EEN54290.1| hypothetical protein



BRAFLDRAFT_204526 [Branchiostoma floridae]


XP_001943294.1
PREDICTED: helicase POLQ-like isoform 1



[Acyrthosiphon pisum] >ref|XP_003240510.1 PREDICTED: helicase



POLQ-like isoform 2 [Acyrthosiphon pisum]


XP_002803889.1
PREDICTED: activating signal cointegrator 1 complex subunit 3-like [Macaca



mulatta]


XP_001651546.1
DNA polymerase theta [Aedes aegypti] >gb|EAT42599.1| DNA polymerase theta



[Aedes aegypti]


CAA11679.1
RNA helicase [Homo sapiens]


XP_002837795.1
hypothetical protein [Tuber melanosporum Mel28] >emb|CAZ81986.1| unnamed



protein product [Tuber melanosporum]


EGT47882.1
hypothetical protein CAEBREN_02542 [Caenorhabditis brenneri]


EDL99655.1
activating signal cointegrator 1 complex subunit 3 (predicted), isoform CRA_b



[Rattus norvegicus]


NP_932124.2
activating signal cointegrator I complex subunit 3 [Mus musculus]


EDL05054.1
mCG119534 [Mus musculus]


gi|352115865
DEAD/DEAH box helicase domain protein


ZP_08963952.1
[Natrinema pellirubrum DSM 15624]









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







TABLE 5







More preferred Hel308 helicases and most preferred Hel308 motifs and extended Hel308 motifs













SEQ


%
%




ID 


Identity
Identity

Extended


NO:
Helicase
Names
Hel308 Pfu
Hel308 Mbu
Hel308 motif
Hel308 motif





10
Hel308 Mbu
Methanococcoides
37%
—
QMAGRAGR
QMAGRAGRP




burtonii


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


13
Hel308 Pfu
Pyrococcus furiosus
—
37%
QMLGRAGR
QMLGRAGRP




DSM 3638


(SEQ ID NO: 14)
(SEQ ID NO: 15)


 


16
Hel308 Hvo
Haloferax volcanii
34%
41%
QMMGRAGR
QMMGRAGRP







(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


19
Hel308 Hla
Halorubrum
35%
42%
QMCGRAGR
QMCGRAGRP




lacusprofundi


(SEQ ID NO: 20)
(SEQ ID NO: 21)


 


22
Hel308 Csy
Cenarchaeum
34%
34%
QLCGRAGR
QLCGRAGRP




symbiosum


(SEQ ID NO: 23)
(SEQ ID NO: 24)


 


25
Hel308 Sso
Sulfolobus
35%
33%
QMSGRAGR
QMSGRAGRP




solfataricus


(SEQ ID NO: 26)
(SEQ ID NO: 27)


 


28
Hel308 Mfr
Methanogenium
37%
44%
QMAGRAGR
QMAGRAGRP




frigidum


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


29
Hel308 Mok
Methanothermococcus
37%
34%
QCIGRAGR
QCIGRAGRP




okinawensis


(SEQ ID NO: 30)
(SEQ ID NO: 31)


 


32
Hel308 Mig
Methanotorris
40%
35%
QCIGRAGR
QCIGRAGRP




igneus Kol 5


(SEQ ID NO: 30)
(SEQ ID NO: 31)


 


33
Hel308 Tga
Thermococcus
60%
38%
QMMGRAGR
QMMGRAGRP




gammatolerans EJ3


(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


34
Hel308 Tba
Thermococcus
57%
35%
QMIGRAGR
QMIGRAGRP




barophilus MP


(SEQ ID NO: 35)
(SEQ ID NO: 36)


 


37
Hel308 Tsi
Thermococcus
56%
35%
QMMGRAGR
QMMGRAGRP




sibiricus MM 739


(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


38
Hel308 Mba
Methanosarcina
39%
60%
QMAGRAGR
QMAGRAGRP




barkeri str. Fusaro


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


39
Hel308 Mac
Methanosarcina
38%
60%
QMAGRAGR
QMAGRAGRP




acetivorans


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


40
Hel308 Mmah
Methanohalophilus
38%
60%
QMAGRAGR
QMAGRAGRP




mahii DSM 5219


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


41
Hel308 Mmaz
Methanosarcina
38%
60%
QMAGRAGR
QMAGRAGRP




mazei


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


42
Hel308 Mth
Methanosaeta
39%
46%
QMAGRAGR
QMAGRAGRP




thermophila PT


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


43
Hel308 Mzh
Methanosalsum
39%
57%
QMAGRAGR
QMAGRAGRP




zhilinae DSM 4017


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


44
Hel308 Mev
Methanohalobium
38%
61%
QMAGRAGR
QMAGRAGRP




evestigatum Z-7303


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


45
Hel308 Mma
Methanococcus
36%
32%
QCIGRAGR
QCIGRAGRP




maripaludis


(SEQ ID NO: 30)
(SEQ ID NO: 31)


 


46
Hel308 Nma
Natrialba magadii
37%
43%
QMMGRAGR
QMMGRAGRP







(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


47
Hel308 Mbo
Methanoregula
38%
45%
QMAGRAGR
QMAGRAGRP




boonei 6A8


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


48
Hel308 Fac
Ferroplasma
34%
32%
QMIGRAGR
QMIGRAGRP




acidarmanus


(SEQ ID NO: 35)
(SEQ ID NO: 36)


 


49
Hel308 Mfe
Methanocaldococcus
40%
35%
QCIGRAGR
QCIGRAGRP




fervens AG86


(SEQ ID NO: 30)
(SEQ ID NO: 31)


 


50
Hel308 Mja
Methanocaldococcus
24%
22%
QCIGRAGR
QCIGRAGRP




jannaschii


(SEQ ID NO: 30)
(SEQ ID NO: 31)


 


51
Hel308 Min
Methanocaldococcus
41%
33%
QCIGRAGR
QCIGRAGRP




infernus


(SEQ ID NO: 30)
(SEQ ID NO: 31)


 


52
Hel308 Mhu
Methanospirillum
36%
40%
QMAGRAGR
QMAGRAGRP




hungatei JF-1


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


53
Hel308 Afu
Archaeoglobus
40%
40%
QMAGRAGR
QMAGRAGRP




fulgidus DSM 4304


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


54
Hel308 Htu
Haloterrigena
35%
43%
QMAGRAGR
QMAGRAGRP




turkmenica


(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


55
Hel308 Hpa
Haladaptatus
38%
45%
QMFGRAGR
QMFGRAGRP




paucihalophilus DX253


(SEQ ID NO: 56)
(SEQ ID NO: 57)


 


58
ski2-like
Halobacterium sp.
  36.8%
  42.0%
QMFGRAGR
QMFGRAGRP



helicase
NRC-1


(SEQ ID NO: 56)
(SEQ ID NO: 57)









