The invention relates to mutant forms of CsgG. The invention also relates to analyte detection and characterisation using CsgG.

Background of the invention

Nanopore sensing is an approach to sensing that relies on the observation of individual binding or interaction events between analyte molecules and a receptor. Nanopore sensors can be created by placing a single pore of nanometer dimensions in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of analyte molecules. The identity of an analyte is revealed through its distinctive current signature, notably the duration and extent of current block and the variance of current levels. Such nanopore sensors are commercially available, such as the MinlON™ device sold by Oxford Nanopore

Technologies Ltd, comprising an array of nanopores integrated with an electronic chip.

There is currently a need for rapid and cheap nucleic acid (e.g. DNA or RNA) sequencing 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 nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection. Nanopore sensing has the potential to provide rapid and cheap nucleic acid sequencing by reducing the quantity of nucleotide and reagents required.

Two of the essential components of sequencing nucleic acids using nanopore sensing are (1) the control of nucleic acid movement through the pore and (2) the discrimination of nucleotides as the nucleic acid polymer is moved through the pore. In the past, to achieve nucleotide discrimination the nucleic acid has been passed through a mutant of hemolysin. This has provided current signatures that have been shown to be sequence dependent. It has also been shown that a large number of nucleotides contribute to the observed current when a hemolysin pore is used, making a direct relationship between observed current and polynucleotide challenging.

While the current range for nucleotide discrimination has been improved through mutation of the hemolysin pore, a sequencing system would have higher performance if the current differences between nucleotides could be improved further. In addition, it has been observed that when the nucleic acids are moved through a pore, some current states show high variance. It has also been shown that some mutant hemolysin pores exhibit higher variance than others. While the variance of these states may contain sequence specific information, it is

SUBSTITUTE SHEET RULE 26

desirable to produce pores that have low variance to simplify the system. It is also desirable to reduce the number of nucleotides that contribute to the observed current.

Summary of the invention

The inventors have surprisingly demonstrated that CsgG and novel mutants thereof may be used to characterise analytes, such as polynucleotides. The invention concerns mutant CsgG monomers. The inventors have surprisingly demonstrated that pores comprising the novel mutant monomers have an enhanced ability to estimate the characteristics of analytes, such as the sequence of polynucleotides. The inventors have made mutant pores that surprisingly provide more consistent movement of a target polynucleotide with respect to, such as through, the pores. The inventors have made mutant pores surprisingly display improved characterisation accuracy. In particular, the mutant pores made by the inventors surprisingly display an increased current range, which makes it easier to discriminate between different nucleotides, and a reduced variance of states, which increases the signal-to-noise ratio. In addition, the mutant pores surprisingly capture nucleotides and polynucleotides more easily.

All amino-acid substitutions, deletions and/or additions disclosed herein are with reference to a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 2, unless stated to the contrary.

Reference to a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 2 encompasses mutant CsgG monomers comprising variants of sequences as set out in the further SEQ ID NOS as disclosed below. Amino-acid substitutions, deletions and/or additions may be made to CsgG monomers comprising a variant of the sequence other than shown in SEQ ID NO:2 that are equivalent to those substitutions, deletions and/or additions disclosed herein with reference to a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO:2.

A mutant monomer may be considered as an isolated monomer.

In particular, the invention concerns mutant CsgG monomers in which the arginine (R) at position 192 has been substituted with aspartic acid (D), glutamine (Q), phenylalanine (F), serine (S) or threonine (T). The inventors have surprisingly demonstrated that such monomers, and in particular a monomer comprising a R192D substitution, are much easier to express than monomers without a substitution at position 192. An increased yield is obtained when the CsgG monomer comprises this mutation.

The mutatnt csgG monomer may be:

a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 2 which comprises R97W;

a mutant CsgG monomer which comprises (a) Rl 92D; (b) R97W/Y and/or R93 W/Y, preferably R97W, R93W or R93Y and R97Y; (c) K94Q/N; (d) G103K/R and/or T104K/R; and/or (e) F191T, deletion of V105, A106 and 1107 and/or deletion of F193, 1194, D195, Y196, Q197, R198 and L199.

The mutant CsgG monomer preferably further comprises Y51A and F56Q.

Particular mutant CsgG monomers comprise variants of the sequence shown in SEQ ID NO: 2 that comprise the following mutations:

(1) Y51A, F56Q and R192D;

(2) Y51 A, F56Q and R97W.

(3) Y51A, F56Q, R192D and R97W;

(4) Y51A, F56Q, R192D and R93W;

(5) Y51A, F56Q, R192D, R93Y and R97Y; or

(6) Y51A, F56Q, R192D and R93W.

(7) the mutations of any one of (l)-(6) and:

(a) deletion of V105, A106 and 1107.

(b) K94Q or K94N;

(c) deletion of D195, Y196, Q197, R198 and L199 or deletion of F193, 1194, D195, Y196, Q197, R198 and L199; and/or

(d) F191T.

(8) the mutations of any one of (l)-(6) and:

(i) K94Q and deletion of V105, A106 and 1107;

(ii) K94N and deletion of V105, A106 and 1107;

(iii) F191T and deletion of V105, A106 and 1107;

(iv) K94Q and F191T;

(v) K94N and F191T;

(vi) K94Q, F191T and deletion of V105, A106 and 1107; or

(vii) K94N, F191T and deletion of V105, A106 and 1107.

(9) the mutations of any one of (l)-(8) and:

- T104K or T104R;

- L90R;

- N91R;

- I95R;

- A99R;

- E101K, E101N, E101Q, E101T or E101H;

- E44N or E44Q; and/or

The invention also provides:

a construct comprising two or more covalently attached CsgG monomers, wherein at least one of the monomers is a mutant monomer of the invention;

- a polynucleotide which encodes a mutant monomer of the invention or a construct of the invention;

a homo-oligomeric pore derived from CsgG comprising identical mutant monomers of the invention or identical constructs of the invention;

a hetero-oligomeric pore derived from CsgG comprising at least one mutant monomer of the invention or at least one construct of the invention;

a method for determining the presence, absence or one or more characteristics of a target analyte, comprising:

(a) contacting the target analyte with a pore of the invention such that the target analyte moves with respect to the pore; and

(b) taking one or more measurements as the analyte moves with respect to the pore and thereby determining the presence, absence or one or more characteristics of the analyte;

a method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore of the invention and a polynucleotide binding protein and thereby forming a sensor for characterising the target polynucleotide;

- a sensor for characterising a target polynucleotide, comprising a complex between a pore of the invention and a polynucleotide binding protein;

use of a pore of the invention to determine the presence, absence or one or more characteristics of a target analyte;

a kit for characterising a target analyte comprising (a) a pore of the invention and (b) the components of a membrane;

an apparatus for characterising target analytes in a sample, comprising (a) a plurality of a pores of the invention and (b) a plurality of membranes;

a method of characterising a target polynucleotide, comprising:

a) contacting the polynucleotide with a pore of the invention, a polymerase and labelled nucleotides such that phosphate labelled species are sequentially added to the target polynucleotide by the polymerase, wherein the phosphate species contain a label specific for each nucleotide; and

b) detecting the phosphate labelled species using the pore and thereby characterising the polynucleotide; and

a method of producing a mutant monomer of the invention or a construct of the invention, comprising expressing a polynucleotide of the invention in a suitable host cell and thereby producing a mutant monomer of the invention or a construct.

Description of the Figures

Figure 1: Illustrates CsgG from E. coli.

Figure 2: Illustrates the dimensions of CsgG.

Figure 3: Illustrates single G translocation at 10 A/ns. There is a large barrier for entry of guanine into F56 ring in CsgG-Eco. * = G enters F56 ring. A = G stops interacting with 56 ring. B = G stops interacting with 55 ring. C= G stops interacting with 51 ring.

Figure 4: Illustrates ssDNA translocation at 100 A/ns. A larger force is required to pull the DNA through the constriction for CsgG-Eco.

Figure 5: Illustrates ss DNA translocation at 10 A/ns. CsgG-F56A-N55S and CsG-F56A-N55S-Y51A mutants both have a lower barrier for ssDNA translocation.

Figures 6 to 8: Mutant pores showing increased range compared with wild-type (WT).

Figures 9 and 10: Mutant pores showing increased throughput compared with wild-type

(WT).

Figures 11 and 12: Mutant pore showing increased insertion compared with wild-type

(WT).

Figure 13: shows the DNA construct X used in Example 2. The region labelled 1 corresponded to 30 SpC3 spacers. The region labelled 2 corresponded to SEQ ID NO: 42. The region labelled 3 corresponded to four iSpl8 spacers. The region labelled 4 corresponded to SEQ ID NO: 43. The section labelled 5 corresponded to four 5-nitroindoles. The region labelled 6 corresponded to SEQ ID NO: 44. The region labelled 7 corresponded to SEQ ID NO: 45. The region labelled 8 corresponded to SEQ ID NO: 46 which had four iSpl8 spacers (the region labelled 9) attached at the 3' end of SEQ ID NO: 46. At the opposite end of the iSpl8 spacers was a 3' cholesterol tether (labelled 10). The region labelled 11 corresponded to four SpC3 spacers.