The Hel308 helicase more preferably comprises (a) the sequence of Hel308 Mbu (i.e. SEQ ID NO: 10) or a variant thereof, (b) the sequence of Hel308 Pfu (i.e. SEQ ID NO: 13) or a variant thereof, (c) the sequence of Hel308 Mok (i.e. SEQ ID NO: 29) or a variant thereof, (d) the sequence of Hel308 Mma (i.e. SEQ ID NO: 45) or a variant thereof, (e) the sequence of Hel308 Fac (i.e. SEQ ID NO: 48) or a variant thereof or (f) the sequence of Hel308 Mhu (i.e. SEQ ID NO: 52) or a variant thereof. The Hel308 helicase more preferably comprises the sequence shown in SEQ ID NO: 10 or a variant thereof.
The Hel308 helicase more preferably comprises (a) the sequence of Hel308 Tga (i.e. SEQ ID NO: 33) or a variant thereof, (b) the sequence of Hel308 Csy (i.e. SEQ ID NO: 22) or a variant thereof or (c) the sequence of Hel308 Mhu (i.e. SEQ ID NO: 52) or a variant thereof. The Hel308 helicase most preferably comprises the sequence shown in SEQ ID NO: 33 or a variant thereof.
A variant of a Hel308 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 particular, a variant of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 is an enzyme that has an amino acid sequence which varies from that of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 and which retains polynucleotide binding activity. A variant of SEQ ID NO: 10 or 33 is an enzyme that has an amino acid sequence which varies from that of SEQ ID NO: 10 or 33 and which retains polynucleotide binding activity. The variant retains helicase activity. 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 Hel308 motif or extended Hel308 motif discussed above. However, variants may include modifications within these motif(s).
Over the entire length of the amino acid sequence of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58, such as SEQ ID NO: 10 or 33, a variant will preferably be at least 30% homologous to that sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58, such as SEQ ID NO: 10 or 33, 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”). 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.
A variant of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 preferably comprises the Hel308 motif or extended Hel308 motif of the relevant wild-type sequence. For instance, a variant of SEQ ID NO: 10 preferably comprises the Hel308 motif of SEQ ID NO: 10 (QMAGRAGR; SEQ ID NO: 11) or extended Hel308 motif of SEQ ID NO: 10 (QMAGRAGRP; SEQ ID NO: 12). The Hel308 motif and extended Hel308 motif of each of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 are shown in Table 5. However, a variant of any one SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 may comprise the Hel308 motif or extended Hel308 motif from a different wild-type sequence. For instance, a variant of SEQ ID NO: 10 may comprise the Hel308 motif of SEQ ID NO: 13 (QMLGRAGR; SEQ ID NO: 14) or extended Hel308 motif of SEQ ID NO: 13 (QMLGRAGRP; SEQ ID NO: 15). A variant of any one SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 may comprise any one of the preferred motifs shown in Table 5. Variants of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 may also include modifications within the Hel308 motif or extended Hel308 motif of the relevant wild-type sequence. Suitable modifications at X1 and X2 are discussed above when defining the two motifs.
A variant of SEQ ID NO: 10 may lack the first 19 amino acids of SEQ ID NO: 10 and/or lack the last 33 amino acids of SEQ ID NO: 10. A variant of SEQ ID NO: 10 preferably comprises a sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more preferably at least 95%, at least 97% or at least 99% homologous based on amino acid identity with amino acids 20 to 211 or 20 to 727 of SEQ ID NO: 10.
The helicase may be covalently attached to the pore. The helicase is preferably not covalently attached to the pore. The application of a voltage to the pore and helicase typically results in the formation of a sensor that is capable of sequencing target polynucleotides. This is discussed in more detail below.
Any of the proteins described herein, i.e. the transmembrane protein pores or Hel308 helicases, may be modified to assist their identification or purification, for example by the 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 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 July; 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. 125I, 35S, enzymes, antibodies, antigens, 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 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.
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 aldehyde followed by reduction with NaBH4, 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 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 Laboratory Press, Cold Spring Harbor, N.Y.
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.
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 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 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 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 Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman K R 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 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 measured using the methods described below. For instance, methylcytosine 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 include: current measurements, impedance measurements, tunneling measurements (Ivanov A P et al., Nano Lett. 2011 Jan. 12; 11(1):279-85), and FET measurements (International Application WO 2005/124888). Optical measurements may be combined 10 with electrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January; 81(1):014301). The measurement may be a transmembrane current measurement such as measurement of ionic current flowing through 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 K R 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 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 Hel308 helicase such that the helicase controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and
(b) measuring the current passing through the pore during one or more interactions to measure 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 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.
The methods may be carried out using the apparatus described in International Application No. PCT/GB08/000,562 (WO 2008/102120).
The methods may involve measuring the current passing through the pore during one or more interactions with the nucleotide(s). Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. 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 during one or more interactions with the nucleotide. Suitable conditions for measuring ionic currents through transmembrane protein pores are known in the art and disclosed in the Example. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically from +2 V to −2 V, typically −400 mV to +400 mV. 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, −20 mV and 0 mV and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably in the range 100 mV to 240 mV 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 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. The salt concentration may be at saturation. The salt concentration may be 3M or lower and is 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, Hel308 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 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 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.
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° C., 19° C. to 70° C., or from 20° C. to 60° 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 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 (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). 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 Mg2+, Mn2+, Ca2+ or Co2+. The enzyme cofactor is most preferably Mg2+.
The target polynucleotide may be contacted with the Hel308 helicase and the pore in any order. In is preferred that, when the target polynucleotide is contacted with the Hel308 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.
As discussed above, Hel308 helicases may work in two modes with respect to the nanopore. First, the method is preferably carried out using the Hel308 helicase such that it moves the target sequence through the pore with the field resulting from the applied voltage. In this mode the 3′ end of the DNA is first captured in the nanopore, and the enzyme moves the DNA into the nanopore such that the target sequence is passed through the nanopore with the field until it finally translocates 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 5′ end of the DNA is first captured in the nanopore, and the enzyme moves the DNA through the nanopore such that the target sequence is pulled out of the nanopore against the applied field until finally ejected back to the cis side of the bilayer.
The method of the invention most preferably involves a pore derived from MspA and a helicase comprising the sequence shown in SEQ ID NO: 8 or 10 or a variant thereof. Any of the embodiments discussed above with reference to MspA and SEQ ID NO: 8 and 10 may be used in combination.
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 Hel308 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 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/GB09/001,679 (published as WO 2010/004265) and PCT/GB10/000,133 (published as WO 2010/086603). The complex is a sensor for characterising the target polynucleotide. The method preferably comprises forming a complex between a pore derived from Msp and a Hel308 helicase. Any of the embodiments discussed above with reference to the method of the invention equally apply to this method.
Kits
The present invention also provides kits for characterising a target polynucleotide. The kits comprise (a) a pore and (b) a Hel308 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 a lipid bilayer.
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 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.
Apparatus
The invention also provides an apparatus for characterising a target polynucleotide. The apparatus comprises a plurality of pores and a plurality of a Hel308 helicase. The apparatus preferably further comprises instructions for carrying out the method of the invention. The apparatus may be any conventional apparatus for polynucleotide analysis, such as an array or a 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 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 configured to supply the samples selectively from the containers to the sensor device. The apparatus may be any of those described in International Application No. PCT/GB08/004,127 (published as WO 2009/077734), PCT/GB10/000,789 (published as WO 2010/122293), International Application No. PCT/GB10/002,206 (not yet published) or International Application No. PCT/US99/25679 (published as WO 00/28312).
Internally Binding Molecular Motors
Molecular motors are commonly used as a means for controlling the translocation of a polymer, particularly a polynucleotide, through a nanopore. Surprisingly, the inventors have found that molecular motors which are capable of binding to a target polynucleotide at an internal nucleotide, i.e. a position other than a 5′ or 3′ terminal nucleotide, can provide increased read lengths of the polynucleotide as the molecular motor controls the translocation of the polynucleotide through a nanopore. The ability to translocate an entire polynucleotide through a nanopore under the control of a molecular motor allows characteristics of the polynucleotide, such as its sequence, to be estimated with improved accuracy and speed over known methods. This becomes more important as strand lengths increase and molecular motors are required with improved processivity. The molecular motor used in the invention is particularly effective in controlling the translocation of target polynucleotides of 500 nucleotides or more, for example 1000 nucleotides, 5000, 10000 or 20000 or more.
The invention thus provides a method of characterising a target polynucleotide, comprising:
(a) contacting the target polynucleotide with a transmembrane pore and a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide such that the molecular motor controls the movement of the target polynucleotide through the pore and nucleotides in the target polynucleotide interact with the pore; and
(b) measuring one or more characteristics of the target polynucleotide during one or more interactions and thereby characterising the target polynucleotide.
Any of the embodiments discussed above in relation to the Hel308 methods of the invention equally apply to this method of the invention.
A problem which occurs in sequencing polynucleotides, particularly those of 500 nucleotides or more, is that the molecular motor which is controlling translocation of the polynucleotide may disengage from the polynucleotide. This allows the polynucleotide to be pulled through the pore rapidly and in an uncontrolled manner in the direction of the applied field. Multiple instances of the molecular motor used in the invention bind to the polynucleotide at relatively short distances apart and thus the length of polynucleotide which can be pulled through the pore before a further molecular motor engages with the pore is relatively short.
An internal nucleotide is a nucleotide which is not a terminal nucleotide in the target polynucleotide. For example, it is not a 3′ terminal nucleotide or a 5′ terminal nucleotide. All nucleotides in a circular polynucleotide are internal nucleotides.
Generally, a molecular motor which is capable of binding at an internal nucleotide is also capable of binding at a terminal nucleotide, but the tendency for some molecular motors to bind at an internal nucleotide will be greater than others. For a molecular motor suitable for use in the invention, typically at least 10% of its binding to a polynucleotide will be at an internal nucleotide. Typically, at least 20%, at least 30%, at least 40% or at least 50% of its binding will be at an internal nucleotide. Binding at a terminal nucleotide may involve binding to both a terminal nucleotide and adjacent internal nucleotides at the same time. For the purposes of the invention, this is not binding to the target polynucleotide at an internal nucleotide. In other words, the molecular motor used in the invention is not only capable of binding to a terminal nucleotide in combination with one or more adjacent internal nucleotides. The molecular motor must be capable of binding to an internal nucleotide without concurrent binding to a terminal nucleotide.
A molecular motor which is capable of binding at an internal nucleotide may bind to more than one internal nucleotide. Typically, the molecular motor binds to at least 2 internal nucleotides, for example at least 3, at least 4, at least 5, at least 10 or at least 15 internal nucleotides. Typically the molecular motor binds to at least 2 adjacent internal nucleotides, for example at least 3, at least 4, at least 5, at least 10 or at least 15 adjacent internal nucleotides. The at least 2 internal nucleotides may be adjacent or non-adjacent.
The ability of a molecular motor to bind to a polynucleotide at an internal nucleotide may be determined by carrying out a comparative assay. The ability of a motor to bind to a control polynucleotide A is compared to the ability to bind to the same polynucleotide but with a blocking group attached at the terminal nucleotide (polynucleotide B). The blocking group prevents any binding at the terminal nucleotide of strand B, and thus allows only internal binding of a molecular motor. An example of this type of assay is disclosed in Example 4.
Suitable molecular motors are well known in the art and typically include, but are not limited to, single and double strand translocases, such as polymerases, helicases, topoisomerases, ligases and nucleases, such as exonucleases. Preferably the molecular motor is a helicase, for example a Hel308 helicase. Examples of Hel308 helicases which are capable of binding at an internal nucleotide include, but are not limited to, Hel308 Tga, Hel308 Mhu and Hel308 Csy. Hence, the molecular motor preferably comprises (a) the sequence of Hel308 Tga (i.e. SEQ ID NO: 33) or a variant thereof or (b) the sequence of Hel308 Csy (i.e. SEQ ID NO: 22) or a variant thereof or (c) the sequence of Hel308 Mhu (i.e. SEQ ID NO: 52) or a variant thereof. The variant typically has at least 40% homology to SEQ ID NO: 33, 22 or 52 based on amino acid identity over the entire sequence and retains helicase activity. Further possible variants are discussed above.
The molecular motor used in the invention may be made by any of the methods discussed above and may be modified or labelled as discussed above. The molecular motor may be used in the methods described herein or as part of the apparatus described herein. The invention further provides a method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore and a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide and thereby forming a sensor for characterising the target polynucleotide. The invention also provides use of a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide to control the movement of a target polynucleotide through a pore. The invention also provides a kit for characterising a target polynucleotide comprising (a) a pore and (b) a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The invention also provides an analysis apparatus for characterising target polynucleotides in a sample, comprising a plurality of pores and a plurality of a molecular motor which is capable of binding to the target polynucleotide at an internal nucleotide.
The following Examples illustrate the invention.
Example 1
This Example illustrates the use of a Hel308 helicase (Hel308 MBu) to control the movement of intact DNA strands through a nanopore. The general method and substrate 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 of these primers included a 50 nucleotide non-complimentary region, either a homopolymeric 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′-O-Methyl-Uracil (mU) nucleotides and the 5′-end of the reverse primer was 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 PO4 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 PAGE purified as previously described.
The DNA substrate design used in all the experiments described here is shown in FIG. 6. 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: 59) Annealed to this strand just after the 50T leader is a primer (SEQ ID NO: 60) containing a 3′ cholesterol tag to enrich the DNA on the surface of the bilayer, and thus improve capture efficiency.
Buffered solution: 400 mM-2 M KCl, 10 mM Hepes pH 8.0, 1 mM ATP, 1 mM MgCl2, 1 mM DTT
Nanopore: E. coli MS (B2)8 MspA ONLP3271 MS-(L88N/D90N/D91N/D93N/D118R/D134R/E139K)8
Enzyme: Hel308 Mbu (ONLP3302,  ̃7.7 μM) 12.5 μl→100 nM final.
Electrical measurements were acquired from single MspA nanopores inserted in 1,2-diphytanoyl-glycero-3-phosphocholine lipid (Avanti Polar Lipids) bilayers. Bilayers were formed across  ̃100 μm diameter apertures in 20 μm 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. Ag/AgCl 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 100 μL of buffer and pre-incubated for 5 mins (DNA=1.5 nM, Enzyme=1 μM). This pre-incubation mix was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate capture of the helicase-DNA complexes in the MspA nanopore (to give final concentrations of DNA=0.15 nM, Enzyme=0.1 μM). Helicase ATPase activity was initiated as required by the addition of divalent metal (1 mM MgCl2) and NTP (1 mM ATP) to the cis compartment. Experiments were carried out at a constant potential of +180 mV.
Results and Discussion
The addition of Helicase-DNA substrate to MspA nanopores as shown in FIG. 1 produces characteristic current blocks as shown in FIG. 2. DNA without helicase bound interacts transiently with the nanopore producing short-lived blocks in current (<<1 second). DNA with helicase bound and active (ie. moving along the DNA strand under ATPase action) produces long characteristic blocks levels with stepwise changes in current as shown in FIG. 2. 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).
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. However, as long as the enzyme does not dissociate, the strands will all end in the same way at the 50T leader (FIG. 1). As FIG. 2 shows, we observe the same characteristic ending to most strands, with the current transitions ending in a long dwell time polyT level (FIG. 3).
Salt Tolerance
Nanopore strand sequencing experiments of this type require ionic salts. The 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 in 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 mM KCl are ideal, and salt concentrations of 1 M 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 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).
We surprisingly show in this Example that Hel308 from Mbu displays salt tolerance up to very high levels of KCl. We find that the enzyme retains functionality in salt concentrations of 400 mM KCl through to 2 M KCl, either in fluorescence experiments or in nanopore experiments (FIG. 4). FIG. 4 shows the Hel308 Mbu DNA events at 400 mM KCl, 1 M KCl, and 2 M KCl salt conditions carried out using the same system described in FIG. 1. We observe similar movement across the range of salt concentrations. As the salt concentration is increased we observe an increase in the current through the nanopore (1-open) at a fixed voltage. This reflects the increase in the conductivity of the solution and the increased number of ions flowing through the nanopore under the applied field. In addition we also observe an increase in the minimum to maximum range of discrimination in the current levels of the DNA events (see FIG. 4 enlargements and bottom right plot). We observe a  ̃200% increase in DNA discrimination range as the salt concentration is increased from 400 mM KCl to 2M KCl (Table 6 below).







TABLE 6







Effect of increasing salt concentration on pore current and DNA range










Open-pore current



Salt (KCl) (M)
(pA)
DNA range (pA)





0.4
180
25


1.0
440
55


2.0
840
75









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. Although FIG. 1 illustrates the use of this movement to pull threaded DNA out of the nanopore, helicase movement could be exploited in other manners to feed DNA through the nanopore in a controlled fashion. FIG. 5 illustrates the basic ‘forward’ and ‘reverse’ modes of operation. In the forward mode, the 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. For Hel308 Mbu, which is a 3′-5′ helicase, this requires capturing the 3′ 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, finally exiting on the trans side of the bilayer. The reverse mode requires capturing the 5′ end of the DNA, after which the helicase proceeds to pull the threaded DNA back out of the nanopore against the field from the applied potential, finally ejecting it on this cis side of the bilayer. FIG. 5 shows these two modes of operation using Hel308 Mbu, and typical example DNA events.
Example 2
This Example illustrates the salt tolerance of a Hel308 helicase (Hel308 MBu) 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. 6A). As shown in 1) of FIG. 6A, the fluorescent substrate strand (100 nM final) has a 3′ ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strand has a carboxyfluorescein base at the 5′ end, and the hybrised complement has a black-hole quencher (BHQ-1) base at the 3′ end. When hybrised the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. 1 μM 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 MgCl2 (5 mM), helicase (100 nM) added to the substrate binds to the 3′ 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.
Substrate DNA: 5′FAM-SEQ ID NO: 61 and SEQ ID NO: 62-BHQ1-3′. FAM=carboxyfluorescein and BHQ1=Black Hole Quencher-1
Capture DNA: SEQ ID NO: 62.
The graph in FIG. 6 shows the initial rate of activity in buffer solutions (10 mM Hepes pH 8.0, 1 mM ATP, 5 mM MgCl2, 100 nM fluorescent substrate DNA, 1 μM capture DNA) containing different concentrations of KCl from 400 mM to 2 M. The helicase works at 2 M.
Example 3
In this Example, three different Hel308 helicases were used, namely Hel308 Mhu (SEQ ID NO: 52), Hel308 Mok (SEQ ID NO: 29) and Hel308 Mma (SEQ ID NO: 45). All experiments were carried out as previously described in Example 1 under the same experimental conditions (pore=MspA B2, DNA=400 mer SEQ ID NO: 59 and 60, buffer=400 mM KCl, 10 mM Hepes pH 8.0, 1 mM dtt, 1 mM ATP, 0.1 mM MgCl2). The results are shown in FIG. 7.
Example 4
This Example measures the internal binding capabilities of a number of Hel308 helicases using a fluorescence assay.
Custom fluorescent substrates were used to assay the ability of the helicases to initiate on DNA lacking native 3′ ends, allowing them to subsequently displace hybridised dsDNA (FIG. 8). As shown in section A of FIG. 8, the fluorescent substrate strand (50 nM final) has a 3′ ssDNA overhang, and a 40 base section of hybridised dsDNA. The major upper strands are modified with four consecutive “spacer 9” groups, either at the 3′ end, or internally, at the junction between the overhang and the dsDNA (as a negative control). Furthermore, the major upper strand has a carboxyfluorescein base at the 5′ end, and the hybridised complement has a black-hole quencher (BHQ-1) base at the 3′ end. When hybridised, the fluorescence from the fluorescein is quenched by the local BHQ-1, and the substrate is essentially non-fluorescent. A capture strand (1 μM), that is complementary to the shorter strand of the fluorescent substrate, is included in the assay. In the presence of ATP (1 mM) and MgCl2 (1 mM), a Hel308 helicase homologue (20 nM), added to the substrate containing 3′-terminal “spacer 9” groups, can bind to the ssDNA overhang of the fluorescent substrate, move along the major strand, and displace the complementary strand as shown in section B. Once the complementary strand with BHQ-1 is fully displaced (section C) the fluorescein on the major strand fluoresces. An excess of capture strand preferentially anneals to the complementary DNA to prevent re-annealing of initial substrate and loss of fluorescence (section D).
Substrate DNA: SEQ ID NO: 63 with a 5′ FAM; SEQ ID NO: 63 with a 5′ FAM and 3′ spacer ((spacer 9)4); SEQ ID NOs: 64 (with a 5′ FAM) and 65 separated by a spacer ((spacer 9)4); and SEQ ID NO: 62 with a 3′ BHQ1.
Capture DNA: SEQ ID NO: 66.
A number of different Hel308 helicase homologues were investigated for their mid-binding abilities, these included Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mma, Hel308 Mhu, Hel308 Min, Hel308 Mig, Hel308 Mmaz, Hel308 Mac, Hel308 Mok, Hel308 Mth, Hel308 Mba and Hel308 Mzh. The graph in FIG. 9 shows the relative rates of Hel308-mediated dsDNA turnover, comparing 3′-unmodified DNA and 3′-“spacer 9” DNA in 400 mM NaCl, 10 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl2, 50 nM fluorescent substrate DNA, 1 μM capture DNA. Several Hel308 homologues were observed to have greater than 20% relative rates of Hel308-mediated dsDNA turnover including, Hel308 Csy, Hel308 Tga, Hel308 Mma, Hel308 Mhu and Hel308 Min.
Example 5
This Example compares the use of two Hel308 helicases, Hel308 MBu and Hel 308 Tga, and their ability to control the movement of intact long DNA strands (900 mer) through a nanopore. The general method and substrate employed throughout this Example are shown in FIG. 10 and described in the description of the Figure above.
Materials and Methods
The DNA was formed by ligating a 50-polyT 5′ leader to a  ̃900 base fragment of PhiX dsDNA. The leader also contains a complementary section to which SEQ ID NO: 69 with a Chol-tag was hybridized to allow the DNA to be tethered to the bilayer. Finally the 3′ end of the PhiX dsDNA was digested with AatII digestion enzyme to yield a 4 nt 3′-overhang of ACGT.
Sequences used: SEQ ID NO: 67-900 mer sense strand including 5′ leader and tether; SEQ ID NO: 68—anti-sense minus 4 base-pair leader 5′; and SEQ ID NO: 69 with several spacers and a Chol-tag at the 3′ end.
Buffered solution: 400 mM-2 NaCl, 10 mM potassium ferrocyanide, 10 mM potassium ferricyanide, 100 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl2,
Nanopore: MS-(B1-G75S-G77S-L88N-Q126R)8 (ONT Ref B2C)
Enzyme: Hel308 Mbu 1000 nM or Hel308 Tga 400 nM final.
Electrical experiments were set up as described in Example 1 in order to achieve a single pore inserted into a lipid bilayer. After achieving a single pore in the bilayer, ATP (1 mM) and MgCl2 (1 mM) were added to the chamber. A control recording at +140 mV was run for 2 minutes. DNA polynucleotide SEQ ID NOs: 67, 68 and 69 (DNA=0.15 nM) were then added and DNA events observed. Finally, Hel308 helicase (Mbu 1000 nM or Tga, 400 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 +140 mV.
Results and Discussion
The addition of Helicase-DNA substrate to MspA nanopores as shown in FIG. 10 produces characteristic current blocks as the helicase controls the translocation of the DNA through the pore. FIG. 11 shows example event traces which indicate how the position of the 900 mer varied as the Hel308 helicase homologue Mbu controlled the translocation of the DNA strand through the MspA pore. This helicase was found to mediate control of DNA translocation, however, when the helicase detached from the DNA, the strand was observed to move back through the pore, owing to the force exerted by the externally applied potential. In the case of the Hel308 helicase homologue Mbu, the 900 mer strand slipped back a large number of positions (approximately 100-200 bases) each time a helicase disengaged. These rapid changes in position are indicated in FIG. 11 by dotted circles. For this experiment, where Hel308 helicase homologue Mbu was used as the molecular motor, 32% of all of the events detected were found to have read the entire length of the 900 mer strand sequence. FIG. 12 shows similar example event traces indicating how the position of the 900 mer varied as the Hel308 helicase homologue Tga controlled the translocation of the DNA strand through the MspA pore. This enzyme exhibited an greater tendency to bind internally, than the Mbu homologue, because when the Tga helicase disengages (indicated by a change in colour black to grey in FIG. 12), the DNA strand moves back through the pore by a relatively small distance (<50 bases). For this experiment, where Hel308 helicase homologue Tga was used as the molecular motor, 74% of all of the events detected were found to have read the entire length of the 900 mer strand sequence. This means that the Tga helicase homologue can provide increased read lengths of single-stranded DNA in comparison to the Mbu helicase homologue owing to its increased tendency to bind internally.
Example 6
This Example illustrates that by employing the Hel308 helicase homologue Tga it is possible to control the translocation of a 5 kb strand of DNA.
A similar experimental procedure was followed to that described in Example 5. It was observed that by employing the Hel308 Tga it was possible to detect the controlled translocation of an entire 5 kb strand of DNA through MS-(B1-G75S-G77S-L88N-Q126R)8. In an identical experiment using Hel308 Mbu, it was not possible to detect translocation of an entire 5 kB strand.
Example 7
This example compares the enzyme processivity of Hel308 Mbu helicase (SEQ ID NO: 10) with Hel308 Mok (SEQ ID NO: 29) using a fluorescence based assay.
A custom fluorescent substrate was used to assay the ability of the helicase to displace hybridised dsDNA (FIG. 3). The fluorescent substrate (50 nM final) has a 3′ ssDNA overhang, and 80 and 33 base-pair sections of hybridised dsDNA (FIG. 13 section A, SEQ ID NO: 70). The major lower “template” strand is hybridised to an 80 nt “blocker” strand (SEQ ID NO: 71), adjacent to its 3′ overhang, and a 33 nt fluorescent probe, labelled at its 5′ and 3′ ends with carboxyfluorescein (FAM) and black-hole quencher (BHQ-1) bases, respectively (SEQ ID NO: 72). When hybridised, the FAM is distant from the BHQ-1 and the substrate is essentially fluorescent. In the presence of ATP (1 mM) and MgCl2 (10 mM), the helicase (20 nM) binds to the substrate's 3′ overhang (SEQ ID NO: 70), moves along the lower strand, and begins to displace the 80 nt blocker strand (SEQ ID NO: 71), as shown in FIG. 13 section B. If processive, the helicase displaces the fluorescent probe (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) too (FIG. 13 section C). The fluorescent probe is designed in such a way that its 5′ and 3′ ends are self-complementary and thus form a kinetically-stable hairpin once displaced, preventing the probe from re-annealing to the template strand (FIG. 13 section D). Upon formation of the hairpin product, the FAM is brought into the vicinity of the BHQ-1 and its fluorescence is quenched. A processive enzyme, capable of displacing the 80 mer “blocker” (SEQ ID NO: 71) and fluorescent (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) strands will therefore lead to a decrease in fluorescence over time. However, if the enzyme has a processivity of less than 80 nt it would be unable to displace the fluorescent strand (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and, therefore, the “blocker” strand (SEQ ID NO: 71) would reanneal to the major bottom strand (FIG. 13 section E, SEQ ID NO: 70).
Additional custom fluorescent substrates were also used for control purposes. The substrate used as a negative control was identical to that of the one described in FIG. 13 but lacking the 3′ overhang (FIG. 14 section A, (SEQ ID NOs: 71, 72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and 73)). A similar substrate to that described in FIG. 13 but lacking the 80 base pair section, used as a positive control for active, but not necessarily processive, helicases (FIG. 14 section B, (SEQ ID NOs: 72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and 74)).
FIG. 15 shows a graph of the time-dependent fluorescence changes upon testing Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase (SEQ ID NO: 29) against the processivity substrate shown in FIG. 13 in buffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mM MgCl2, 50 nM fluorescent substrate DNA (SEQ ID NOs: 70, 71 and 72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) at its 3′ end). The decrease in fluorescence exhibited by Hel308 Mok denotes the increased processivity of these complexes as compared to Hel308 Mbu (SEQ ID NO: 10). FIG. 16 shows positive controls demonstrating that all helicases were indeed active, as denoted by a fluorescence decrease for all samples.










SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 and 55 (Table 4) are


aligned below The number below the * indicates the SEQ ID NO. The “-” are shown for alignment purposes only and do not form part of the sequences.



















*



1                                                                                            95


10
Hel308
Mbu
(1)
--------------------------------MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLE-KKNLLAAIPTASGKILLAELAMIK


53
Hel308
Afu
(1)
------------------------------MKVEELAESISSYAVGILKEEGIEELFPPQAEAVEKVFS--GKNLLLAMPTAAGKILLAEMAMVR


22
Hel308
Csy
(1)
--------------------------------MRISELDIPRPAIEFLEGEGYKKLYPPQAAAAKAGLID-GKSVLVSAPTASGKILIAAIAMIS


75
Hel308
Dth
(1)
MPGVDELLQQMGQGDLQGLSTVAVKEIPAREAEFSGIEGLPPPLKQALTESGIENFYTHQARAVNLVRK--GRSVVTATPTASGKSLIYNIPVLE


48
Hel308
Fac
(1)
----------------------------------MKLSEITPSEFLKVTDNNDFTLYEHQEEAVAKLREN--KNVIVSVPTASGKTLIGYISIYD


19
Hel308
Hla
(1)
-------------------------------MQPSSLSGLPAGVGEALEAEGVAELYPPQEAAVEAGVAD-GESLVAAVPTASGKILIAELAMLS


55
Hel308
Hpa
(1)
-------------------------------MNVADLTGLPDGVPEHFHAQGIEELYPPQAEAVEAGITE-GESVVASIPTASGKTFIAELAMLS


54
Hel308
Htu
(1)
-------------------------------MNLEELTGLPPGATDHERGEGIEELYPPQADAVEAGATD-GENLVAAVPTASGKIMIAALSMLS


16
Hel308
Hvo
(1)
-------------------------------MRTADLTGLPTGIPEALRDEGIEELYPPQAEAVEAGLID-GESLVAAVPTASGKILIAELAMLS


39
Hel308
Mac
(1)
--------------------------------MKIESLDLPDEVKREYENSGIPELYPPQAEAVEKGLLE-GKNLLAAIPTASGKILLAELAMLK


38
Hel308
Mba
(1)
--------------------------------MKIESLDLPDEVKQFYLNSGIMELYPPQAEAVEKGLLE-GRNLLAAIPTASGKILLAELAMLK


47
Hel308
Mbo
(1)
--------------------------------MQIQDLAIPEPLRQQYLGLGIRELYPPQAACVERGLLD-GKNLLVAIPTASGKILIAEMAMHR


44
Hel308
Mev
(1)
--------------------------------METGKLELPEYVIQFYLDTGIEKLYPPQAEAVEKGLLD-NKNLLAAIPTASGKILISELAMLK


49
Hel308
Mfe
(1)
--------------------------------------MPTNKILEILKDFGIEELRPPQKKALEKGLLDKNKNFLISIPTASGKTLIGEMALIN


28
Hel308
Mfr
(1)
------------------------------------DLSLPKAFIQYYKDKGIESLYPPQSECIENGLLD-GADLLVAIPTASGKILIAEMAMHA


52
Hel308
Mhu
(1)
--------------------------------MEIASLPLPDSFIRACHAKGIRSLYPPQAECIEKGLLE-GKNLLISIPTASGKILLAEMAMWS


32
Hel308
Mig
(1)
-------------------------------------MQKYSHVFEVLKENGIKELRPPQKKVIEKGLLNKEKNFLICIPTASGKTLIGEMALIN


51
Hel308
Min
(1)
-------------------------------------------MDEILKFLGIKELRPPQKKALELGILDKKKNFLISIPTGAGKTVIAEMALIN


45
Hel308
Mma
(1)
-----------------------------------------MHVLDLLKENKITELRPPQKKVIDEGLFDKTKNFLICIPTASGKTLIGEMALLN


40
Hel308
 Mmah
(1)
--------------------------------MKIEELDLPSEAIEVYLQAGIEELYPPQADAVEKGLLQ-GENLLAAIPTASGKILLAEMAMLK


76
Hel308
 Mmar
(1)
-------------------------------MDVADLPGVPEWLPDHLRDDGIEELYPPQAEAVEAGVIE-GENLVASIPTASGKILIAELAMLS


41
Hel308
 Mmaz
(1)
--------------------------------MKIESLDLPDEIKREYENSGILELYPPQAEAVEKGLLE-GKNLLAAIPTASGKILLAELAMLK


29
Hel308
Mok
(1)
----------------------------------------MLMLMEVLKENGIAELRPPQKKVVEGGLLNKNKNFLICIPTASGKTLIGEMAFIN


42
Hel308
Mth
(1)
-----------------------------MLTIRDLIRWLPESVIELYEALGIDELYPPQAEAIERGLLD-GRNMIISVPTAAGKILLAELAMLR


43
Hel308
Mzh
(1)
--------------------------------MNINNLNLPEKVKKYYTDIGIVDLYPPQREAVDKGLLD-GENIVAAIPTASGKILLAELCMLK


46
Hel308
Nma
(1)
-------------------------------MNVEELSGLPPGARSHFQEQGIEELYPPQAEAVEAGATE-GENLVAAVPTASGKIMIAALSMLS


77
Hel308
Nth
(1)
--------------------------------MSETFYLLSERMQKKIWEMGWDEFTPVQDKTIPIVMNT-NKDVVVSSGTASGKTEAVFLPILS


13
Hel308
Pfu
(1)
-----------------------------MRVDELR---VDERIKSTLKERGIESFYPPQAEALKSGILE-GKNALISIPTASGKILIAEIAMVH


25
Hel308
Sso
(1)
-------------------------MSLELEWMPIEDLKLPSNVIEIIKKRGIKKLNPPQTEAVKKGLLE-GNRLLLTSPIGSGKILIAEMGIIS


34
Hel308
Tba
(1)
---------MLSTKPKAYKRESPIG--YAMQVDELSKFGVDERIIRKIKERGISEFYPPQAEALRSGVLN-GENLLLAIPTASGKILVAEIVMLH


37
Hel308
Tga
(1)
-----------------------------MKVDELP---VDERLKAVLKERGIEELYPPQAEALKSGALE-GRNLVLAIPTASGKILVSEIVMVN


50
Hel308
Tsi
(1)
---------MKLNKLKSYINAFLLGMVMSMKVDELKSLGVDERILRLLRERGIEELYPPQADALKTEVLK-GKNLVLAIPTASGKILVAEIVMIN


78
Hel308
Mja
(1)
----------------------------------------MDKILEILKDFGIVELRPPQKKALERGLLDKNKNFLISIPTASGKTLIGEMALIN











Consensus
(1)
                                       LP  V   L E GI ELYPPQAEAVE GLLD GKNLLIAIPTASGKTLIAELAML


 


 















96                                                                                          190



Hel308
Mbu
(63)
AIREGG------KALYIVPLRALASEKFERFK-ELAP----FGIKVGISTGDLDSRADWLGVNDIIVATSEKTDSLLRNGTSWMD-------EIT



Hel308
Afu
(64)
EAIKGG------KSLYVVPLRALAGEKYESFK-KWEK----IGLRIGISTGDYESRDEHLGDCDIIVTTSEKADSLIRNRASWIK-------AVS



Hel308
Csy
(63)
HLSRNR-----GKAVYLSPLRALAAEKFAEFGKIGGIPL-GRPVRVGVSTGDFEKAGRSLGNNDILVLTNERMDSLIRRRPDWMD-------EVG



Hel308
Dth
(94)
SIINDP----ASRALYLFPLKALTRDQLTSLEEFARLLAGKVHVDSAVYDGDTDPQARARIRSKPPNILLTNPDMIHRSFLPYHRSWQKFFSALK



Hel308
Fac
(60)
TYLKGK------KSMYIVPLRSLAMEKFSELL-SLRN----LGVKVTMSIGDYDVPPSFVKNYDVIIATSERADSMLHRDPDILN-------YFG



Hel308
Hla
(64)
SIERGG------KALYIVPLRALASEKKTEFE-RWEE----FGVTVGVSTGNYESDGEWLATRDIIVATSEKVDSLIRNGAPWID-------DLT



Hel308
Hpa
(64)
SVARGG------KALYIVPLRALASEKKEEFE-EFEQ----YGVSIGVSTGNYESDGDWLASRDIIVATSEKVDSLVRNGAKWID-------DLS



Hel308
Htu
(64)
AVQRGG------KALYIVPLRALASEKKEEFE-AYEE----FGVTTGVTTGNYESTDDWLATKDIIVATSEKVDSLVRNGADWLS-------ELT



Hel308
Hvo
(64)
SVARGG------KALYIVPLRALASEKKAEFE-RWEE----YGIDVGVSTGNYESDGEWLSSRDIIVATSEKVDSLVRNNAAWMD-------QLT



Hel308
Mac
(63)
SVLAGG------KALYIVPLRALASEKFRRFQ-DFSE----LGIRVGISTGDYDRRDEGLGINDIIVATSEKTDSLLRNETAWMQ-------EIS



Hel308
Mba
(63)
SILAGG------KALYIVPLRALASEKFRRFR-EFSE----LGIRVGISTGDYDLRDEGLGVNDIIVATSEKTDSLLRNETVWMQ-------EIS



Hel308
Mbo
(63)
HIANGG------KCLYIVPLKALASEKYEEFG-NK-------GVKVGLSTGDLDRRDDALGKNDIIVATSEKVDSLLRNGARWIP-------DIT



Hel308
Mev
(63)
SISNGG------KCLYIVPLRALASEKFERFK-QFSS----IGVNIGISTGDFDSTDEWLGSNDIIVATSEKADSLLRNETSWMK-------DIT



Hel308
Mfe
(58)
HLLDENKNPTNKKGIFIVPLKALASEKYEEFKNKYER----YGLRVALSIGDYD-EDEDLSRYHLIITTAEKLDSLWRHKIDWID-------DVS



Hel308
Mfr
(59)
AIARGG------MCLYIVPLKALATEKAQEFK-GK-------GAEIGVATGDYDQKEKRLGSNDIVIATSEKVDSLLRNGVPWLS-------QVT



Hel308
Mhu
(63)
RIAAGG------KCLYIVPLRALASEKYDEFS-KKG------VIRVGIATGDLDRTDAYLGENDIIVATSEKTDSLLRNRTPWLS-------QIT



Hel308
Mig
(59)
HLLDENKTPTNKKGLFIVPLKALASEKYEEFKRKYEK----YGLKVALSIGDYD-EKEDLSSYNIIITTAEKLDSLMRHEIDWLN-------YVS



Hel308
Min
(53)
HLLLDK----GKKGVYIVPLKALASEKYEEFKKKYEK----FGVRVALSIGDYD-EDEDLENYDLIITTAEKFDSLWRHGIKLS--------DIS



Hel308
Mma
(55)
HILDENKNLTGKKGLFIVPLKALANEKFDEFREKYEK----YGIKVGLSIGDFD-TKENLSKFHIIITTSEKLDSLMRHNVEWIN-------DVS



Hel308
 Mmah
(63)
AIKKGG------KALYIVPLRALASEKFRDFK-RFES----LGIKTAISTGDFDSRDEWLGSNDIIVATSEKTDSLLRNSTPWMK-------DIT



Hel308
 Mmar
(64)
SVARGG------KALYIVPLRALASEKQADFE-EFEQ----YGLDIGVSTGNYESEGGWLADKDIVVATSEKVDSLVRNDAPWIE-------DLT



Hel308
 Mmaz
(63)
SVLNGG------KALYIVPLRALASEKFRRFQ-EFSV----LGMRVGISTGDYDRRDEGLGINDIIVATSEKTDSLLRNETAWMQ-------EIS



Hel308
Mok
(56)
HLLDNNKTPTNKKGLFIVPLKALANEKYEEFKGKYEK----YGLKIALSIGDFD-EKEDLKGYDLIITTAEKLDSLIRHKVEWIK-------DIS



Hel308
Mth
(66)
GALSGK------RSLYIVPLRALASEKFESFS-RFSK----LGLRVGISTGDFEKRDERLGRNDIIIATSEKADSLIRNGASWVR-------RIG



Hel308
Mzh
(63)
SIGMGG------KCLYIVPLKALASEKYSRFR-EFES----LGIKVGIATGDLDSREEWLGKNDIIIATSEKVDSLLRNESSWMK-------EIN



Hel308
Nma
(64)
AVQRGG------KALYIVPLRALASEKKAEFD-AYEE----FGVTTGVATGNYESTSEWLATKDIIVATSEKVDSLVRNGADWLS-------DLT



Hel308
Nth
(63)
QIEKDAT--KDLKILYISPLKALINDQFERIIKLCEKSY-IPIHRWHGDVNQNKKKQLTKNPAGILQITPESIESLFINRTNELNYIL---SDIE



Hel308
Pfu
(63)
RILTQG-----GKAVYIVPLKALAEEKFQEFQ-DWEK----IGLRVAMATGDYDSKDEWLGKYDIIIATAEKFDSLLRHGSSWIK-------DVK



Hel308
Sso
(70)
FLLKNG-----GKAIYVTPLRALTNEKYLTFK-DWEL----IGFKVAMTSGDYDTDDAWLKNYDIIITTYEKLDSLWRHRPEWLN-------EVN



Hel308
Tba
(84)
KLFTGG-----GKAVYLVPLKALAEEKYREFK-TWED----LGVRVAVTTGDYDSSEEWLGKYDIIIATSEKFDSLLRHKSRWIR-------DVT



Hel308
Tga
(63)
KLIQEG-----GKAVYLVPLKALAEEKYREFK-EWEK----LGLKVAATTGDYDSTDDWLGRYDIIVATAEKFDSLLRHGARWIN-------DVK



Hel308
Tsi
(86)
KILREG-----GKTVYLVPLKALAEEKYKEFK-FWEK----LGIRIAMTTGDYDSTEEWLGKYDIIIATSEKFDSLLRHKSPWIK-------DIN



Hel308
Mja
(56)
HLLDGNKNPTNKKGIFIVPLKALASEKYEEFKSKYER----YGLRIALSIGDYD-EDEDLSKYHLIITTAEKLDSLWRHKIDWIN-------DVS











Consensus
(96)
 IL GG      KALYIVPLRALASEKY EFK  FE      GVRVGISTGDYD  DEWLG  DIIVATSEKVDSLLRN   WI        DIT


 


 















191                                                                                         285



Hel308
Mbu
(140)
TVVVDEIHLLDSKNRGPTLEVTITKLMRLNPD----VQVVALSATVGNAREMADWLG---AALVLSEWRPTDLHEGVLFGDAINFPG-SQKKIDR



Hel308
Afu
(141)
CLVVDEIHLLDSEKRGATLEILVTKMRRMNKA----LRVIGLSATAPNVTEIAEWLD---ADYYVSDWRPVPLVEGVLCEGTLELFD----GAFS



Hel308
Csy
(145)
LVIADEIHLIGDRSRGPTLEMVLTKLRGLRSS----PQVVALSATISNADEIAGWLD---CTLVHSTWRPVPLSEGVYQDGEVAMGDGSRHEVAA



Hel308
Dth
(185)
YIVVDEVHTYRG-VMGSNMAWVFRRLRRICAQYGREPVFIFSSATIANPGQLCSALTGHEPEVIQKGGAPAGKKHFLLLDPEMQGAAQS------



Hel308
Fac
(137)
LVIIDEIHMISDPSRGPRLETVISSLLYLNPE----ILLLGLSATVSNIQEIAEWMN---AETVVSNFRAVPLETGIIFKGNLITDG--------



Hel308
Hla
(141)
CVVSDEVHLVDDPNRGPTLEVTLAKLRKVNPG----LQTVALSATVGNADVIAEWLD---AELVESDWRPIDLRMGVHFGNAIDFADGSKREVPV



Hel308
Hpa
(141)
CVVADEVHLVNDAHRGPTLEVTLAKLRRVNPD----LQTVALSATVGNAGEMADWLD---ATLVDSTWRPIDLRKGVLYGQALHFDDGTQQELAR



Hel308
Htu
(141)
CVVSDEVHLIDDRNRGPTLEVTLAKLRRLNPG----MQVVALSATVGNADEIADWLD---ASLVDTDWRPIDLQMGVHYGNALNFDDGSTREVPV



Hel308
Hvo
(141)
CVVADEVHLVDDRHRGPTLEVTLAKLRRLNTN----LQVVALSATVGNAGVVSDWLD---AELVKSDWRPIDLKMGVHYGNAVSFADGSQREVPV



Hel308
Mac
(140)
VVVVDEVHLIDSADRGPTLEVTLAKLRKMNPF----CQILALSATVGNADELAAWLD---AELVLSEWRPTDLMEGVFFDGTFFCKD-KEKLIEQ



Hel308
Mba
(140)
VVVADEVHLIDSPDRGPTLEVTLAKLRKMNPS----CQILALSATVGNADELAVWLE---AELVVSEWRPTELLEGVFFNGTFYCKD-REKTVEQ



Hel308
Mbo
(137)
LVVIDEIHLIDSPDRGPTLEMVIAKMRSKNPG----MQLIGLSATIGNPKVLAGWLD---AELVTSSWRPVDLRQGVFYDNRIQFAE-RMRPVKQ



Hel308
Mev
(140)
TIVVDEIHLLDSADRGPTLEITIAKLLRLNPN----SQIIGLSATIGNAEEIAGWLD---AELVQSQWRPIELYEGVFLEDNINFKQ-SQKPIKN



Hel308
Mfe
(141)
VVVVDEIHLINDESRGGTLEILLTKLKKFN------IQIIGLSATIGNPEELANWLN---AELIVDDWRPVELKKGIYKNGIIEFINGE-----N



Hel308
Mfr
(133)
CLVVDEVHLIDDESRGPTLEMVITKLRHASPD----MQVIGLSATIGNPKELAGWLG---ADLITSDWRPVDLREGICYHNTIYFDN-EDKEIPA



Hel308
Mhu
(138)
CIVLDEVHLIGSENRGATLEMVITKLRYTNPV----MQIIGLSATIGNPAQLAEWLD---ATLITSTWRPVDLRQGVYYNGKIRFSD-SERPIQG



Hel308
Mig
(142)
VAIVDEIHMINDEKRGGTLEVLLTKLKNLD------VQIIGLSATIGNPEELAEWLN---AELIIDNWRPVKLRKGIFFQNKIMYLNGA-----C



Hel308
Min
(131)
VVVVDEIHVIGDSERGGTLEVLLTKLKELD------VQIIGLSATIGNPEELSEWLN---AELLLDNWRPVELRKGIYREGVIEYLDGE------ 



Hel308
Mma
(138)
LAVIDEIHLIGDNERGGTLEVILTKLKNLN------AQIVGLSATIGNPEELSNWLN---AKLIVDGWRPVELKKGIYFENELEFLKNP-----A



Hel308
 Mmah
(140)
AVIVDEVHLLDSANRGPTLEVTLAKLKRLNPG----AQVVALSATVGNAMEIAQWLE---AKLVLSEWRPTYLHEGIFYGDAINFDE-DQTFIER



Hel308
 Mmar
(141)
CVVTDEVHLVDDGERGPTLEVTLAKLRRLNPD----LQTVALSATIGNAEALATWLD---AGLVDSDWRPIDLQKGVHYGQALHLEDGSQQRLSV



Hel308
 Mmaz
(140)
VVVADEVHLIDSPDRGPTLEITLSKLRRMNPS----CQVLALSATVGNADELAAWLD---AELVLSEWRPTDLMEGVFYNGIFYCKD-KEKPVGQ



Hel308
Mok
(139)
VVVIDEIHLIGDESRGGTLEVLLTKLKTKKT-----IQIIGLSATIGNPEELAKWLN---AELIVDEWRPVKLKKGIGYGNKIMFIDDNGNTINE



Hel308
Mth
(143)
VLVVDEIHLLDSANRGPTLEMTMTKLMHLNPE----MQVIGLSATIANGREIADWIK---GEIVSSDWRPVRLREGVLLEDRLVFPD-GEIQLEN



Hel308
Mzh
(140)
TVVADEVHLLNSVNRGPTLEITLAKLIHLNPG----SQIIALSATIGNPEDIAGWLG---ARLVVSEWRPTDLYEGILLDGLLHIGN-IKKDIQD



Hel308
Nma
(141)
CVVSDEVHLIDDRNRGPTLEVTLAKLRRLNPQ----LQVVALSATVGNADELADWLD---AELVDTDWRPIDLQMGVHYGNALNFDDGETREVPV



Hel308
Nth
(152)
FIIIDELHAFLDNERGVHLRSLLSRLENYIKEK---PRYFALSATLNNFKLIKEWIN---YNDIKNVEIIDSNEDDKDLLLSLMHFDKGKDYKKP



Hel308
Pfu
(141)
ILVADEIHLIGSRDRGATLEVILAHMLGKA-------QIIGLSATIGNPEELAEWLN---AELIVSDWRPVKLRRGVFYQGFVTWEDGSIDRFSS



Hel308
Sso
(148)
YFVLDELHYLNDPERGPVVESVTIRAKRRN--------LLALSATISNYKQIAKWLG---AEPVATNWRPVPLIEGVIYPERKKKEYNVIFKDNT



Hel308
Tba
(162)
LIVADEIHLLGSYDRGATLEMILSHMLGKA-------QILGLSATVGNAEELAEWLN---AKLVVSDWRPVKLRKGVFAHGQLIWEDGKVDKFPP



Hel308
Tga
(141)
LVVADEVHLIGSYDRGATLEMILTHMLGRA-------QILALSATVGNAEELAEWLD---ASLVVSDWRPVQLRRGVFHLGTLIWEDGKVESYPE



Hel308
Tsi
(164)
LVIADEIHLLGSYDRGATLEMILAHLDDKA-------QILGLSATVGNAEEVAEWLN---ADLVMSEWRPVALRKGVFYHGELFWEDGSIERFPT



Hel308
Mja
(139)
VVVVDEIHLINDETRGGTLEILLTKLKEFN-------QIIGLSATIGNPDELAEWLN---AELIVDDWRPVELKKGIYKNEAIEFINGEIREIKA











Consensus
(191)
VVVVDEIHLI D  RGPTLEVLLAKLR LNP      LQIIALSATIGNAEELAEWL   AELVVSDWRPVDLR GVFY   L F D     I


 


 















286                                                                                         380