Figure 14: shows an example chromatography trace of Strep trap (GE Healthcare) purification of CsgG protein (x-axis label = elution volume (mL), Y-axis label = Absorbance (mAu)). The sample was loaded in 25mM Tris, 150mM NaCl, 2mM EDTA, 0.01% DDM and eluted with 10 mM desthiobiotin. The elution peak in which CsgG protein eluted is labelled as El .

Figure 15: shows an example of a typical SDS-PAGE visualisation of CsgG protein after the initial strep purification. A 4-20% TGX Gel (Bio Rad) was run at 300 V for 22 minutes in IX TGS buffer. The gel was stained with Sypro Ruby stain. Lanes 1 - 3 show the main elution peak (labelled El in Figure 14) which contained CsgG protein as indicated by the arrow. Lanes 4 - 6 corresponded to elution fractions of the tail of the main elution peak (labelled El in Figure 14) which contained contaminents. M shows the molecular weight marker used which was a Novex Sharp Unstained (unit = kD).

Figure 16: Shows an example of a size exclusion chromatogram (SEC) of CsgG protein (120 mL S200 GE healthcare, x-axis label = elution volume (mL), y-axis label = absorbance (mAu)). The SEC was carried out after strep purification and heating the protein sample. The running buffer for SEC was 25mM Tris, 150mM NaCl, 2mM EDTA, 0.01% DDM, 0.1% SDS, pH 8.0 and the column was run at 1 mL/minute rate. The trace labelled X shows absorbance at 220nm and the trace labelled Y shows absorbance at 280nm. The peak labelled with a star was collected.

Figure 17: shows an example of a typical SDS-PAGE visualisation of CsgG protein after SEC. A 4-20% TGX Gel (Bio Rad) was run at 300V for 22 minutes in IX TGS buffer and the gel was stained with Sypro Ruby stain. Lane 1 shows CsgG protein sample after strep purification and heating but before SEC. Lanes 2 - 8 show fractions collected across the peak running approximately 48mL - 60 mL of figure 16 (mid peak = 55mL) and labelled with a star in figure 16. M shows the molecular weight marker used which was a Novex Sharp Unstained (unit = kD). The bar corresponding to the CsgG-Eco pore is indicated by an arrow.

Figures 18 to 24: Mutant pores showing increased range compared with wild-type (WT).

Figures 25 to 30: Mutant pores showing increased range compared with wild-type (WT). Figure 31 shows snap shots of the enzyme (T4 Dda -(E94C/C109A/C136A/A360C) (SEQ ID NO: 24 with mutations E94C/C109A/C136A/A360C and then (AM1)G1G2)) on top of the pore (CsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51T/F56Q where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus pore mutant No. 20)) taken at 0 and 20 ns during the simulations (Runs 1 to 3).

Figure 32 shows snap shots of the enzyme (T4 Dda -(E94C/C109A/C136A/A360C) (SEQ ID NO: 24 with mutations E94C/C109A/C136A/A360C and then (AM1)G1G2)) on top of the pore (CsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51T/F56Q where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus pore mutant No. 20)) taken at 30 and 40 ns during the simulations (Runs 1 to 3).

Figure 33 shows a snap shot of the enzyme (T4 Dda -(E94C/F98W/C109A/C136A/K194L/A360C) (SEQ ID NO: 24 with mutations

E94C/F98W/C109A/C136A/K194L/A360C and then (AM1)G1G2) on top of the pore CsgG-Eco-(Y51 A/F56Q/R97W)-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51 A/F56Q/R97W

where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus pore mutant No. 26) taken during the simulations described in Example 5.

Figure 34 shows two ten second screen shots of current traces showing translocation of DNA (SEQ ID NO: 51) through MspA mutant x = MspA - ((Del-L74/G75/D118/L119)D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139K)8 (SEQ ID NO: 50 with mutations D56F/E59R/L88N/D90N/D91N/Q126R/D134R/E139K and deletion of the amino acids L74/G75/D118/L119) without the control of an enzyme.

Figure 35 shows two ten second screen shots of current traces showing translocation of DNA (SEQ ID NO: 51) through CsgG-Eco-(Y51A/F56Q/R97W/R192D-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51 A/F56Q/R97W/R192D where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus) without the control of an enzyme.

Figure 36 shows two ten second screen shots of current traces showing translocation of DNA (SEQ ID NO: 51) through CsgG-Eco-(Y51A/F56Q/R97W/E101 S/R192D-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51A/F56Q/R97W/E101 S/R192D where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus) without the control of an enzyme.

Figure 37 shows an overlay of two gel filtration chromatograms (120ml S200 column) of the CsgG mutants pores A) CsgG-Eco-(Y51A/F56Q/R97W)-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51A/F56Q/R97W where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus) and B) CsgG-Eco-(Y51A/F56Q/R97W/R192D)-StrepII(C))9 (SEQ ID NO: 2 with mutations Y51 A/F56Q/R97W/R192D where StrepII(C) is SEQ ID NO: 47 and is attached at the C-terminus). Absorbance at A280 for CsgG-Eco-(Y51A/F56Q/R97W)-StrepII(C))9 is labelled A and for CsgG-Eco-(Y51A/F56Q/R97W/R192D)-StrepII(C))9 is labelled B. Both constructs were grown in 500ml cultures. Expression and purification of both proteins were carried out using exactly the same protocol and same volumes were loaded onto the column. Running Buffer was 25mM Tris, 150mM NaCl, 2mM EDTA, 0.01% DDM, 0.1% SDS pH8. The fractional delay with CsgG-Eco-(Y51A/F56Q/R97W)-StrepII(C))9 pore was due to different connection configuration used on AKTA Purifier 10. The difference in the absorbance values indicated the amount of proteins expressed with higher absorbance values indicating higher amounts of expressed protein.

Figure 38 shows SDS-PAGE analysis of CsgG nanopores. Lanes A-C contained CsgG- Eco-(Y51A/F56Q/R97W)-StrepII(C))9, lanes D-F contained CsgG-Eco-(Y51A/F56Q/R97W/R192D)-StrepII(C))9 and lane M contained the molecular weight marker. The two pores were expressed and purified using exactly the same protocol. The pores were subjected to electrophoresis on a 4-20% TGX gel (Bio rad cat # 5671093) in TGS buffer at 300 V for 22 minutes. The gel was visualised with Sypro Ruby stain (Life Technologies

cat#S1200). The same volumes from each pore sample were loaded on the gel to compare the amount of proteins obtained after purification - lanes A and D contained 5uL, lanes B and E contained lOuL and lanes C and F contained 15uL.

Figure 39 shows the basecall accuracy of eight CsgG mutant pores compared to the basecall accuracy of a baseline pore, mutant 28 (CsgG-(WT-Y51 A/F56Q/R97W/R192D-StrepII)9). The deletion of D195-L199 (Mutant A), F193-L199 (Mutant B) or V105-I107 (Mutant D), or the substitution of F191T (Mutant C) results in a further improvement in accuracy in addition to the improvement in accuracy resulting from the R97W and R192D substitutions in mutant 28. The effect on basecall accuracy of deleting V105-I107 was also tested in a mutant pore containing an additional K94Q mutation (Mutant E) and an improvement in accuracy compared to baseline mutant 28 was still observed. Introducing a R93W mutation (Muatnt F) or both R93 Y and R97W mutations (Mutant H) instead of a R97W mutation (baseline mutant 28) increased the basecall accuracy. Deleting D195-L199 in addition to R93W (Mutant G) resulted in an enhancement of basecall accuracy.

Figure 40 shows the template speed distribution (A) and the template accuracy distribution (B) of the baseline mutant 28 CsgG-(WT-Y51A/F56Q/R97W/R192D-StrepII)9 and Mutant D which comprises an additional deletion of V105-I107. A template DNA was prepared and passed through the mutant pores as described in the Examples. The template speed and accuracy were determined as described in the Examples. Figure 40A shows that the speed distribution was tighter when Mutant D was used compared to the baseline mutant. Figure 40B shows that mutant D has a tighter distribution of template accuracy compared to the baseline mutant.

Figure 41 displays an example "squiggle" that shows the "noisy" pore error mode exhibited by baseline mutant 28 CsgG-(WT-Y51 A/F56Q/R97W/R192D-StrepII)9. The top panel of Figure 41 shows the difference in flow of current through the pore during the "good" and "noisy" pore states. The bottom panel of Figure 41 shows an expanded view of the transition from "good" state to "noisy" state.

Figure 42 shows the reduction in noisy pore state of mutant pores having the same sequence as the baseline mutant 28, which contains Y51A/F56Q/R97W/R192D mutations, with an additional K94N mutation (Mutant I) or an additional K94Q mutation (Mutant J) when compared to baseline mutant 28, averaged over at least 5 runs.

Figure 43 illustrates the structure of the template strand having an adapter ligated to each end thereof. The adapter has a T4 Dda helicase enzyme prebound thereto. The sequences of the various parts of the adaptor used in the Examples are shown in SEQ ID NOs: 52 to 55.

Figure 44 shows the median time between Thrombin Binding Aptamer (TBA) events for mutant CsgG nanopores comprising one of the following substitutions: Q42K (Mutant K), E44N (Mutant L), E44Q (Mutant M), L90R (Mutant N), N91R (Mutant O), I95R (Mutant P), A99R (Mutant Q), E101H (Mutant R), E101K (Mutant S), E101N (Mutant T), E101Q (Mutant U), E101T (Mutant V) and Ql 14K (Mutant W). The median time was significantly reduced compared to the baseline pore comprising the mutations Y51A/F56Q/K94Q/R97W/R192D-del(Vl 05-1107) (Baseline mutant E), all of which are also included in each of the 13 mutants tested. Figure 44 shows that each of the Q42K, E44N, E44Q, L90R, N91R, I95R, A99R, E101H, E101K, E101N, E101Q, E101T and Q114K substitutions increase template DNA capture rates. Figure 45 shows sequence alignments of the 21 CsgG homologues corresponding to SEQ ID Nos 2, 5, 6, 7, 27, 28, 29, 30, 32, 36, 3, 35, 31, 40, 33, 34, 37, 39, 38, 41 and 4

Figure 46 shows the same relative sequence alignments as Figure 45 with predicted alpha helical secondary structure regions additionally shaded.

Figure 47 shows the same relative sequence alignments as Figure 45 with predicted beta sheet secondary structure regions additionally shaded.

Figure 48 shows two examples of raw electrical data for poreAQ and pore97W.

Description of the Sequence Listing

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encoding the wild-type CsgG monomer from Escherchia coli Str. K-12 substr. MC4100. This monomer lacks the signal sequence.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of the wild-type CsgG monomer from Escherchia coli Str. K-12 substr. MC4100. This monomer lacks the signal sequence. The abbreviation used for this CsgG = CsgG-Eco.

SEQ ID NO: 3 shows the amino acid sequence of YP 001453594.1 : 1-248 of hypothetical protein CKO 02032 [Citrobacter koseri ATCC BAA-895], which is 99% identical to SEQ ID NO: 2.

SEQ ID NO: 4 shows the amino acid sequence of WP 001787128.1 : 16-238 of curli production assembly/transport component CsgG, partial [Salmonella enterica], which is 98% to SEQ ID NO: 2.

SEQ ID NO: 5 shows the amino acid sequence of KEY44978.1 |: 16-277 of curli production assembly/transport protein CsgG [Citrobacter amalonaticus], which is 98% identical to SEQ ID NO: 2.

SEQ ID NO: 6 shows the amino acid sequence of YP 003364699.1 : 16-277 of curli production assembly/transport component [Citrobacter rodentium ICC 168], which is 97% identical to SEQ ID NO: 2.

SEQ ID NO: 7 shows the amino acid sequence of YP 004828099.1 : 16-277 of curli production assembly/transport component CsgG [Enterobacter asburiae LF7a], which is 94% identical to SEQ ID NO: 2.

SEQ ID NO: 8 shows the polynucleotide sequence encoding the Phi29 DNA polymerase.

SEQ ID NO: 9 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 10 shows the codon optimised polynucleotide sequence derived from the sbcB gene from E. coli. It encodes the exonuclease I enzyme (EcoExo I) from E. coli.

SEQ ID NO: 11 shows the amino acid sequence of exonuclease I enzyme (EcoExo I) from . coli.

SEQ ID NO: 12 shows the codon optimised polynucleotide sequence derived from the xthA gene from E. coli. It encodes the exonuclease III enzyme from E. coli.

SEQ ID NO: 13 shows the amino acid sequence of the exonuclease III enzyme from E. coli. This enzyme performs distributive digestion of 5' monophosphate nucleosides from one strand of double stranded DNA (dsDNA) in a 3' - 5' direction. Enzyme initiation on a strand requires a 5' overhang of approximately 4 nucleotides.

SEQ ID NO: 14 shows the codon optimised polynucleotide sequence derived from the rec J gene from T. thermophilics. It encodes the RecJ enzyme from T. thermophilics (Jt/zRecJ-cd).

SEQ ID NO: 15 shows the amino acid sequence of the RecJ enzyme from T.

thermophilus (Jt/zRecJ-cd). This enzyme performs processive digestion of 5' monophosphate nucleosides from ssDNA in a 5' - 3' direction. Enzyme initiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 16 shows the codon optimised polynucleotide sequence derived from the bacteriophage lambda exo (redX) gene. It encodes the bacteriophage lambda exonuclease.

SEQ ID NO: 17 shows the amino acid sequence of the bacteriophage lambda

exonuclease. The sequence is one of three identical subunits that assemble into a trimer. The enzyme performs highly processive digestion of nucleotides from one strand of dsDNA, in a 5'-3 'direction (http://www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on a strand preferentially requires a 5' overhang of approximately 4 nucleotides with a 5' phosphate.

SEQ ID NO: 18 shows the amino acid sequence of Hel308 Mbu.

SEQ ID NO: 19 shows the amino acid sequence of Hel308 Csy.

SEQ ID NO: 20 shows the amino acid sequence of Hel308 Tga.

SEQ ID NO: 21 shows the amino acid sequence of Hel308 Mhu.

SEQ ID NO: 22 shows the amino acid sequence of Tral Eco.

SEQ ID NO: 23 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 24 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 25 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 26 shows the amino acid sequence of WP 006819418.1 : 19-280 of transporter [Yokenella regensburgei], which is 91% identical to SEQ ID NO: 2.

SEQ ID NO: 27 shows the amino acid sequence of WP_024556654.1 : 16-277 of curli production assembly/transport protein CsgG [Cronobacter pulveris], which is 89% identical to SEQ ID NO: 2.

SEQ ID NO: 28 shows the amino acid sequence of YP 005400916.1 : 16-277 of curli production assembly/transport protein CsgG [Rahnella aquatilis HX2], which is 84% identical to SEQ ID NO: 2.

SEQ ID NO: 29 shows the amino acid sequence of KFC99297.1 : 20-278 of CsgG family curli production assembly/transport component [Kluyvera ascorbata ATCC 33433], which is 82% identical to SEQ ID NO: 2.

SEQ ID NO: 30 shows the amino acid sequence of KFC86716.1 |: 16-274 of CsgG family curli production assembly/transport component [Hafnia alvei ATCC 13337], which is 81% identical to SEQ ID NO: 2.

SEQ ID NO: 31 shows the amino acid sequence of YP_007340845.1 |: 16-270 of uncharacterised protein involved in formation of curli polymers [Enter ob acted aceae bacterium strain FGI 57], which is 76% identical to SEQ ID NO: 2.

SEQ ID NO: 32 shows the amino acid sequence of WP 010861740.1 : 17-274 of curli production assembly/transport protein CsgG [Plesiomonas shigelloides], which is 70% identical to SEQ ID NO: 2.

SEQ ID NO: 33 shows the amino acid sequence of YP 205788.1 : 23-270 of curli production assembly/transport outer membrane lipoprotein component CsgG [Vibrio fischeri ESI 14], which is 60% identical to SEQ ID NO: 2.

SEQ ID NO: 34 shows the amino acid sequence of WP_017023479.1 : 23-270 of curli production assembly protein CsgG [Aliivibrio logei], which is 59% identical to SEQ ID NO: 2.

SEQ ID NO: 35 shows the amino acid sequence of WP_007470398.1 : 22-275 of Curli production assembly/transport component CsgG [Photobacterium sp. AK15], which is 57% identical to SEQ ID NO: 2.

SEQ ID NO: 36 shows the amino acid sequence of WP 021231638.1 : 17-277 of curli production assembly protein CsgG [Aeromonas veronii], which is 56% identical to SEQ ID NO: 2.

SEQ ID NO: 37 shows the amino acid sequence of WP_033538267.1 : 27-265 of curli production assembly/transport protein CsgG [Shewanella sp. ECSMB14101], which is 56% identical to SEQ ID NO: 2.

SEQ ID NO: 38 shows the amino acid sequence of WP_003247972.1 : 30-262 of curli production assembly protein CsgG [Pseudomonas putida], which is 54% identical to SEQ ID NO: 2.

SEQ ID NO: 39 shows the amino acid sequence of YP_003557438.1 : 1-234 of curli production assembly/transport component CsgG [Shewanella violacea DSS12], which is 53% identical to SEQ ID NO: 2.

SEQ ID NO: 40 shows the amino acid sequence of WP 027859066.1 : 36-280 of curli production assembly/transport protein CsgG [Marinobacterium jannaschii], which is 53% identical to SEQ ID NO: 2.

SEQ ID NO: 41 shows the amino acid sequence of CEJ70222.1 : 29-262 of Curli production assembly/transport component CsgG [Chryseobactenum oranimense G311], which is 50% identical to SEQ ID NO: 2.

SEQ ID NO: 42 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 43 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 44 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 45 shows a polynucleotide sequence used in Example 2.

SEQ ID NO: 46 shows a polynucleotide sequence used in Example 2. Attached to the 3' end of SEQ ID NO: 46 is six iSpl8 spacers which are attached at the opposite end to two thymines and a 3' cholesterol TEG.

SEQ ID NO: 47 shows the polynucleotide sequence of StrepII(C).

SEQ ID NO: 48 shows the polynucleotide sequence of Pro.

SEQ ID NO: 49 shows the codon optimised polynucleotide sequence encoding the wild-type MspA monomer. This mutant lacks the signal sequence.