Hel308
Mbu
(227)
LEK-----DDAVNLVLDTIKAEGQ-----CLVFESSRRNCAGFAKTASS---KVAKILDNDIMIKLAGIAEEVES--TGETDTAIVLANCIRKGV



Hel308
Afu
(225)
TSRR----VKFEELVEECVAENGG-----VLVFESTRRGAEKTAVKLSA---ITAKYVEN------EGLEKAILE--ENEGEMSRKLAECVRKGA



Hel308
Csy
(233)
TGGG-----PAVDLAAESVAEGGQ-----SLIFADTRARSASLAAKASA---VIPEAKGADAAKLAAAAKKIISS--GGETKLAKTLAELVEKGA



Hel308
Dth
(273)
----------AIRVLQKALELGLR-----TIVYTQSRKMTELIAMWASQRAGRLKKYISAYRAGFLPEQRREIEQKLASGELLAVVSTSALELGI



Hel308
Fac
(217)
-EKKHLGRDDEVSLIKESIESGGQ-----ALVFRNSRRNAEKYAQSMVN----------------FFDFQNDFEKLEIPPDLFNEAQANMVAHGV



Hel308
Hla
(229)
ERGE----DQTARLVADALDTEEDGQGGSSLVFVNSRRNAESSARKLTD---VTGPRLTDDERDQLRELADEIRS--GSDTDTASDLADAVEQGS



Hel308
Hpa
(229)
-GNE----KETAALVRDTLEDGGS-----SLVFVNSRRNAEAAAKRLAD---VTKTHLTDDERRDLLDIADQIRD--VSDTETSDDLATAIEKGA



Hel308
Htu
(229)
EGSE----KQEAALVRDILREGGS-----SLVFVNSRRNAEGAAKRLGQ---VSSREITEDERAELAELADDIRD--DSDTETSADLADCVERGA



Hel308
Hvo
(229)
GRGE----RQTPALVADALEGDGEGDQGSSLVFVNSRRNAESAARRMAD---VTERYVTGDERSDLAELAAEIRD--VSDTETSDDLANAVAKGA



Hel308
Mac
(227)
PTK-----DEAINLVLDTLREGGQ-----CLVFESSRKNCMGFAKKATS---AVKKTLSAEDKEKLAGIADEILE--NSETDTASVLASCVRAGT



Hel308
Mba
(227)
STK-----DEAVNLALDTLKKDGQ-----CLVFESSRKNCMAFAKKAAS---TVKKTLSAEDRNALAGIADEILE--NSETDTSTNLAVCIRSGT



Hel308
Mbo
(224)
VSKN----YDDLNLCLDTIAEGGQ-----CLVFVSSRRNAEAFAKRAAG---AIKSEDA-----ALAACAERLLE--GTPTEMVKTLAACVAKGA



Hel308
Mev
(227)
IVK-----DTAVNLVLDTIDENGQ-----CLVFESSRRNCAGFAKKAKS---KVGKSLDKGLLAELNNIAEEVLE--TSDTETTKELASCIKRGT



Hel308
Mfe
(222)
REIKAINNNDIYNLVVDCVKDGGC-----CIVFCNTKRGAVNEAKKLN-----LKKFLTNEEKRKLKEVAEEILSILEPPTEMCKTLAECILNGS



Hel308
Mfr
(220)
PAK-----TEDINLLLDCVADGGQ-----CLVFVSSRRNAEGYAKRAAT---ALKCSHA-----ALDSIAEKLEA--AAETDMGRVLATCVKKGA



Hel308
Mhu
(225)
KTK-----HDDLNLCLDTIEEGGQ-----CLVFVSSRRNAEGFAKKAAG---ALKAGSP-----DSKALAQELRR--LRDRDEGNVLADCVERGA



Hel308
Mig
(223)
KELPNFSNNPMLNLVLDCVKEGGC-----CLVFCNSKNGAVSEAKKLN-----LKKYLSNSEKYELQKLKEEILSILDPPTETCKTLAECLEKGV



Hel308
Min
(211)
-------VKECQDIVKEVVKDNGS-----VIIFCPTKKKAENRALSLD-----LSDLLKKSEKRKLEEISEELLSLFDPPTELCKKLASCVRKGI



Hel308
Mma
(219)
KKIKQVSRNNLTDLIVDSVEEKGS-----CLIFCNSKRNAVGEAKKHN-----LAKYLTRTEQHELNKLSEEILSILDRPVETCKALSKCIQNGV



Hel308
 Mmah
(227)
RHK-----EDSVNLVIDTVIQGGQ-----CLVFDSSRRNCVGFAKKCAP---AVGELLDRQNRNELEEVAKEVLE--NGETKLTETLAYCIKKGV



Hel308
 Mmar
(229)
QNNE----KQTAAIVRDTLEDDGS-----TLVFVNSRRNAEAAAGRLAN---TVRPHLSTEERDQLADIAEEIRD--VSDTETSDDLADAVADGA



Hel308
 Mmaz
(227)
PTK-----DEAVNLVLDTIKEGGQ-----CLVFESSRKNCMGFAKKAVS---AVKKTLSNEDRETLAGIADEIIE--NSETDVSSVLATCVRSGT



Hel308
Mok
(226)
VIVDEISKNNMFNLVVDSILKDGS-----CIIFCNSKRGAVGEAKKLN-----LKKYLSPDEISELRHLKEEVLSVLDNPTKTCKDLAECIEKGV



Hel308
Mth
(230)
RNR-----DPVLNLVLDTVDQGGQ-----MLIFESTRRNAESMAKKVSG---ALQESGE------TIELAERLS----GEGKTAKKLAMCLRHGA



Hel308
Mzh
(227)
ESR-----DDAVNLVIDTVKDKGQ-----CLVFESSRRNCMGFAKKAGK---WVSKILDEHDTIQLKSLSQEIGE--AGETEIADVLSRCVRQGV



Hel308
Nma
(229)
EAGE----KQEAALVRDILQEGGS-----SLVFVNSRRNAEAAARRLGQ---VSSRELTAGEQNDLAALATEIRE--DSDTETSQDLADCVERGA



Hel308
Nth
(241)
----------ID-LYQDLRELTKN---VHSLIFCNSRAEVEETTLYLNR---LANREVNTELYLAHHSSIDKKER-EYVEKTMANSKSPKSVVTT



Hel308
Pfu
(226)
----------WEELVYDAIRKKKG-----ALIFVNMRRKAERVALELSK---KVKSLLTKPEIRALNELADSLE-----ENPTNEKLAKAIRGGV



Hel308
Sso
(232)
TKKVHG-DDAIIAYTLDSLSKNGQ-----VLVFRNSRKMAESTALKIAN---YMNFVSLDEN--ALSEILKQLDDIEEGGSDEKELLKSLISKGV



Hel308
Tba
(247)
Q---------WDSLVIDAVKKGKQ-----ALVFVNTRRSAEKEAGMLGK---KVRRLLTKPEARRLKELAESLE-----SNPTNDKLKEVLVNGA



Hel308
Tga
(226)
N---------WYSLVVDAVKRGKG-----ALVFVNTRRSAEKEALALSK---LVSSHLTKPEKRALESLASQLE-----DNPTSEKLKRALRGGV



Hel308
Tsi
(249)
Q---------WDSLVIDALKKGKQ-----ALVFVNTRRSAEKEALLLAG---KIQRFLTKPEERKLKQLADGLD-----TTPTNQKLKEALTKGV



Hel308
Mja
(225)
VDN-----NDIYNLVVDCVKEGGC-----CLVFCNTKRNAVNEAKKLN-----LKKFLTEEEKIRLKEIAEEILSILEPPTEMCKTLAECILNGS











Consensus
(286)
             LVLDTV EGGQ      LVF NSRRNAE  AKKLA     V K LT  E   L  LAEEI      ETETS  LA CV KG


 


 















381                                                                                         475



Hel308
Mbu
(307)
AFHHAGLNSNH------------RKLVENGFRQNLIKVISSTPTLAA------------------------------------------------



Hel308
Afu
(300)
AFHHAGLLNGQ------------RRVVEDAFRRGNIKVVVATPTLAA------------------------------------------------



Hel308
Csy
(313)
AFHHAGLNQDC------------RSVVEEEFRSGRIRLLASTPTLAA------------------------------------------------



Hel308
Dth
(353)
DIGHLDLCLLVGYPGSVMATMQRGGRVGRSGRDSAIMLIGHEDALDQYLLRNPREFFSLEPESAVINPDNPSIMRRHLVCAAAEKPIALQEMMLD



Hel308
Fac
(290)
MFHHAGLSNDQ------------RTMIEKLFKQGYIKILTATPTLAA------------------------------------------------



Hel308
Hla
(315)
AFHHAGLRSED------------RARVEDAFRDRLIKCISATPTLAA------------------------------------------------



Hel308
Hpa
(309)
AFHHAGLASDH------------RSLVEDAFRDKLIKVISATPTLAA------------------------------------------------



Hel308
Htu
(310)
AFHHAGLSSTQ------------RSLVEDAFRDRLLKVISATPTLAA------------------------------------------------



Hel308
Hvo
(315)
AFHHAGLAAEH------------RTLVEDAFRDRLIKCICATPTLAA------------------------------------------------



Hel308
Mac
(307)
AFHHAGLTSPL------------RELVETGFREGYVKLISSTPTLAA------------------------------------------------



Hel308
Mba
(307)
AFHHAGLTTPL------------RELVEDGFRAGRIKLISSTPTLAA------------------------------------------------



Hel308
Mbo
(300)
AFHHAGLSRKE------------RSIVEEAFRKNLLKCISSTPTLAA------------------------------------------------



Hel308
Mev
(307)
AFHHAGLNSAQ------------RKIVEDNFRNNKIKVISSTPTLAA------------------------------------------------



Hel308
Mfe
(307)
AFHHAGLTYQH------------RKIVEDAFRNKLIKVICCTPTLSV------------------------------------------------



Hel308
Mfr
(295)
AFHHAGMNRMQ------------RTLVEGGFRDGFIKSISSTPTLAA------------------------------------------------



Hel308
Mhu
(300)
AFHHAGLIRQE------------RTIIEEGFRNGYIEVIAATPTLAA------------------------------------------------



Hel308
Mig
(308)
AFHHAGLTYEH------------RKIVEEGFRNKLIKVICCTPTLSA------------------------------------------------



Hel308
Min
(289)
AFHHSGLTYEH------------RKIIEKAFRERILKVICSTTTLAF------------------------------------------------



Hel308
Mma
(304)
AFHHAGLTYKH------------RKIVEDGFRNRLIKVICCTPTLSA------------------------------------------------



Hel308
 Mmah
(307)
AFHHAGLNSAH------------RRIVEDAFRNNLIKMICSTPTLAA------------------------------------------------



Hel308
 Mmar
(310)
AFHHAGLSRGH------------RELVEDAFRDRLVKVVCATPTLAA------------------------------------------------



Hel308
 Mmaz
(307)
AFHHAGLTTPL------------RELVENGFREGRIKIISSTPTLAA------------------------------------------------



Hel308
Mok
(311)
AFHHAGLTYEQ------------RKIVEEGFRKKLIKAICCTPTLSA------------------------------------------------



Hel308
Mth
(302)
AFHHAGLLPEQ------------RRLIELGFRQNVVKVIACTPTLAA------------------------------------------------



Hel308
Mzh
(307)
AFHHAGLNSEH------------RRMVEEGFRKNLIKMISSTPTLAA------------------------------------------------



Hel308
Nma
(310)
AFHHAGLSSTQ------------RSLVEDAFRDRLLKVISATPTLAA------------------------------------------------



Hel308
Nth
(318)
SSLELGIDIGA------------IDYVVQIDDTHTVSSLKQRLGRSG------------------------------------------------



Hel308
Pfu
(298)
AFHHAGLGRDE------------RVLVEENFRKGIIKAVVATPTLSA------------------------------------------------



Hel308
Sso
(316)
AYHHAGLSKAL------------RDLIEEGFRQRKIKVIVATPTLAA------------------------------------------------



Hel308
Tba
(320)
AFHHAGLGRAE------------RTLIEDAFREGLIKVLTATPTLAM------------------------------------------------



Hel308
Tga
(299)
AFHHAGLSRVE------------RTLIEDAFREGLIKVITATPTLSA------------------------------------------------



Hel308
Tsi
(322)
AFHHAGLGRTE------------RSIIEDAFREGLIKVITATPTLSA------------------------------------------------



Hel308
Mja
(305)
AFHHAGLTYQH------------RKIVEDAFRKRLIKVICCTPTLSA------------------------------------------------











Consensus
(381)
AFHHAGL                R LVEDAFR  LIKVI ATPTLAA


 


 















476                                                                                         570



Hel308
Mbu
(342)
--------------------------------------GLNLPARRVIIRSYRRFDS-NFG--------------------------MQPIPVLE



Hel308
Afu
(335)
--------------------------------------GVNLPARRVIVRSLYRFDG-YSK----------------------------RIKVSE



Hel308
Csy
(348)
--------------------------------------GVNLPARRVVISSVMRYNS-SSGM-------------------------SEPISILE



Hel308
Dth
(448)
NEAGKCIKSLEKDGELLASRDRSFYYTRARYPHKDVDLRGIGQTYNIFEHSTGEYLGEVDGVRAFKETHPGAVYLHMGETYVVQDLDLETFAVYA



Hel308
Fac
(325)
--------------------------------------GVNLPARTVIIRDITRFSD---GY-------------------------SKPISGIE



Hel308
Hla
(350)
--------------------------------------GVNTPARRVIVRDWRRYDG-EFGG-------------------------MKPLDVLE



Hel308
Hpa
(344)
--------------------------------------GVNTPSRRVIVRDWRRYDG-DIGG-------------------------MQPLDVLE



Hel308
Htu
(345)
--------------------------------------GVNTPARRVIVRDWRRFDP-SAGG-------------------------MAPLDVLE



Hel308
Hvo
(350)
--------------------------------------GVNTPSRRVVVRDWQRYDG-DYGG-------------------------MKPLDVLE



Hel308
Mac
(342)
--------------------------------------GLNLPARRVIIRSYRRYSS-DSG--------------------------MQPIPVLE



Hel308
Mba
(342)
--------------------------------------GLNLPARRVIIRNYRRYSS-EDG--------------------------MQPIPVLE



Hel308
Mbo
(335)
--------------------------------------GLNLPARRVIIRDYLRFSA-GEG--------------------------MQPIPVSE



Hel308
Mev
(342)
--------------------------------------GLNLPARRVIVRNYKRYDP-NFG--------------------------MQPIPVLD



Hel308
Mfe
(342)
--------------------------------------GLNLPCRRAIVKDLTRYT--NRG--------------------------MRYIPIME



Hel308
Mfr
(330)
--------------------------------------GLNLPARRVIIRDYLRYSG-GEG--------------------------MRPIPVRE



Hel308
Mhu
(335)
--------------------------------------GLNLPARRVIIRDYNRFAS-GLG--------------------------MVPIPVGE



Hel308
Mig
(343)
--------------------------------------GINIPCRRAIVRDLMRFS--NGR--------------------------MKPIPIME



Hel308
Min
(324)
--------------------------------------GLNLPCRRVIISELKRYT--RRG--------------------------LTYIPIME



Hel308
Mma
(339)
--------------------------------------GLNLPCRRAIVRDIKRYS--QNG--------------------------LVDIPRME



Hel308
 Mmah
(342)
--------------------------------------GLNLPARRVIIRSYKRYDP-NAG--------------------------MQPIPVLD



Hel308
 Mmar
(345)
--------------------------------------GVNTPSRRVVVRDWRRYDG-SAGG-------------------------MAPLSVLE



Hel308
 Mmaz
(342)
--------------------------------------GLNLPARRVIIRSYRRYSS-DSG--------------------------MQPIPVLE



Hel308
Mok
(346)
--------------------------------------GINMPCRRAIIRDLKRFS--SRG--------------------------YIPIPKME



Hel308
Mth
(337)
--------------------------------------GLNLPARRVLIRSYKRYEA-GLG--------------------------TRPIPVME



Hel308
Mzh
(342)
--------------------------------------GLNLPARRVIIRSYKRYDP-NFG--------------------------MKPIPVLE



Hel308
Nma
(345)
--------------------------------------GVNTPARRVIVRDWRRFDP-SAGG-------------------------MAPLDVLE



Hel308
Nth
(353)
--------------------------------------RKLGTNQVLQVYSTTNDSLVQSLA-------------------------VIDLLLEK



Hel308
Pfu
(333)
--------------------------------------GINTPAFRVIIRDIWRYS--DFG--------------------------MERIPIIE



Hel308
Sso
(351)
--------------------------------------GVNLPARTVIIGDIYRFNKKIAGY-------------------------YDEIPIME



Hel308
Tba
(355)
--------------------------------------GVNLPSFRVIIRDTKRYS--TFG--------------------------WSDIPVLE



Hel308
Tga
(334)
--------------------------------------GVNLPSFRVIIRDTKRYA--GFG--------------------------WTDIPVLE



Hel308
Tsi
(357)
--------------------------------------GVNLPAYRVIIRDTKRYS--NFG--------------------------WVDIPVLE



Hel308
Mja
(340)
--------------------------------------GLNLPCRRAIVKDLTRFIN----KG------------------------MRYIPIME











Consensus
(476)
                                      GLNLPARRVIIRDYKRY      G                         M PIPVLE