SEQ ID NO: 50 shows the amino acid sequence of the mature form of the wild-type

MspA monomer. This mutant lacks the signal sequence.

SEQ ID NO: 51 shows the polynucleotide sequence of Thrombin Binding Aptamer used in Examples 7 and 11.

SEQ ID NO: 52 shows the polynucleotide sequence of a Y-adaptor top strand.

SEQ ID NO: 53 shows the polynucleotide sequence of a Y-adaptor blocker strand.

SEQ ID NO: 54 shows the polynucleotide sequence of a Y-adaptor cholesterol tether strand.

SEQ ID NO: 55 shows the polynucleotide sequence of a Y-adaptor bottom strand.

SEQ ID NO: 56 shows the polynucleotide sequence of a 3.6kb double stranded DNA target sequence used in the Examples.

Detailed description of the invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology 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 polynucleotide" includes two or more polynucleotides, reference to "a polynucleotide binding protein" includes two or more such proteins, reference to "a

helicase" includes two or more helicases, reference to "a monomer" refers to two or more monomers, reference to "a pore" includes two or more pores and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Mutant CsgG monomers

An aspect of the invention provides mutant CsgG monomers. The mutant CsgG monomers may be used to form the pores of the invention. A mutant CsgG monomer is a monomer whose sequence varies from that of a wild-type CsgG monomer and which retains the ability to form a pore. Methods for confirming the ability of mutant monomers to form pores are well-known in the art and are discussed in more detail below.

Pores constructed from the CsGG monomers of some embodiments of the invention comprising the modification R97W display an increased accuracy as compared to otherwise identical pores without the modification at 97 when characterizing (or sequencing) target polynucleotides. An increased accuracy is also seen when instead of R97W the CsgG monomers of the invention comprise the modification R93W or the modifications R93 Y and R97Y.

Accordingly, pores may be constructed from one or more mutant CsgG monomers that comprise a modification at R97 or R93 of SEQ ID NO: 2 such that the modification increases the hydrophobicity of the amino acid. For example, such modification may include an amino acid substitution with any amino acid containing a hydrophobic side chain, including, e.g., but not limited to W and Y.

The CsgG monomers of the invention that comprise R192D/Q F/S/T are easier to express than monomers which do not have a substitution at position 192 which may be due to the reduction of positive charge. Accordingly position 192 may be substituted with an amino-acid which reduces the positive charge. The monomers of the invention that comprise

R192D/Q F/S/T may also comprise additional modifications which improve the ability of mutant pores formed from the monomers to interact with and characterise analytes, such as

polynucleotides.

The CsgG monomers of some embodiments of the invention that comprise a deletion of V105, A106 and 1107, a deletion of F193, 1194, D195, Y196, Q197, R198 and L199 or a deletion of D195, Y196, Q197, R198 and L199, and/or F191T display an increased accuracy when characterizing (or sequencing) target polynucleotides.

Pores comprising the CsgG monomers of some embodiments of the invention that comprise K94Q or K94N show a reduction in the number of noisy pores (namely those pores that give rise to an increased signal :noise ratio) as compared to identical pores without the mutation at 94 when characterizing (or sequencing) target polynucleotides. Position 94 is found within the vestibule of the pore and was found to be a particularly sensitive position in relation to the noise of the current signal. Pores comprising the CsgG monomers of some embodiments of the invention that comprise T104K or T104R, N91R, E101K/N/Q/T/H, E44N/Q, Ql 14K, A99R, I95R, N91R, L90R, E44Q/N and/or Q42K all demonstrate an imporved ability to capture target polynucleotides when used to characterize (or sequence) target polynucleotides as compared to identical pores without substitutions at these positions.

Characterisation, such as sequencing, of a polynucleotide using a transmembrane pore may be carried out such as disclosed in International Application No. PCT/GB2012/052343 (published as WO 2013/041878). As the target polynucleotide moves with respect to, or through the pore, the analyte may be characterised from the distinctive ion current signature produced, typically by measuring the ion current flow through the pore. The level of current measured at any particular time is typically dependent on a group of k polymer (for example nucleotide) units where k is a positive integer and the typical current signature may be represented as a series of current levels indicative of a particular k-mer. The movement of the polynucleotide with respect to, such as through, the pore can be viewed as movement from one k-mer to another or from k-mer to k-mer. Analytical techniques to characterise the polynucleotide may for example involve the use of an HMM, a neural network and for example a Forwards Backwards algorithm or Viterbi algorithm to determine the likelihood of the series of measurements corresponding to a particular sequence. Alternatively the polynucleotide may be characterised by determining a feature vector and comparing the feature vector to another feature vector, which may be known,

such as disclosed in International Application No. PCT/GB2013/050381 (published as WO 2013/121224). However, the analytical techniques used to characterise the polynucleotide are not necessarily restricted to the above examples.

When a monomer of the invention forms a transmembrane pore and is used with a polynucleotide binding protein to characterise a target polynucleotide, some of the modified positions interact with the polynucleotide binding protein. For example, when the monomer forms a transmembrane pore and is used with a polynucleotide binding protein to characterise a target polynucleotide, R97W interacts with the polynucleotide binding protein. Modifying the CsgG monomer in accordance with the invention typically provides more consistent movement of the target polynucleotide with respect to, such as through, a transmembrane pore comprising the monomer. The modification(s) typically provide more consistent movement from one k-mer to another or from k-mer to k-mer as the target polynucleotide moves with respect to, such as through, the pore. The modification(s) typically allow the target polynucleotide to move with respect to, such as through, the transmembrane pore more smoothly. The modification(s) typically provide more regular or less irregular movement of the target polynucleotide with respect to, such as through, the transmembrane pore.

Modifying the CsgG monomer in accordance with the invention (e.g. R97W) typically reduces the amount of slipping forward associated with the movement of the target

polynucleotide with respect to, such as through, a pore comprising the monomer. Some helicases including the Dda helicase used in the Example move along the polynucleotide in a 5' to 3' direction. When the 5'end of the polynucleotide (the end away from which the helicase moves) is captured by the pore, the helicase works with the direction of the field resulting from the applied potential and moves the threaded polynucleotide into the pore and into the trans chamber. Slipping forward involves the DNA moving forwards relative to the the pore (i.e. towards its 3' and away from its 5' end) at least 4 consecutive nucleotides and typically more than 10 consecutive nucleotides. Slipping forward may involve movement forward of 100 consecutive nucleotides or more and this may happen more than once in each strand.

Modifying the CsgG monomer in accordance with the invention (e.g. R97W) typically reduces the noise associated with the movement of the target polynucleotide with respect to, such as through, a transmembrane pore comprising the monomer. Unwanted movement of the target polynucleotide in any dimension as the signal is being analysed typically results in noise in the current signature or level for the k-mer. The modification may reduce this noise by reducing unwanted movement associated with one or more k-mers, such as each k-mer, in the target polynucleotide. The modification may reduce the noise associated with the current level or signature for one or more k-mers, such as each k-mer, in the target polynucleotide.

The enzyme motors employed for moving the polynucleotide have multiple sub-steps in the full catalytic cycle where ATP is hydrolysed to move the polynucleotide forward one base (eg. binding ATP.Mg, hydrolysing to produce ADP.P.Mg, moving the polynucleotide one base forward, and releasing the ADP/P/Mg by-products). Each sub-step process has a characteristic dwell time distribution determined by the kinetics of the process. If any of these sub-steps of the catalytic cycle move the position of the polynucleotide in the reader (e.g. by moving the polynucleotide relative to the enzyme, or by changing the position of the enzyme on the top of the pore) then this may be observed as a change in current through the pore, as long the change lasts sufficiently long to be detected by the acquisition electronics. If the sub-step processes result in no change of conformation or shift in polynucleotide, or occur too quickly to observe, then in an ideal system the full catalytic cycle will result in only one step change in current for the polynucleotide moving one integer base forward.

For pores that do not contain R97W (eg Pro-CPl-Eco-(WT-Y51A/F56Q-StrepII(C))9), we observe long dwell time levels where predicted by the model, with an approximately exponential dwell distribution that is dependent on ATP.Mg concentration. For poreAQ we also short-lived substeps current levels in between the major levels, as marked in Figure 48. Because the sub-step current levels are short-lived, they are most easily observed in the gap between two widely separated current levels. The sub-steps levels correspond to an intermediate

approximately 0.5base movement of the polynucleotide, and under these conditions have an ATP.Mg independent dwell time of approximately 3 milliseconds.

Pores containing R97W (e.g. Pro-CPl-Eco-(WT-Y51A/F56Q/R97W-StrepII(C))9) shows similar longer lived main levels with ATP.Mg dependent dwell times, but shows no signs of distinct intermediate sub-step current levels under these conditions or at this acquisition frequency (possible explanations being that they do not occur, occur too quickly to be observed, or that the substeps do occur and are slow enough in principle to be observed but that in practice they are not observed due to for example the way in which the enzyme interacts with the pore).