 


 















571                                                                                         665



Hel308
Mbu
(372)
YKQMAGRAGRPHLDPYGESVLLAKTYDEF--AQLMENYVEADAEDIWSKLGTENALRTHVLSTIVNGFASTRQELFDFFGATFFAYQQ-DKWMLE



Hel308
Afu
(363)
YKQMAGRAGRPGMDERGEAIIIVGKRDR---EIAVKRYIFGEPERITSKLGVETHLRFHSLSIICDGYAKTLEELEDFFADTFFFKQN--EISLS



Hel308
Csy
(379)
YKQLCGRAGRPQYDKSGEAIVVGGVNAD----EIFDRYIGGEPEPIRSAMVDDRALRIHVLSLVTTSPGIKEDDVTEFFLGTLGGQQS-GESTVK



Hel308
Dth
(543)
AKSEANYYTRPITEKYTEIVEVQATRATAAGELCLGRLKVTEHVSAYEKRLVRGQARIGLIPLDLPPLVFETQGMWFTLDSQVRRDVEDRRLHFM



Hel308
Fac
(354)
IQQMIGRAGRPKYDKKGYGYIYAASPG---MIRVAEGYLTGELEPVISRMDSNSLIRFNVLALISSGIATDLKGIQDFYGKTLLAAQN-DIDGYE



Hel308
Hla
(381)
VHQMCGRAGRPGLDPYGEAVLLANDADTK--EELFERYLWADPEPVRSKLAAEPALRTHVLATVASGFASTRDGLLSFLDNTLYATQTDDEGRLA



Hel308
Hpa
(375)
VHQMFGRAGRPGLDPHGEAVLIAKSHDEL--QELFDQYVWADPEPVHSKLAAEPALRTHILATVASGFAGTEEELLDFLERTLYATQTDETGRLE



Hel308
Htu
(376)
VHQMMGRAGRPGLDPYGEAVLLAKSHDES--EELFDRYIWADPEPVRSKLAAEPALRTHVLATIASGFARTRGGLLEFLEATLYASQSSEAGRLE



Hel308
Hvo
(381)
VHQMMGRAGRPGLDPYGEAVLLAKDADAR--DELFERYIWADAEDVRSKLAAEPALRTHLLATVASGFAHTREGLLEFLDQTLYATQTDDPERLG



Hel308
Mac
(372)
YKQMAGRAGRPRLDPYGEAVLLAKSYEEL--LFLFEKYIEAGAEDIWSKLGTENALRTHVLSTISNGFARTKEELMDFLEATFFAYQY-SNFGLS



Hel308
Mba
(372)
YKQMAGRAGRPRLDPYGEAVLVAKSYKEF--VFLFENYIEANAEDIWSKLGTENALRTHVLSTISNGFARTYDELMDFLEATFFAFQY-SNFGLS



Hel308
Mbo
(365)
YRQMAGRAGRPRLDPYGEAVLIAKEAEQV--PELFEVYIEAEAEDVHSRIAEPTALYTHVLSLVASGFAGTRGELTEFMNRSFYVHEHKQGRLIH



Hel308
Mev
(372)
YKQMAGRAGRPSLDPYGESVLISHTYNEF--TDLLDRYIDAEPEDILSKLGTENALRTHVLSTIVNGFATTRQGMVDFMGSSFFAYQQ-QKWSLI



Hel308
Mfe
(371)
IQQCIGRAGRLGLDPYGEGIIVAKNDR---DYLRSYQVLTQKPEPIYSKLSNQAVLRTQLLGLIATIEIRDEYDLEWFIRNTFYAYQYGNLREVA



Hel308
Mfr
(360)
YRQMAGRAGRPHLDPYGEAILIAKTEYAV--NDLHEEYVEAPDEDVTSRCGEKGVLTAHILSLIATGYARSYDELMAFLEKTLYAYQHTGKKALT



Hel308
Mhu
(365)
YHQMAGRAGRPHLDPYGEAVLLAKDAPSV--ERLFETFIDAEAERVDSQCVDDASLCAHILSLIATGFAHDQEALSSFMERTFYFFQHPKTRSLP



Hel308
Mig
(372)
IHQCIGRAGRPGLDPYGEGIIFVKNER---DLERAEQYLEGKPEYIYSKLSNQAVLRTQLLGMIATREIENEFDLISFIKNTFYAHQYGNLGGVL



Hel308
Min
(353)
VQQCIGRAGRPGLDEYGEGILVAKDER---DYLRALQCLTQKPEPIYSKLSNDSVLRTQILGLIATRYVLDEYDLEEFIKNTFYAYQYKNLDEIK



Hel308
Mma
(368)
IQQCIGRAGRPGLDPYGEGIIYIKNER---DAEKAYEILTGSVENIYSKLANQKVLRIHILGLISTGEIKDGQNLVNFMKNTFYAHQFGNIGAVL



Hel308
 Mmah
(372)
YKQMAGRAGRPHLDPYGEAVVIVKTYEEF--TDVLERYISASAEDIWSKLGTENALRTHILSTIASGFANCHREILTFLGSTFFAHQQ-QSWNFE



Hel308
 Mmar
(376)
VHQMMGRAGRPGLDPYGEAVLIASSHDEV--DELFERYVWADPEPVRSKLAAEPALRTHILATVASGFARSRKGLLEFLEQTLYASQTDDSGQLE



Hel308
 Mmaz
(372)
YKQMAGRAGRPRLDPYGEAVLLAKSYEEF--VFLFEKYIEAGAEDIWSKLGTENALRTHILSTISNGFARTREELMDFLEATFFAFQY-SNFGLS



Hel308
Mok
(375)
IHQCIGRAGRPNLDPYGEGIIYINNTENPELIENAKNYLIGNVEEIYSKLSNQKVLRTHMLGLITTGDIKNKNDLEEFIKNTFYAYQYQNTKKIL



Hel308
Mth
(367)
YRQMAGRAGRPGLDPYGESLIMARSESEL--QKLMDHYVMGEPEDIWSKLASERALRTHVLATIASRFADSVDSLSRLMASTFYARQQ-DPSYLG



Hel308
Mzh
(372)
YKQMAGRAGRPHLDPYGESVLIARSYDEF--MDIMENYVNADPEDIWSKLGTENALRTHVLSTIVNGFAYTYRGLMDFVKMTFFAYQK-EASDLH



Hel308
Nma
(376)
VHQMMGRAGRPGLDPYGEAVLLAKSHDES--QELFDRYVWADPEPVRSKLAAEPALRTHVLATIASGFARTREGLLEFLEATLYASQSSEGGRLE



Hel308
Nth
(385)
WIEPATEYPLPLDILFHQIISICHEANGVRLDPLIDNIKANAAFYKLKEEDINHVINYMIENDFLQLIRNSAELIVGLEGERLLRGKEFYAVFMT



Hel308
Pfu
(362)
VHQMIGRAGRPKYDEVGEGIIVSTSDD---PREVMNHYIFGKPEKLFSQLSNESNLRSQVLALIATFGYSTVEEILKFISNTFYAYQRKDTYSLE



Hel308
Sso
(383)
YKQMSGRAGRPGFDQIGESIVVVRDKEDV--DRVFKKYVLSDVEPIESKLGSERAFYTFLLGILSAEGNLSEKQLENFAYESLLAKQL-----VD



Hel308
Tba
(384)
IQQMIGRAGRPKYDKEGEAIIVAKTEK---PEELMEKYIFGKPEKLFSMISNDAAFRSQVLALITNFGVESFRELIGFLEKTFYYHQRKDLEILE



Hel308
Tga
(363)
IQQMMGRAGRPRYDKYGEAIIVARTDE---PGKLMERYIRGKPEKLFSMLANEQAFRSQVLALITNFGIRSFPELVRFLERTFYAHQRKDLSSLE



Hel308
Tsi
(386)
IQQMMGRAGRPKYDIEGQAIIIAKTEK---PEDLMKRYVLGKPEKLFSMISNEASFRSQVLALITNFGVGNFKELVNFLERTFYYHQRKNLEALE



Hel308
Mja
(369)
IQQCIGRAGRPGLDPYGEGIIVAKNDRDY---LRAYQALTQKPEPIYSKLSNQAVLRTQLLGLIATGEIRDEYDLEWFIRNTFYAHQYGNLREVA











Consensus
(571)
I QM GRAGRP LDPYGEAVLIAKS D     EL E YI ADPE IWSKLA E ALRTHVLALIASGFA T  ELLDFL  TFYAYQ      L


 


 















666                                                                                         760



Hel308
Mbu
(464)
EVINDCLEFLIDKAMVSET-E--------------------------------------DIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGF



Hel308
Afu
(453)
YELERVVRQLENWGMVVEAAH-----------------------------------------------LAPTKLGSLVSRLYIDPLTGFIFHDVL



Hel308
Csy
(469)
FSVAVALRFLQEEGMLGRR----------------------------------------------GGRLAATKMGRLVSRLYMDPMTAVTLRDAV



Hel308
Dth
(638)
GGLHALEHGLIGCMPLIILTDRNDLGGIASPVHEQLHKG--------------AVFIYDGTPGGIGLCRQAFELGDRLVARAMGILSSCTCENGC



Hel308
Fac
(445)
LAFESALYFLKDNDFITEEN----------------------------------------------DIYSATKFGRLTSDLYIDPVSSLILKKCL



Hel308
Hla
(474)
AVTDTVLDYLAVNDFIERDRD------------------------------------------GGSESLTATGIGHTVSRLYLDPMSAAEMIDGL



Hel308
Hpa
(468)
TVTQHVLDYLDRNGFLERDD-----------------------------------------------RLRATGLGHRVSQLYLDPMSAAEIIDGL



Hel308
Htu
(469)
SVTDDVLDYLERNDFIERSR--DDEAEDSGEDDGPFTSAADLAEQ---------QAAK------REETLEATSLGHTVSRLYLDPMSAAEIVHGL



Hel308
Hvo
(474)
QVTDRVLDYLEVNGFVEFEG----------------------------------------------ETIQATPVGHTVSRLYLDPMSAAEIIDGL



Hel308
Mac
(464)
VVVDECLNFLRQEGMLEQDS----------------------------------------------DALISTMFGKLVSRLYIDPLSAALIAKGL



Hel308
Mba
(464)
TVVNECLNFLRQEGMLEKD-----------------------------------------------DALIPTSFGKLVSRLYIDPLSAARIAKGL



Hel308
Mbo
(458)
RAIDEALQFLITAEMVVEV----------------------------------------------GEHIGATELGTLVSRMYIDPRSAFAIVTTL



Hel308
Mev
(464)
DVVDDCIEFLQDNEMIKD-----------------------------------------DG--ER---LYATRLGQVISTLYIDPLSGAIIIDKL



Hel308
Mfe
(463)
KNINEVIRFLEEK--------------------------------------------------EFMIDFIPTELGKRVAELYIDPLSAKYMIDGL



Hel308
Mfr
(453)
RTLDDALGFLTEAEMVTDL----------------------------------------------SGMLHATEYGDLTSRLYIDPHSAEIITTAL



Hel308
Mhu
(458)
RLVADAIRFLTTAGMVEER----------------------------------------------ENTLSATRLGSLVSRLYLNPCTARLILDSL



Hel308
Mig
(464)
RNIKEVINFLEEN--------------------------------------------------DFIADYFPTKLGKRVSELYIDPLSAKIIIDGL



Hel308
Min
(445)
KKIKEIIEFLEDCN--------------------------------------------------FIKNFEVTPLGKKVSNLYLDPLSAKIMIDNI



Hel308
Mma
(460)
LNVSEVVEFLEKNKFLETTIHKKTENKVRELSFDS------S-NN---LVLDSKETSFDLTNPNSNIEFRSTKLGKRISELYIDPMSSEIIIEEL



Hel308
 Mmah
(464)
ELLEDCLIFLKNEGMLEQD-N--------------------------------------ET-------IRATELGKMISSLYIDPLSASKIIRGL



Hel308
 Mmar
(469)
RVVDDVLTYLQRNDFLEIEAG----------------------------------------------ELDATSLGHTVSRLYLDPMSAAEIVDGL



Hel308
 Mmaz
(464)
AVVDECLDFLRREGMLEKDP----------------------------------------------DALVSTVFGKLVSRLYIDPLSAALIAKGL



Hel308
Mok
(470)
ENIYEITNFLEKNGFIELNYRRDENKDKSNNSHNNKKNISNTNNSIKMLVLDNNNSLTIKSRHEEDVYYNITPLGKKVSELYIDPLSAEYIIDGL



Hel308
Mth
(459)
ETIASVLEFLVRSDMIDKD-------------------------------------------------LTPTPLGALVSRLYIDPLSAMVMIQEI



Hel308
Mzh
(464)
DVIEECVRFLIDNEMIISD-S--------------------------------------NDILPES-AFRSTATGKLISMLYIDPLSGSLIMDGI



Hel308
Nma
(469)
RVTDDVLSYLERNDFIERSGGPEDTLNSEADAASAFTSAADLADS---------DGGDSGGTTGQEEDLEATSLGHTVSRLYLDPMSAAEIVHGL



Hel308
Nth
(480)
QEEFEVREGIRKIGSIDKS-------------------------------------------LMVSEGDNIILAGQLWTIKNIDIERDIIYVAKA



Hel308
Pfu
(454)
EKIRNILYFLLEN---------------------------------------------EFIEISLEDKIRPLSLGIRTAKLYIDPYTAKMFKDKM



Hel308
Sso
(471)
VYFDRAIRWLLEHSFIKEE----------------------------------------------GNTFALTNFGKRVADLYINPFTADIIRKGL



Hel308
Tba
(476)
GKAKSIVYFLLEN---------------------------------------------EFIDIDLNDSFIALPFGIRTSQLYLDPLTAKKFKDAL



Hel308
Tga
(455)
YKAKEVVYFLIEN---------------------------------------------EFIDLDLEDRFIPLPFGKRTSQLYIDPLTAKKFKDAF



Hel308
Tsi
(478)
GKAKSIVYFLFEN---------------------------------------------EFIDIDLNDQFMPLPLGIRTSQLYLDPVTAKKFKDAF



Hel308
Mja
(461)
KNINEVIRFLEENEFI--------------------------------------------------IDFMPTELGKRVSELYIDPLSAKFIIDGL











Consensus
(666)
  I EVL FL  N  I                                                    L AT LG  VS LYIDPLSA  IIDGL


 


 















761                                                                                         855



Hel308
Mbu
(520)
KDIGKSTGGNMGSLEDDKG----------------------------------------------------DDITVTDMTLLHLVCSTPDMRQLY



Hel308
Afu
(501)
SRMELS-----------------------------------------------------------------------DIGALHLICRTPDMERLT



Hel308
Csy
(518)
GEASPGR--------------------------------------------------------------------MHTLGFLHLVSECSEFMPRF



Hel308
Dth
(719)
PGCIHSPKCGSGNR------------------------------------------------------------PLDKEAAMHMLAVLAGERCGE



Hel308
Fac
(494)
DLEFS------------------------------------------------------------------------EELYLYYISKTPDMLTFN



Hel308
Hla
(527)
RSVARDAADTGASAEADNG-EFVRTGDADDASGGDEPGFGTYTRAGDDESGER------ETENEETDEEETEASEVTPLGLYHLISRTPDMYELY



Hel308
Hpa
(516)
RDADG---------------------------------------------------------------------KPTALGLYHLVSRTPDMYQLY



Hel308
Htu
(547)
ERADER---------------------------------------------------------------------PTALGLYQLVSRTPDMYELY



Hel308
Hvo
(523)
EWAADHRTEKLRALAGETPEKPTRDRSESDESGGFQRASEMVADDGDGGGGEDGVGANGDGDSDDADGVETDRTYPTPLGLYHLVCRTPDMYQLY



Hel308
Mac
(513)
REAGT----------------------------------------------------------------------LTELTLLHLVCSTPDMRLMY



Hel308
Mba
(512)
KGAKS----------------------------------------------------------------------LSELTLLHLVCSTPDMRLLY



Hel308
Mbo
(507)
REQEK----------------------------------------------------------------------YADLGLIQLICTTPDMPTLY



Hel308
Mev
(513)
KKADK----------------------------------------------------------------------VTDMTMLHIICSTPDMRQLY



Hel308
Mfe
(508)
NEMENED----------------------------------------------------------------------DIYYLYLISKTLEMMPNL



Hel308
Mfr
(502)
REEGE----------------------------------------------------------------------LTDLALLQLLCMTPDMFTLY



Hel308
Mhu
(507)
KSCKT----------------------------------------------------------------------PTLIGLLHVICVSPDMQRLY



Hel308
Mig
(509)
KEMGNVDNE--------------------------------------------------------------------ELYYLYLISKTLEMMPLL



Hel308
Min
(490)
EVKDDLH-------------------------------------------------------------------------LLYILCKCIEMKPLL



Hel308
Mma
(545)
HELKKKCDQLDR--------------------------------------------------------------SKIDQYLFYLISKTNEMRPLL



Hel308
 Mmah
(513)
EKTTH----------------------------------------------------------------------VTDMTLLQLICSTPDMRLLY



Hel308
 Mmar
(518)
RDWERGASDSTSASGSPAD----AQAEP-PANSGFTTASELAEDADESDADRD-------------------PDDISALGLYHLVSRTPDMYQLY



Hel308
 Mmaz
(513)
REAGT----------------------------------------------------------------------LTELTLLHLICSTPDMRIMY



Hel308
Mok
(565)
KNLHKKTLSNPKNM------------------------------------------------------------ECYILHILYIISKTTEMQPVL



Hel308
Mth
(505)
RGIRR----------------------------------------------------------------------PTVLTLLHVITMTPDMELLF



Hel308
Mzh
(519)
RKADY----------------------------------------------------------------------FEDITMMHLICSTPDMKNLY



Hel308
Nma
(555)
EDADER---------------------------------------------------------------------PTALGLYQLVSRTPDMYELY



Hel308
Nth
(532)
VDGKPPK--------------------------------------------------------------------YSGGGFILNPKIPERMHKIL



Hel308
Pfu
(504)
EEVVKDPN---------------------------------------------------------------------PIGIFHLISLTPDITPFN



Hel308
Sso
(520)
EGHKAS----------------------------------------------------------------------CELAYLHLLAFTPDGPLVS



Hel308
Tba
(526)
PQIEENPN---------------------------------------------------------------------PLGIFQLLASTPDMGTLS



Hel308
Tga
(505)
PAIERNPN---------------------------------------------------------------------PFGIFQLIASTPDMATLT



Hel308
Tsi
(528)
EKLEKNPN---------------------------------------------------------------------PLGIFQLLASTPDMSSLR



Hel308
Mja
(506)
EEMENEE----------------------------------------------------------------------EIYYLYLISKTLEMMPNL











Consensus
(761)
                                                                              LGLLHLIS TPDM  LY


 


 















856                                                                                         950



Hel308
Mbu
(563)
LRNTDYTIVNEYIVAHSDEFH---EIPDKLKETDYEWFMGEVKTAMLLEEW---------------VTEVSAEDITRHFNVGEGDIHALADTSEW



Hel308
Afu
(525)
VRKTDSWVEEEAFRLRKELSY----YPSDFS-VEYDWFLSEVKTALCLKDW---------------IEEKDEDEICAKYGIAPGDLRRIVETAEW



Hel308
Csy
(545)
ALRQKDHEVAEMMIEAGRGELLR---P--------VYSYECGRGLLALHRW---------------IGESPEAKLAEDLKFESGDVHRMVESSGW



Hel308
Dth
(754)
AKRKDVSCRIETDEGSMEIDSG-YTKSDQAELPYAVLDIETRYSAQEVGGWGNCHRMGVSFAVVFDSRNQEFVTFDQEQAADLGSFLEDFSLVVG



Hel308
Fac
(517)
YRASDYEYLEEFLDRHNISDFS-------------EESMGAAKTAIILNEW---------------INEVPINTIAETFGIGPGDIQAKASSADW



Hel308
Hla
(615)
LKSGDRETYTELCYERETEFLG--DVPSEYEDVRFEDWLASLKTARLLEDW---------------VNEVDEDRITERYGVGPGDIRGKVDTAEW



Hel308
Hpa
(542)
LRSGDRERYTEIAYEREPEFLG--HMPSEFEDNAFEDWLSALKTARLLEDW---------------ASELDEDRITERYAIGPGDIRGKVETAQW



Hel308
Htu
(573)
LRSGEDEKFGELFYERETELLG--DAPSEYEEDRFEDWLAALKTGKLLEDW---------------ADETDEETITDRYKIGPGDLRGKVDTAEW



Hel308
Hvo
(618)
LKSGDRETYTELCYEREPEFLG--RVPSEYEDVAFEDWLSALKTAKLLEDW---------------VGEVDEDRITERYGVGPGDIRGKVETSEW



Hel308
Mac
(538)
MRSQDYQDINDFVMAHAEEFS---KVPSPFNIVEYEWFLSEVKTSLLLMDW---------------IHEKPENEICLKFGTGEGDIHTTADIAEW



Hel308
Mba
(537)
MRSHDYQDINDYVMAHASEFV---KVPSPFDTTEYEWFLGEVKTSLLLLDW---------------IHEKSENEICLKFGTGEGDIHSIADIAEW



Hel308
Mbo
(532)
AKNADLPALSRMIEVRGADIW---LPP-PLDDDAAETYYRAVKTAMLLSDW---------------TDELSEEKICERYGVGPGDVFGMVENINW



Hel308
Mev
(538)
LRSKEYEKINEYVMTHSDEFV---EVPNPFKSIEYEWFLGEVKTALLINEW---------------IDEKTLDDITAEFGVGEGDINALSDISEW



Hel308
Mfe
(533)
RVYKSEE--LNLIDEMENLG------IKSFE----IEDLEAFKTAKMLYDW---------------ISEVPEDEILKKYKIEPGILRYKVENAVW



Hel308
Mfr
(527)
VKKNDLGTLEKFFFEHEEEFR---T---EFSYDEMEDFFRSLKTAMLLSDW---------------TDEIGDDTICTRFGVGPGDIFNAVQGISW



Hel308
Mhu
(532)
LKAADTQLLRTFLFKHKDDLI---LPL-PFEQEEEELWLSGLKTALVLTDW---------------ADEFSEGMIEERYGIGAGDLYNIVDSGKW



Hel308
Mig
(536)
RVNSFEE--LDLILEMEEAG------IYDRT----YDDLAAFKNAKMLYDW---------------INEVPEDEILKKYKIEPGILRYKVEQAKW



Hel308
Min
(512)
RVYRKEE--EELAEELLNYE------I-FIS----YENLEEFKTAKMLYDW---------------INEVPEDEILKTYKVEPGILRYKVEVAKW



Hel308
Mma
(578)
RIRPNEE--LDLILEMDKMG------LKDYS----IENIEAFKNSKMFCDW---------------VSEIPEEIILEKYGVEPGILRYKVEQAKW



Hel308
 Mmah
(538)
LRNRDYEIINDYVMNHTEEFI---EVPSPFKQIEYEWFLSEVKTALLLLEW---------------INEKSLEKIVENYQVGEGDIYASSDIAEW



Hel308
 Mmar
(589)
LRSGDREEYEMELFEREEELLG--PTPSEFEEGRFEDWLSALKTARLLEDW---------------ATEVDEATITDRYGVGPGDIRGKVETAQW



Hel308
 Mmaz
(538)
MRSQDYQEVNDYVMAHAGEFS---KVPNPFNIAEYEWFLGEVKTSLLLMDW---------------IHEKPENEICLKFGIGEGDIHATADIAEW



Hel308
Mok
(600)
RVRRKEE--NDLINDMIKLDIDVDDVIYGIS----SENLEYFKNAKLFYDW---------------INEIPEEELLLGYNIEPGILRYNVEQAKW



Hel308
Mth
(530)
VQQS-DNWLEDFISEHSSELG---NEKN------FDWLLREVKTASMLMDW---------------INEVHEDRIEDRYSISPGDLVRIAETAEW



Hel308
Mzh
(544)
MRSSDYENVNMYVLQNKDKFI---SMPSPFKMIEYEWFLGEVKTALLLLDW---------------INEVPADDICKKYGIGEGDIRMFSETAVW



Hel308
Nma
(581)
LRSGEDEKFGELYYERERELLG--DAPSEFEEERFEDWLAALKTGKLLEDW---------------ATEDDEEQITERYKIGPGDLRGKVDTAEW



Hel308
Nth
(559)
CERKNFEFIDNMAQNHLEEQR---------------------KPFELYNIK---------------PNERVIWNNGDEILFETYTGTKIFQTLAW



Hel308
Pfu
(530)
YSKREFERLEEEYYEFKDRLYFDDPYISGYDPYLERKFFRAFKTALVLLAW---------------INEVPEGEIVEKYSVEPGDIYRIVETAEW



Hel308
Sso
(545)
VGRNEEEELIELLEDLDCELL----IEEPYEEDEYSLYINALKVALIMKDW---------------MDEVDEDTILSKYNIGSGDLRNMVETMDW



Hel308
Tba
(552)
IKRKEQESYLDYAYEMEDYLYRSIPYWEDYE---FQKFLSEVKTAKLLLDW---------------INEVSEAKLIEAYGIDTGDLYRIIELADW



Hel308
Tga
(531)
ARRREMEDYLDLAYELEDKLYASIPYYEDSR---FQGFLGQVKTAKVLLDW---------------INEVPEARIYETYSIDPGDLYRLLELADW



Hel308
Tsi
(554)
VKRKEQEDLLDYAYEMEEYLYQNIPYWEDYK---FEKFLGETKTAKLLLDW---------------INEVNDVKILETYEIDTGDLYRILELVDW



Hel308
Mja
(531)
RVYNSEE--LNLIDEMDSLGIK----------SFEIEDLEAFKTAKMLYDW---------------INEVPEDEILKRYKIEPGILRYKVENAVW











Consensus
(856)
LR  D E L E I E   E          F    FE FL  VKTA LL DW               I EV ED I ERYGIGPGDL   VE AEW


 


 















951                                                                                        1045



Hel308
Mbu
(640)
LMHAAAKLAELLGVEYSS--------HAYSLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVS---------VAKLKGADISVLSKLVGP-