The raw data traces (Figure 48) show the ionic current (y-axis, pA) vs. time (x-axis, seconds) trace of an enzyme controlled DNA strand translocation through a nanopore for the pores Pro-CPl-Eco-(WT-Y51A/F56Q/R97W-StrepII(C))9 (Pore 97W) and Pro-CP l-Eco-(WT-Y51 A/F56Q-StrepII(C))9 (Pore AQ) . Each current level is the result of the sequence held in the nanopore reader altering the flow of ions, and step-wise changes in current are observed when the polynucleotide changes position in the nanopore, for example when the enzyme moves the entire strand forward one base. In this case the DNA strand contains in part a repeating sequence (GGTT)n. The data was acquired by loading a Dda enzyme onto synthetic DNA

polynucleotides and running on a MinlON recording raw data output (Cis buffer: 500mM KC1,

25mM HEPES, pH8, 0.6mM MgC12, 0.6mM ATP, 140m V, 37degC, 5kHz acquisition frequency). Pore97W only shows the main current levels from integer step-wise movements of the polynucleotide, with no significant data density between the levels. In comparison, PoreAQ has significant intermediate sub-step levels, as marked by the arrows in Figure 48.

The mutant monomers preferably have improved polynucleotide reading properties i.e. display improved polynucleotide capture and nucleotide discrimination. In particular, pores constructed from the mutant monomers preferably capture nucleotides and polynucleotides more easily than the wild type. In addition, pores constructed from the mutant monomers preferably display an increased current range, which makes it easier to discriminate between different nucleotides, and a reduced variance of states, which increases the signal-to-noise ratio.

In addition, the number of nucleotides contributing to the current as the polynucleotide moves through pores constructed from the mutants is preferably decreased. This makes it easier to identify a direct relationship between the observed current as the polynucleotide moves through the pore and the polynucleotide sequence. In addition, pores constructed from the mutant monomers may display an increased throughput, i.e. are more likely to interact with an analyte, such as a polynucleotide. This makes it easier to characterise analytes using the pores. Pores constructed from the mutant monomers may insert into a membrane more easily. A mutant monomer of the invention comprises a variant of the sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is the wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. The ability of a variant to form a pore can be assayed using any method known in the art. For instance, the variant may be inserted into an amphiphilic layer along with other appropriate subunits and its ability to oligomerise to form a pore may be determined. Methods are known in the art for inserting subunits into membranes, such as amphiphilic layers. For example, subunits may be suspended in a purified form in a solution containing a triblock copolymer membrane such that it diffuses to the membrane and is inserted by binding to the membrane and assembling into a functional state.

In all of the discussion herein, the standard one letter codes for amino acids are used. These are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V). Standard substitution notation is also used, i.e. Q42R means that Q at position 42 is replaced with R.

In one embodiment of the mutant monomers of the invention, the variant of SEQ ID NO: 2 comprises (a) one or more mutations at the following positions (i.e. mutations at one or more of the following positions) 141, R93, A98, Q100, G103, T104, A106, 1107, N108, LI 13, SI 15, Tl 17, Y130, K135, E170, S208, D233, D238 and E244 and /or (b) one or more of D43S, E44S, F48S/N/Q/Y/W/I/V/H/R/K, Q87N/R/K, N91K/R, K94R/F/Y/W/L/S/N, R97F/Y/W/V/I/K/S/Q/H, E101I/L/A/H, N102K/Q/L/I/V/S/H, R110F/G/N, Q114R/K, R142Q/S, T150Y/A/V/L/S/Q/N, R192D/Q/F/S/T and D248S/N/Q/K/R. The variant may comprise (a); (b); or (a) and (b).

In some embodiments of the invention, the variant of SEQ ID NO: 2 comprises R97W.

In some embodiments of the invention, the variant of SEQ ID NO: 2 comprises

R192D/Q/F/S/T, preferably R192D/Q. more preferably R192D. In (a), the variant may comprise modifications at any number and combination of the positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the positions. In (a), the variant preferably comprises one or more of 14 IN, R93F/Y/W/L/I/V/N/Q/S, A98K/R, Q100K/R, G103F/W/S/N/K/R, T104R/K, A106R/K, I107R/K/W/F/Y/L/V, N108R/K, L113K/R, S115R/K, T117R/K, Y130W/F/H/Q/N, K135L/V/N/Q/S, E170S/N/Q/K/R, S208V/I/F/W/Y/L/T, D233S/N/Q/K/R, D238S/N/Q/K/R and E244S/N/Q/K/R.

In (a), the variant preferably comprises one or more modifications which provide more consistent movement of a target polynucleotide with respect to, such as through, a

transmembrane pore comprising the monomer. In particular, in (a), the variant preferably comprises one or more mutations at the following positions (i.e. mutations at one or more of the following positions) R93, G103 and 1107. The variant may comprise R93; G103; 1107; R93 aand G103; R93 and 1107; G103 and 1107; or R93, G103 and 1107. The variant preferably comprises one or more of R93F/Y/W/L/I/V/N/Q/S, G103F/W/S/N/K/R and

I107R/K/W/F/Y/L/V. These may be present in any combination shown for the positions R93, G103 and 1107.

In (a), the variant preferably comprises one or modifications which allow pores constructed from the mutant monomers preferably capture nucleotides and polynucleotides more easily. In particular, in (a), the variant preferably comprises one or more mutations at the following positions (i.e. mutations at one or more of the following positions) 141, T104, A106, N108, LI 13, SI 15, T117, E170, D233, D238 and E244. The variant may comprise

modifications at any number and combination of the positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of the positions. The variant preferably comprises one or more of I41N, T104R/K, A106R/K, N108R/K, L113K/R, S115R/K, T117R/K, E170S/N/Q/K/R, D233S/N/Q/K/R, D238S/N/Q/K/R and E244S/N/Q/K/R. Additionally or alternatively the variant may comprise (c) Q42K/R, E44N/Q, L90R/K, N91R/K, I95R/K, A99R/K, E101H/K/N/Q/T and/or Ql 14K/R.

In (a), the variant preferably comprises one or more modifications which provide more consistent movement and increase capture. In particular, in (a), the variant preferably comprises one or more mutations at the following positions (i.e. mutations at one or more of the following positions) (i) A98, (ii) Q100, (iii) G103 and (iv) 1107. The variant preferably comprises one or more of (i) A98R/K, (ii) Q100K/R, (iii) G103K/R and (iv) I107R/K. The variant may comprise {i}; {ii}; {iii}; {iv}; {i,ii}; {i,iii}; {i,iv}; {ii,iii}; {H,iv}; {iii,iv}; {i,ii,iii}; {i,H,iv}; {i,iii,iv}; {ii,iii,iv}; or {i,ii,iii,iv} .

Particularly preferred mutant monomers which provide for increased capture of analytes, such as a polynucleotides include a mutation at one or more of positions Q42, E44, E44, L90, N91, 195, A99, E101 and Ql 14, which mutation removes the negative charge and/or increases the positive charge at the mutated positions. In particular, the following mutations may be included in a mutant monomer of the invention to produce a CsgG pore that has an improved ability to capture an analyte, preferably a polynucleotide: Q42K, E44N, E44Q, L90R, N91R, I95R, A99R, E101H, E101K, E101N, E101Q, E101T and Q114K. Examples of particular mutant monomers which comprise one of these mutations in combination with other beneficial mutations are described in Example 11.

In (a), the variant preferably comprises one or more modifications which provide increased characterisation accuracy. In particular, in (a), the variant preferably comprises one or more mutations at the following positions (i.e. mutations at one or more of the following positions) Y130, K135 and S208, such as Y130; K135; S208; Y130 and K135; Y130 and S208; K135 and S208; or Y130, K135 and S208. The variant preferably comprises one or more of Y130W/F/H/Q/N, K135L/V/N/Q/S and R142Q/S. These substitutions may be present in any number and combination as set out for Y130, K135 and S208.

In (b), the variant may comprise any number and combination of the substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the substitutions. In (b), the variant preferably comprises one or more modifications which provide more consistent movement of a target polynucleotide with respect to, such as through, a transmembrane pore comprising the monomer. In particular, in (b), the variant preferably comprises one or more one or more of (i) Q87N/R/K, (ii) K94R/F/Y/W/L/S/N, (iii) R97F/Y/W/V/I/K/S/Q/H, (iv) N102K/Q/L/I/V/S/H and (v) Rl 10F/G/N. More preferably, the variant comprises K94D or K94Q and/or R97W or R97Y. The variant may comprise {i}; {ii}; {iii}; {iv}; {v}; {i,ii}; {i,iii}; {i,iv}; {i,v}; {ii,iii}; {H,iv}; {ii,v}; {iii,iv}; {iii,v}; {iv,v}; {i,ii,iii}; {i,H,iv}; {i,ii,v}; {i,iii,iv}; {i,iii,v}; {i,iv,v}; {ii,iii,iv}; {ii,iii,v}; {ϋ,ίν,ν}; {iii,iv,v}; {i,ii,iii,iv}; {i,ii,iii,v}; {i,ii,iv,v}; {i,iii,iv,v}; {ii,iii,iv,v}; or {i,ii,iii,iv,v}. Other preferred variants that are modified to provide more consistent movement of a target polynucleotide with respect to, such as through, a transmembrane pore comprising the monomer include (vi) R93W and R93 Y. A preferred variant may comprise R93W and R97W, R93Y and R97W, R93W and R97W, or more preferably R93Y and R97Y. The variant may

comprise {vi}; {I, vi}; {Ii, vi}; {Iii, vi}; {Iv, vi}; {V, w}; {I, ii, vi}; {I, iii, vi}; {I, iv, vi}; {T, v, w};

{Ii, iii, vi}; {Ii, iv, vi}; {P, v, w}; {Iii, iv, vi}; {Iii, v, vi}; {Iv, v, w}, {i, ii, iii, vi}; {I, ii, iv, vi}; {T, u, v, w}, {i, iii, iv, vi}; {I, iii, v, vi}; {I, iv, v, vi}; {Ii, iii, iv, vi}; {, Iii, v, vi}; {Ii, iv, v, vi}; {iii, iv, v, vi},

{I, ii, iii, iv, vi}, {i, ii, iii, v, vi}; {I, ii, iv, v, vi}; {I, iii, iv, v, vi}; {Ii, iii, iv, v, vi}; or {i, ii, iii, iv, v, vi}.

In (b), the variant preferably comprises one or modifications which allow pores constructed from the mutant monomers preferably capture nucleotides and polynucleotides more easily. In particular, in (b), the variant preferably comprises one or more of (i) D43S, (ii) E44S, (iii) N91K/R, (iv) Ql 14R/K and (v) D248S/N/Q/K/R. The variant may comprise {i}; {ii}; {iii}; {iv}; {v}; {i,ii}; {i,iii}; {i,iv}; {i,v}; {ii,iii}; {H,iv}; {ii,v}; {iii,iv}; {iii,v}; {iv,v}; {i,ii,iii}; {i,ii,iv}; {i,ii,v}; {i,iii,iv}; {i,iii,v}; {i,iv,v}; {ϋ,ίϋ,ίν}; {ϋ,ίϋ,ν}; {ϋ,ίν,ν}; {iii,iv,v}; {i,ii,iii,iv}; {i,ii,iii,v}; {i,ii,iv,v}; {i,iii,iv,v}; {ii,iii,iv,v}; or {i,ii,iii,iv,v} .

In (b), the variant preferably comprises one or more modifications which provide more consistent movement and increase capture. In particular, in (b), the variant preferably comprises one or more of Q87R/K, E101I/L/A/H and N102K, such as Q87R/K; E101I/L/A/H; N102K; Q87R/K and ElOlI/L/A/H; Q87R/K and N102K; E101I/L/A/H and N102K; or Q87R/K, E101I/L/A/H and N102K.

In (b), the variant preferably comprises one or more modifications which provide increased characterisation accuracy. In particular, in (a), the variant preferably comprises F48S/N/Q/Y/W/I/V.

In (b), the variant preferably comprises one or more modifications which provide increased characterisation accuracy and increased capture. In particular, in (a), the variant preferably comprises F48H/R/K.

The variant may comprise modifications in both (a) and (b) which provide more consistent movement. The variant may comprise modifications in both (a) and (b) which provide increased capture.

The invention provides variants of SEQ ID NO: 2 which provide an increased throughput of an assay for characterising an analyte, such as a polynucleotide, using a pore comprising the variant. Such variants may comprise a mutation at K94, preferably K94Q or K94N, more preferably K94Q. Examples of particular mutant monomers which comprise a K94Q or K94N mutation in combination with other beneficial mutations are described in Examples 10 and 11.

The invention provides variants of SEQ ID NO: 2 which provide increased

characterisation accuracy in an assay for characterising an analyte, such as a polynucleotide, using a pore comprising the variant. Such variants include varaints that comprise: a mutation at F191, preferably F191T; deletion of V105-I107; deletion of F193-L199 or of D195-L199; and/or a mutation at R93 and/or R97, preferably R93Y, R97Y, or more preferably, R97W, R93W or

both R97Y and R97Y. Examples of particular mutant monomers which comprise one or more of these mutations in combination with other beneficial mutations are described in Example 9.

In another embodiment of the mutant monomers of the invention, the variant of SEQ ID NO: 2 comprises (A) deletion of one or more positions R192, F193, 1194, D195, Y196, Q197, Rl 98, L 199, L200 and E201 and /or (B) deletion of one or more of

V139/G140/D149/T150/V186/Q187/V204/G205 (called band 1 herein),

G137/G138/Q151/Y152/Y184/E185/Y206/T207 (called band 2 herein) and

A141/R142/G147/A148/A188/G189/G202/E203 (called band 3 herein).

In (A), the variant may comprise deletion of any number and combination of the positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the positions. In (A), the variant preferably comprises deletion of

- D195, Y196, Q197, R198 and L199;

- R192, F193, 1194, D195, Y196, Q197, R198, L199 and L200;

- Q197, R198, LI 99 and L200;

- 1194, D195, Y196, Q197, R198 and L199;

- D195, Y196, Q197, R198, L199 and L200;

- Y196, Q197, R198, LI 99, L200 and E201;

- Q197, R198, LI 99, L200 and E201;

- Q197, R198, L199; or

- F193, 1194, D195, Y196, Q197, R198 and L199.

More preferably, the variant comprises deletion of D195, Y196, Q197, R198 and L199 or F193, 1194, D195, Y196, Q197, R198 and L199. In (B), any number and combination of bands 1 to 3 may be deleted, such as band 1; band 2; band 3; bands 1 and 2; bands 1 and 3; bands 2 and 3; or bands 1, 2 and 3.

The variant may comprise deletions according to (A); (B); or (A) and (B).

The variants comprising deletion of one or more positions according to (A) and/or (B) above may further comprise any of the modifications or substitutions discussed above and below. If the modifications or substitutions are made at one or more positions which appear after the deletion positions in SEQ ID NO: 2, the numbering of the one or more positions of the modifications or subtitutions must be adjusted accordingly. For instance, if L199 is deleted, E244 becomes E243. Similarly, if band 1 is deleted, R192 becomes R186.

In another embodiment of the mutant monomers of the invention, the variant of SEQ ID NO: 2 comprises (C) deletion of one or more positions V105, A106 and 1107. The deletions in accordance with (C) may be made in addition to deletions according to (A) and/or (B).

The above-described deletions typically reduce the noise associated with the movement of the target polynucleotide with respect to, such as through, a transmembrane pore comprising the monomer. As a result the target polynucleotide can be characterised more accurately.

In the paragraphs above where different amino acids at a specific positon are separated by the / symbol, the / symbol means "or". For instance, Q87R/K means Q87R or Q87K.

The invention provides variants of SEQ ID NO: 2 which provide increased capture of an an analyte, such as a polynucleotide. Such variants may comprise a mutation at T104, preferably T104R or T104K, a mutation at N91, preferably N91R, a mutation at E101, preferably

E101K/N/Q/T/H, a mutation at position E44, preferably E44N or E44Q and/or a mutation at position Q42, preferably Q42K.

The mutations at different positions in SEQ ID NO: 2 may be combined in any possible way. In particular, a monomer of the invention may comprise one or more mutation that improves accuracy, one ore more mutation that reduces noise and/ore one or more mutation that enhances capture of an analyte.

In the mutant monomers of the invention, the variant of SEQ ID NO: 2 preferably comprises one or more of the following (i) one or more mutations at the following positions (i.e. mutations at one or more of the following positions) N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192, such as one or more mutations at the following positions (i.e. mutations at one or more of the following positions) N40, D43, E44, S54, S57, Q62, E101, E131 and T150 or N40, D43, E44, E101 and E131; (ii) mutations at Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56; (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi) deletion of one or more of the following positions F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57. The variant may comprise any combination of (i) to (xi). In particular, the variant may comprise: (where each variant in parentheses { } separated by a space represents an optional variant from the list of variants, namely {i} or {ii} or {iii} or {iv} or {v} and so on) {i} {ii} {iii} {iv} {v} {vi} {vii} {viii} {ix} {x} {xi} {i,ii} {i,iii} {i,iv} {i,v} {i,vi} {i,vii} {i,viii} {i,ix} {i,x} {i,xi} {ii,iii} {ii,iv} {ii,v} {ii,vi} {ii,vii} {ii,viii} {ii,ix} {ii,x} {ii,xi} {iii,iv} {iii,v} {iii,vi} {iii,vii} {iii,viii} {iii,ix} {iii,x} {iii,xi} {iv,v} {iv,vi} {iv,vii} {iv,viii} {iv,ix} {iv,x} {iv,xi} {v,vi} {v,vii} {v,viii} {v,ix} {v,x} {v,xi} {vi,vii} {vi,viii} {vi,ix} {vi,x} {vi,xi} {νϋ,νίϋ} {vii,ix} {vii,x} {vii,xi} {viii,ix} {viii,x} {viii,xi} {ix,x} {ix,xi} {x,xi} {i,ii,iii} {i,ii,iv} {i,ii,v} {i,ii,vi} {i,ii,vii} {i,ii,viii} {i,ii,ix} {i,H,x} {i,ii,xi} {i,iii,iv} {i,iii,v} {i,iii,vi} {i,iii,vii} {i,iii,viii} {i,iii,ix} {i,iii,x} {i,iii,xi} {i,iv,v} {i,iv,vi} {i,iv,vii} {i,iv,viii} {i,iv,ix} {i,iv,x} {i,iv,xi} {i,v,vi} {i,v,vii} {i,v,viii} {i,v,ix} {i,v,x} {i,v,xi} {i,vi,vii} {i,vi,viii} {i,vi,ix} {i,vi,x} {i,vi,xi}