Hel308
Afu
(600)
LSNAMNRIAEEVG-N--T--------SVSGLTERIKHGVKEELLELVRIRHIGRVRARKLYNAGIRN---------AEDIVRHREKVASLIGRG-



Hel308
Csy
(614)
LLRCIWEISKHQERPDLLG-------ELDVLRSRVAYGIKAELVPLVSIKGIGRVRSRRLFRGGIKG---------PGDLAAVPVERLSRVEGIG



Hel308
Dth
(848)
FNLLKFDYRVIQGLSDYDFSSLPILDMIREIEARLGHRLSLDHLARHTLGINKSANGLMALKWWKEGELDKIVEYCRQDVSVIRDLYLFGRDKGY



Hel308
Fac
(584)
ISYSLYRLGSMFDKENEN--------NLLHLNIRIKEGVKEEIIRIIEIPQVGRVRGRRLYNNGFKS---------IDDIANARVEDISRIFGFS



Hel308
Hla
(693)
LLRAAETLARDVEGVDGDVVV-----AVREARKRIEYGVREELLDLAGVRNVGRKRARRLFEAGIET---------RADLREADKAVVLGALRGR



Hel308
Hpa
(620)
LLNAAERLAAELQRDDAEGIPSATTTAVREARKRVEYGVEEELLDLAGVRNVGRKRARRLYEAGIES---------RADLREADKSVVLGALRGR



Hel308
Htu
(651)
LLGAAESLAAEIDSEWTV--------AVREARARVEHGVGEELLELVSVGGVGRKRARRLYDAGIEE---------PADLRSADKGIVLSVLKG-



Hel308
Hvo
(696)
LLGAAERLATELD---LDSVY-----AVREAKKRVEYGVREELLDLAGVRGVGRKRARRLFEAGVET---------RADLREADKPRVLAALRGR



Hel308
Mac
(615)
IMHVATQLARLLDLKGAK--------EAAELEKRIHYGAGPELMDLLDIRGIGRVRARKLYGAGFKS---------TADLAGATPEKVAALVGP-



Hel308
Mba
(614)
IMHVTSQLAGLLDLKGAR--------EAAELEKRIHYGAAPELIDLLNIRGIGRVRARKLYEAGFKS---------SAELAEVDPEKVAALLGP-



Hel308
Mbo
(608)
LLHATSQLARMFVPKFYG--------QIADCEICMKNGIRRELLPLVRLRGIGRVRARRLFNNGITS---------PEELSRHKKEDLVKILGS-



Hel308
Mev
(615)
LMHSAVNLANLTDLDAD---------KAQELEKRIHHGVNKDLIQLVSISNIGRVRARKLYEAGIQS---------VSDIKNTKLHILSNYLGR-



Hel308
Mfe
(601)
LMHALKEMAKIIGKN---------SEIPEKLEIRLEYGAKEDIIELLNVKYIGRVRARKLYNAGIRN---------VEDIINNPSK---VASIIG



Hel308
Mfr
(601)
LLHASGRLARLVAPEHRD--------AVEETTLRVRHGIRRELIPLVRVKGIGRVRARRLFNNGITG---------PELLAAADPSVVGHIVGG-



Hel308
Mhu
(608)
LLHGTERLVSVEMPEMSQ--------VVKTLSVRVHHGVKSELLPLVALRNIGRVRARTLYNAGYPD---------PEAVARAGLSTIARIIGE-



Hel308
Mig
(604)
MIYSTKEIAKLLNRN---------IDTLSKLEIRLEYGAKEDIIELLKIKYVGRARARKLYDAGIRS---------VEDIINNPKK---VASLLG



Hel308
Min
(579)
LSYSLKEIAKILNKEVP------------NLELRLEYGAKEELLELLKIKYIGRVRARKLYSAGIRN---------REDIIKNPKK---VANILG



Hel308
Mma
(646)
MIYSTKEIAKLIHLDNSE-----IYKSLLKMEVRIEYGAKEELIELLNVKNVGRIRSRKLYDAGIRS---------KIEINKNPEK---ILELFG



Hel308
 Mmah
(615)
LMHATQRIASRINPQLET--------ECAKLEKRIHYGAGSELIELVEIPNVGRARARKLFKKGYRS---------RQKLATADEKQLAGIVGP-



Hel308
 Mmar
(667)
LLGAAESLASEVDLDAAR--------AISEARIRVEHGVREELVDLAGVRGVGRKRARRLFQAGITD---------RAQLRDADKAVVLAALRGR



Hel308
 Mmaz
(615)
IMHVTAQLAGLLDLKGAK--------EASELEKRIRYGAAPELMDLLDIRSVGRVRARKLYEAGFKS---------TAELAAASPEHIAVLVGP-



Hel308
Mok
(674)
MIHSAKEIFNLLNIDNKV-----IKDCLNDLEIRMEYGAKQDIIELLKIKHIGRARARILYNAGIKN---------ANDIINNQKN---IINLLG



Hel308
Mth
(600)
LMSALHRISKHMDLGVTY--------LAERLALRIHYGAGDELLQLLELKGIGRVRARKLYQAGYRS---------LEDLKAADKSTLSEILGP-



Hel308
Mzh
(621)
LMHATSRLSGLLKVSEASE-------KSKELEKRLSYGINSELVNIVALKGIGRVRARKIYENGYRS---------IDDLKKADPLKLSKIVGS-



Hel308
Nma
(659)
LLGAAESLASEIDSEWAV--------AVREARARVEHGVGEELLELVSVSGIGRKRARRLYAAGIEE---------PAALRSADKGVILHVLKG-



Hel308
Nth
(618)
ILRSYNVNIKEIDGIGRIN-----IEGGIDLPGVLQDIKETDWRPEYLLDFTLEQEKFKSKFSPYLP---------KDLQDKMHIAHLVDIEGVK



Hel308
Pfu
(610)
LVYSLKEIAKVLG-AYE------IVDYLETLRVRVKYGIREELIPLMQLPLVGRRRARALYNSGFRS---------IEDISQARPEELLKIEGIG



Hel308
Sso
(621)
LTYSAYHLSRELKLNEHAD-------KLRILNLRVRDGIKEELLELVQISGVGRKRARLLYNNGIKE---------LGDVVMNPDKVKNLLGQK-



Hel308
Tba
(629)
LMYSLIELAKVLNAGGE------TIKYLRRLHLRLKHGVREELLELVELPMIGRRRARALYNAGFKN---------VNDIVKAKPSELLAVEGIG



Hel308
Tga
(608)
LMYSLIELYKLFEPKEE------ILNYLRDLHLRLRHGVREELLELVRLPNIGRKRARALYNAGFRS---------VEAIANAKPAELLAVEGIG



Hel308
Tsi
(631)
LMYSLIELYKLFDPKPE------VLDFLKKLHIRVKHGVREELLELITLPMIGRKRARALYNAGFKG---------IDDIVRAKASELLKVEGIG



Hel308
Mja
(599)
IMHALKEIAKLIGKSSDI---------PEKLEIRLEYGAKEDIIELLSIKYIGRVRARKLYNAGIRS---------IEDIINNPSK---VASIIG











Consensus
(951)
LMHA   LAKLL               L EL IRI YGVKEELLELV IR IGRVRARKLY AGIRS           DL  A    L  ILG


 


 















1046                                                                                       1140



Hel308
Mbu
(717)
-KVAYNILSGIGVRVNDKHFNSAPISSNTLD--------------------TLLDKNQKTFNDFQ------------------------------



Hel308
Afu
(674)
--IAERVVEGISVKSLNPESAAALEHHHHHH----------------------------------------------------------------



Hel308
Csy
(693)
ATLANNIKSQLRKGG--------------------------------------------------------------------------------



Hel308
Dth
(943)
LLFKNKAGKKVRIPVSWQDTAFQV-----------------------------------------------------------------------



Hel308
Fac
(662)
TKLAKDIIENAGKLNNRYYR---------------------------------------------------------------------------



Hel308
Hla
(774)
ERTAERILEHAGREDPSMDDVRPDKSASAAATAGS--------------ASDEDGEGQASLGDFR------------------------------



Hel308
Hpa
(706)
KKTAENILENVGRQDPSLDDVEADAET---------AA---------TSARATNDGGQQSLGDFE------------------------------



Hel308
Htu
(728)
EKTAENILENAGREDPSMDGVEPADGGPAVGAATNGSSGGSETDETGRADAAESDDSQSSLGDF-------------------------------



Hel308
Hvo
(774)
RKTAENILEAAGRKDPSMDAVDEDDAPDDAVPDDA---G--------FETAKERADQQASLGDFEGS----------------------------



Hel308
Mac
(692)
-KIAERIFRQIGRREAVSEISDSERLEKS------------------------SQDGQSTISDF-------------------------------



Hel308
Mba
(691)
-KIADRIFKQIRGRGTSSGIIASEPPEKS------------------------PYSGQKTISDY-------------------------------



Hel308
Mbo
(685)
-GIAEQVLEQLHPSKDTGKKEPPSGDKNTN------------------------PG-QSTLFHFG------------------------------



Hel308
Mev
(691)
-KTAYKVLEQLGVEPEDNQQIDEEPESIKSY--------------------SGNDQGQKTFNDF-------------------------------



Hel308
Mfe
(675)
EKITKKILEDLG--IKFGQ---------------------------------------QKLIF--------------------------------



Hel308
Mfr
(678)
-KTAESII---------------------------------------------------------------------------------------



Hel308
Mhu
(685)
-GIARQVIDEITGVKRSGIHSSDDDYQQKT------------------------PE-LLTDIPGIGKKMAEKLQNAGIITVSDLLTADEVLLSDV



Hel308
Mig
(678)
EKIAKKILGELG--MKFGQ---------------------------------------QTLQI--------------------------------



Hel308
Min
(650)
EKISKKIFEELG--VRYGQ---------------------------------------QRLI---------------------------------



Hel308
Mma
(724)
EKIGKKILGEHG--MKYGQ---------------------------------------QTLLNFN------------------------------



Hel308
 Mmah
(692)
-KIAQKILSYLGRETDSNGYVEPETLENK-------------------------KQ-QKTFQDFI------------------------------



Hel308
 Mmar
(745)
RKTAENVLENAGHRDPSMEGVEPAPDVSVDLNDGADGD---------ASAESTANDDQASLGDF-------------------------------



Hel308
 Mmaz
(692)
-KITERIFKQIGRREAVSEFSDIEPLEKG------------------------SSDGQRTISDY-------------------------------



Hel308
Mok
(752)
EKIARKILSELGVDTKFGQ---------------------------------------MRLSI--------------------------------



Hel308
Mth
(677)
-KIAEGVISQLK-EPGVSA----------------------------------------------------------------------------



Hel308
Mzh
(699)
-KISQKILKQLDIDVDISEIKEKDSDTVP-E--------------------P--ESSQKTISDFT------------------------------



Hel308
Nma
(736)
EKTAENILENAGREEPSMDGVEPIPVEGGSGSGSSNSSGSSEPNADANATEDDADDNQSSLGDF-------------------------------



Hel308
Nth
(699)
TFLENKKIKEIKL----------------------------------------------------------------------------------



Hel308
Pfu
(689)
VKTVEAIFKFLGKNVKISE-----------------------------------KPRKSTLDYFLKS----------------------------



Hel308
Sso
(699)
-LGEKVVQEAARLLNRFH-----------------------------------------------------------------------------



Hel308
Tba
(709)
VKVLERIYRHFGVELPLLKNIKDPDKPEDKPKEKP-------------------KPKKGTLDYFLK-----------------------------



Hel308
Tga
(688)
AKILDGIYRHLGIEKRVTE-----EK----------------------------PKRKGTLEDFLR-----------------------------



Hel308
Tsi
(711)
IGVIEKIYQHFGVELPTNE-----KK--------K-------------------KVKKGTLDEFFK-----------------------------



Hel308
Mja
(673)
EKIAKKILDELGVKFGQQKLSFSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK----KL----------------------------------











Consensus
(1046) 
 KIAEKIL  LG                                               TL  F


 


 















1141                                      1186



Hel308
Mbu
(761)
----------------------------------------------



Hel308
Afu
(703)
----------------------------------------------



Hel308
Csy
(708)
----------------------------------------------



Hel308
Dth
(967)
----------------------------------------------



Hel308
Fac
(682)
----------------------------------------------



Hel308
Hla
(825)
----------------------------------------------



Hel308
Hpa
(753)
----------------------------------------------



Hel308
Htu
(792)
----------------------------------------------



Hel308
Hvo
(830)
----------------------------------------------



Hel308
Mac
(731)
----------------------------------------------



Hel308
Mba
(730)
----------------------------------------------



Hel308
Mbo
(724)
----------------------------------------------



Hel308
Mev
(734)
----------------------------------------------



Hel308
Mfe
(697)
----------------------------------------------



Hel308
Mfr
(685)
----------------------------------------------



Hel308
Mhu
(754)
LGAARARKVLAFLSNSEKENSSSDKTEEIPDTQKIRGQSSWEDFGC



Hel308
Mig
(700)
----------------------------------------------



Hel308
Min
(671)
----------------------------------------------



Hel308
Mma
(748)
----------------------------------------------



Hel308
 Mmah
(730)
----------------------------------------------



Hel308
 Mmar
(800)
----------------------------------------------



Hel308
 Mmaz
(731)
----------------------------------------------



Hel308
Mok
(776)
----------------------------------------------



Hel308
Mth
(694)
----------------------------------------------



Hel308
Mzh
(740)
----------------------------------------------



Hel308
Nma
(800)
----------------------------------------------



Hel308
Nth
(712)
----------------------------------------------



Hel308
Pfu
(721)
----------------------------------------------



Hel308
Sso
(716)
----------------------------------------------



Hel308
Tba
(756)
----------------------------------------------



Hel308
Tga
(721)
----------------------------------------------



Hel308
Tsi
(745)
----------------------------------------------



Hel308
Mja
(730)
----------------------------------------------











Consensus
(1141) 
----------------------------------------------

## Claims

1. A method of characterising a target polynucleotide, comprising:
(a) providing in an aqueous solution a transmembrane pore and a membrane, wherein the transmembrane pore is present in the membrane, and wherein the aqueous solution comprises a salt at a concentration in a range of 0.3 M to 3 M;
(b) combining, in the aqueous solution of step (a), the target polynucleotide and a Hel308 helicase, wherein the helicase binds to the target polynucleotide to form a polynucleotide helicase complex, thereby controlling the movement of the target polynucleotide through the transmembrane pore; and
(c) measuring, during an application of a potential across the transmembrane pore, an ionic current as the target polynucleotide moves through the transmembrane pore, to determine one or more characteristics of the target polynucleotide and thereby characterising the target polynucleotide.

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 the target polynucleotide and (v) whether or not the target polynucleotide is modified.

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. A method according to claim 1, wherein at least a portion of the polynucleotide is double stranded.

5. A method according to claim 1, wherein the transmembrane pore is a protein pore.

6. A method according to claim 5, wherein the transmembrane protein pore is selected from the group consisting of: α-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.

7. A method according to claim 1, wherein the Hel308 helicase comprises the amino acid motif Q-X1-X2 G R A G R (SEQ ID NO: 8), wherein X1 is C, M or L and X2 is any amino acid residue.

8. A method according to claim 7, wherein X2 is A, F, M, C, V, L, I, S, T or P.

9. A method according to claim 1, wherein the Hel308 helicase is capable of binding to the target polynucleotide at an internal nucleotide.

10. A method according to claim 1, wherein the salt is KCl.

11. A method according to claim 10, wherein the salt concentration is at least 1.0 M.