i, vii, viii} {i, vii, ix} {i, vii, x} {i, vii, xi} {i, viii, ix} {i, viii, x} {i, viii, xi} {t , ix, x} {i, ix, xi} {t, x, xi} ii, iii, iv} {ii, iii, v} {ii, iii, vi} {ii, iii, vii} {ii, iii , viii} {ii, iii, ix} {ii, iii, x} {ii, iii, xi} {ii, iv, v} {ii, iv, vi} ii, iv, vii} {ii, iv, viii } {ii, iii, ix} {ii, iv, x} {ii, iv, xi} {u, v, w} {u, v, vii} {u, v, viii} {u, v, ix} {u, v, x} u, v, xi} {ii, vi, vii} {ii, vi, viii} {ii, vi, ix} {D, w, x} {ii, vi, xi} {D , vii, viii} {ii, vii, ix} {ii, vii, x} ii, vii, xi} {ii, viii, ix} {ii, viii, x} {ii, viii, xi} {D, ix , x} {D, ix, xi} {D, x, xi} {iii, iv, v} {iii, iv, vi} iii, iv, vii} {iii, iv, viii} {iii, iv, ix } {iii, iv, x} {iii, iv, xi} {iii, v, vi} {iii, v, vii} {iii, v, viii} {iii, v, ix} iii, v, x} { iii, v, xi} {iii, vi, vii} {iii, vi, viii} {iii, vi, ix} {iii, vi, x} {iii, vi, xi} {iii, vii, viii} {iii , vii, ix} iii, vii, x} {iii, vii, xi} {iii, viii, ix} {iii, viii, x} {i, ii, iii, iv} {i, ii, iii, v} {i, ii, iii, vi} {i, ii, iii, vii} i, ii, iii, viii} {i, ii, iii, ix} {i, ii, iii, x} {i, ii, iii, xi} {t, J , iv, v} {i, ii, iv, vi} {i, ii, iv, vii} {i, ii, iv, viii} i, ii, iii, ix} {i, ii, iv, x} { i, ii, iv, xi} {t, u, v, w} {t, u, v, vii} {t, u, v, viii} {t, u, v, ix} {t, u, v , x} {t, u, v, xi} i, ii, vi, vii} {i, ii, vi, viii} {i, ii, vi, ix} {i, ii, vi, x} {t, ii, vi, xi} {i, ii, vii, viii} {i, ii, vii, ix} {i, ii, vii, x} i, ii, vii, xi} {i, ii, viii, ix} {i, ii, viii, x} {i, ii, viii, xi} {i, ii, ix, x} {i, ii, ix, xi} {t, b, x, xi} {i, iii, iv, v} i, iii, iv, vi} {i, iii, iv, vii} {i, iii, iv, viii} {i, iii, iv, ix} {i, iii, iv, x} {t , iii, iv, xi} {i, iii, v, vi} {i, iii, v, vi} i, iii, v, viii} {i, iii, v, ix} {i, iii, v, x {} i, iii, v, xi} {i, iii, vi, vii} {i, iii, vi, viii} {i, iii, vi, ix} {t, iii, vi, x} i, iii, vi, xi} {i, iii, vii, viii} {i, iii, vii, ix} {i, iii, vii, x} {i, iii, vii, xi} {i, iii, viii, ix} {i, iii, viii, x} i, iii, viii, xi} {i, iii, ix, x} {i, iii, ix, xi} {i, iii, x , xi} {i, iv, v, vi} {i, iv, v, vii} {i, iv, v, viii} {i, iv, v, ix} i, iv, v, x} {t, iv, v, xi} {i, iv, vi, vii} {i, iv, vi, viii} {i, iv, vi, ix} {i, iv, vi, x} {i, iv, vi, xi } {i, iv, vii, viii} i, iv, vii, ix} {i, iv, vii, x} {i, iv, vii, xi} {i, iv, viii, ix} {i, iv, viii, x} {i, iv, viii, xi} {i, iii, ix, x} {i, iii, ix, xi} i, iv, x, xi} {t, v, vi, vii} {t , v, vi, viii} {t, v, vi, ix} {t, v, w, x} {t, v, vi, xi} {t, v, vii, viii} {t, v, vii, ix} i, v, vi, x} {t, v, vii, xi} {t, v, viii, ix} {t, v, viii, x} {t, v, viii, xi} {i, v , ix, x} {t, v, ix, xi} {t, v, x, xi} i, vi, vii, viii} {t, vi, vii, ix} {t, vi, vii, x} { i, vi, vii, xi} {t, vi, viii , Ix} {t, vi, viii, x} {t, vi, viii, xi} {t, vi, ix, x} i, vi, ix, xi} {i, vi, x, xi} {t, vii, viii, ix} {i, vii, viii, x} {i, vii, viii, xi} {i, vii, ix, x} {i, vii, ix, xi} {i, vi, x, xi } i, viii, ix, x} {i, viii, ix, xi} {i, viii, x, xi} {t, ix, x, xi} {ii, iii, iv, v} {ii, iii, iv, vi} {ii, iii, iv, vii}

ii, iii, iv, viii} {ii, iii, iv, ix} {ii, iii, iv, x} {ii, iii, iv, xi} {ii, iii, v, vi} {ii, iii, v , vii} {ii, iii, v, viii} ii, iii, v, ix} {ii, iii, v, x} {ii, iii, v, xi} {ii, iii, vi, vii} {D, iii, vi, viii} {ii, iii, vi, ix} {ii, iii, vi, x} {ii, iii, vi, xi} ii, iii, vii, viii} {ii, iii, vii, ix} {ii, iii, vii, x} {ii, iii, vii, xi} {ii, iii, viii, ix} {ii, iii, viii, x} {ii, iii, viii, xi} ii, iii, ix , x} {ii, iii, ix, xi} {ii, iii, x, xi} {ii, iv, v, vi} {ii, iv, v, vii} {ii, iv, v, viii} {D , iv, v, ix} {ii, iv, v, x} ii, iv, v, xi} {ii, iv, vi, vii} {ii, iv, vi, viii} {ii, iv, vi, ix } {ii, iv, vi, x} {ii, iv, vi, xi} {ii, iv, vii, viii} ii, iv, vii, ix} {ii, iv, vi, x} {ii, iv, vii, xi} {ii, iv, viii, ix} {ii, iv, viii, x} {ii, iv, viii, xi} {ii, iii, ix, x} ii, iii, ix, xi} {D , iv, x, xi} {u, v, vi, vii} {u, v, vi, viii} {u, v, vi, ix} {u, v, w, x} {u, v, vi, xi} {u, v, vii, viii} , V, vii, ix} {u, v, vii, x} {u, v, vii, xi} {u, v, viii, ix} {u, v, viii, x} {u, v, viii, xi} {u, v, ix, x} {u, v, ix, xi} u, v, x, xi} {ii, vi, vii, viii} {ii, vi, vii, ix} {D, vi , vii, x} {ii, vi, vii, xi} {ii, vi, viii, ix} {ii, vi, viii, x} ii, vi, viii, xi} {ii, vi, ix, x} { ii, vi, ix, xi} {D, w, x, xi} {ii, vii, viii, ix} {ii, vii, viii, x} {ii, vii, viii, xi} ii, vii, ix, x} {ii, vii, ix, xi} {ii, vii, x, xi} {ii, viii, ix, x} {ii, viii, ix, xi} {ii, viii, x, xi} {D, ix, x, xi} iii, iv, v, vi} {iii, iv, v, vii} {iii, iv, v, viii} {iii, iv, v, ix} {iii, iv, v, x} {iii, iv, v, xi} {iii, iv, vi, vii}

{Iii, iv, vi, viii} {iii, iv, vi, ix} {iii, iv, vi, x} {iii, iv, vi, xi} {iii, iv, vii, viii} {iii, iv, vii, ix} {iii, iv, vii, x} {iii, iv, vii, xi} {iii, iv, viii, ix} {iii, iv, viii, x} {iii, iv, viii, xi} { iii, iv, ix, x} {iii, iv, ix, xi} {iii, iv, x, xi} {iii, v, vi, vii} {iii, v, vi, viii} {iii, v, vi , ix} {iii, v, vi, x} {iii, v, vi, xi} {iii, v, vii, viii} {iii, v, vii, ix} {iii, v, vii, x} {iii , v, vii, xi} {iii, v, viii, ix} {iii, v, viii, x} {iii, v, viii, xi} {iii, v, ix, x} {iii, v, ix, xi}

{Iii, v, x, xi} {iii, vi, vii, viii} {iii, vi, vii, ix} {iii, vi, vii, x} {iii, vi, vii, xi} {iii, vi, viii, ix} {iii, vi, viii, x} {iii, vi, viii, xi} {iii, vi, ix, x} {iii, vi, ix, xi} {iii, vi, x, xi} { iii, vii, viii, ix} {iii, vii, viii, x} {iii, vii, viii, xi} {iii, vii, ix, x} {iii, vii, ix, xi} {iii, vii, x , xi} {iii, viii, ix, x} {iii, viii, ix, xi} {iii, viii, x, xi} {iii, ix, x, xi} {iv, v, vi, vii} {iv , v, vi, viii} {iv, v, vi, ix} {iv, v, vi, x} {iv, v, vi, xi} {iv, v, vii, viii} {iv, v, vii, ix}

{Iv, v, vii, x} {iv, v, vii, xi} {iv, v, viii, ix} {iv, v, viii, x} {iv, v, viii, xi} {iv, v, ix, x} {iv, v, ix, xi}

{Iv, v, x, xi} {iv, vi, vii, viii} {iv, vi, vii, ix} {iv, vi, vii, x} {iv, vi, vii, xi} {iv, vi, viii, ix} {iv, vi, viii, x} {iv, vi, viii, xi} {iv, vi, ix, x} {iv, vi, ix, xi} {iv, vi, x, xi} { iv, vii, viii, ix} {iv, vii, viii, x} {iv, vii, viii, xi} {iv, vii, ix, x} {iv, vii, ix, xi} {iv, vii, x , xi} {iv, viii, ix, x} {iv, viii, ix, xi} {iv, viii, x, xi} {iii, ix, x, xi} {v, vi, vii, viii} {v , vi, vii, ix} {v, vi, vii, x} {v, vi, vii, xi} {v, vi, viii, ix} {v, vi, viii, x} {v, vi, viii, xi} {v, vi, ix, x} {v, vi, ix, xi} {v, w, x, xi} {v, vii, viii, ix} {v, vii, viii, x} {w, vii, viii, xi} {v, vii, ix, x}

{V, vii, ix, xi} {v, vi, x, xi} {v, viii, ix, x} {v, viii, ix, xi} {v, viii, x, xi} {v, ix, x, xi} {vi, vii, viii, ix} {vi, vii, viii, x} {vi, vii, viii, xi} {vi, vii, ix, x} {vi, vii, ix, xi} { vi, vii, x, xi} {vi, viii, ix, x} {vi, viii, ix, xi} {vi, viii, x, xi} {vi, ix, x, xi} {vii, viii, ix , x} {vii, viii, ix, xi} {vii, viii, x, xi} {vii, ix, x, xi} {viii, ix, x, xi} {i, ii, iii, iv, v} {i, ii, iii, iv, vi} {i, ii, iii, iv, vii} {i, ii, iii, iv, viii} {i, ii, iii, iv, ix} {i, ii, iii , iv, x} {i, ii, iii, iv, xi} {i, ii, iii, v, vi} {i, ii, iii, v, vii} {i, ii, iii, v, viii} { i, ii, iii, v, ix} {i, ii, iii, v, x} {i, ii, iii, v, xi} {i, ii, iii, vi, vii} {i, ii, iii, vi, viii} {i, ii, iii, vi, ix} {i, ii, iii, vi, x} {i, ii, iii, vi, xi} {i, ii, iii, vii, viii} {i , ii, iii, vii, ix}

{Ί, ϋ, ίϋ, νϋ, χ} {i, ii, iii, vii, xi} {i, ii, iii, viii, ix} {i, ii, iii, viii, x} {i, ii, iii , viii, xi} {i, ii, iii, ix, x}

{I, ii, iii, ix, xi} {i, ii, iii, x, xi} {i, ii, iv, v, vi} {i, ii, iv, v, vii} {i, ii, iv , v, viii} {i, ii, iv, v, ix} {i, ii, iv, v, x} {i, ii, iv, v, xi} {i, ii, iv, vi, vii} { i, ii, iv, vi, viii} {i, ii, iv, vi, ix} {i, ii, iv, vi, x} {i, ii, iv, vi, xi} {i, ii, iv, vii, viii} {i, ii, iv, vii, ix} {i, ii, iv, vii, x} {i, ii, iv, vii, xi} {i, ii, iv, viii, ix} {i , ii, iv, viii, x} {i, ii, iv, viii, xi}

{I, ii, iv, ix, x} {i, ii, iv, ix, xi} {i, ii, iv, x, xi} {t, u, v, vi, vii} {i, ii, v , vi, viii} {t, u, v, vi, ix} {t, u, v, w, x} {t, u, v, vi, xi} {t, u, v, vii, viii} { t, u, v, vii, ix} {t, u, v, vii, x} {t, u, v, vii, xi} {t, u, v, viii, ix} {t, u, v, viii, x} {t, u, v, viii, xi} {t, u, v, ix, x} {t, u, v, ix, xi} {t, u, v, x, xi} {t , ii, vi, vii, viii} {i, ii, vi, vii, ix} {i, ii, vi, vii, x} {i, ii, vi, vii, xi} {i, ii, vi, viii , ix} {i, ii, vi, viii, x} {i, ii, vi, viii, xi} {i, ii, vi, ix, x} {i, ii, vi, ix, xi} {t, D, w, x, xi} {i, ii, vii, viii, ix} {i, ii, vii, viii, x} {i, ii, vii, viii, xi} {i, ii, vii, ix, x} {i, ii, vii, ix, xi} {i, ii, vii, x, xi}

{I, ii, viii, ix, x} {i, ii, viii, ix, xi} {i, ii, viii, x, xi} {i, ii, ix, x, xi} {i, iii, iv , v, w} {i, iii, iv, v, vii}

{I, iii, iv, v, viii} {i, iii, iv, v, ix} {i, iii, iv, v, x} {i, iii, iv, v, xi} {i, iii, iv , vi, vii} {i, iii, iv, vi, viii}

{I, iii, iv, vi, ix} {i, iii, iv, vi, x} {i, iii, iv, vi, xi} {i, iii, iv, vii, viii} {i, iii, iv , vii, ix} {i, iii, iv, vii, x}

{I, iii, iv, vii, xi} {i, iii, iv, viii, ix} {i, iii, iv, viii, x} {i, iii, iv, viii, xi} {i, iii, iv , ix, x} {i, iii, iv, ix, xi}

{I, iii, iv, x, xi} {i, iii, v, vi, vii} {i, iii, v, vi, viii} {i, iii, v, vi, ix} {i, iii, v , vi, x} {i, iii, v, vi, xi} {i, iii, v, vii, viii} {i, iii, v, vii, ix} {i, iii, v, vii, x} { i, iii, v, vii, xi} {i, iii, v, viii, ix} {i, iii, v, viii, x} {i, iii, v, viii, xi}

{Ί, ίϋ, ν, ίχ, χ} {i, iii, v, ix, xi} {ί, ίϋ, ν, χ, χί} {i, iii, vi, vii, viii} {i, iii, vi , vii, ix} {i, iii, vi, vii, x} {i, iii, vi, vii, xi} {i, iii, vi, viii, ix} {i, iii, vi, viii, x} { i, iii, vi, viii, xi} {i, iii, vi, ix, x} {i, iii, vi, ix, xi}

{I, iii, vi, x, xi} {i, iii, vii, viii, ix} {i, iii, vii, viii, x} {i, iii, vii, viii, xi} {i, iii, vii , ix, x} {i, iii, vii, ix, xi} {i, iii, vii, x, xi} {i, iii, viii, ix, x} {i, iii, viii, ix, xi} { i, iii, viii, x, xi} {i, iii, ix, x, xi} {i, iv, v, vi, vii}

{I, iv, v, vi, viii} {i, iv, v, vi, ix} {i, iv, v, vi, x} {i, iv, v, vi, xi} {i, iv, v , vii, viii} {i, iv, v, vii, ix} {i, iv, v, vii, x} {i, iv, v, vii, xi} {i, iv, v, viii, ix} { i, iv, v, viii, x} {i, iv, v, viii, xi} {i, iv, v, ix, x} {i, iv, v, ix, xi} {i, iv, v, x, xi} {i, iv, vi, vii, viii} {i, iv, vi, vii, ix} {i, iv, vi, vii, x} {i, iv, vi, vii, xi} {i , iv, vi, viii, ix} {i, iv, vi, viii, x} {i, iv, vi, viii, xi} {i, iv, vi, ix, x} {i, iv, vi, ix , xi} {i, iv, vi, x, xi} {i, iv, vii, viii, ix} {i, iv, vii, viii, x}

{I, iv, vii, viii, xi} {i, iv, vii, ix, x} {i, iv, vii, ix, xi} {i, iv, vii, x, xi} {i, iv, viii , ix, x} {i, iv, viii, ix, xi} {i, iv, viii, x, xi} {i, iii, ix, x, xi} {t, v, vi, vii, viii} { i, v, vi, vii, ix} {t, v, vi, vii, x} {t, v, vi, vii, xi}

{I, v, vi, viii, ix} {t, v, vi, viii, x} {i, v, vi, viii, xi} {t, v, vi, ix, x} {t, v, vi , ix, xi} {t, v, w, x, xi} {t, v, vii, viii, ix} {t, v, vii, viii, x} {t, v, vii, viii, xi} { i, v, vii, ix, x} {t, v, vii, ix, xi} {t, v, vi, x, xi} {t, v, viii, ix, x}

{T, v, viii, ix, xi} {t, v, viii, x, xi} {t, v, ix, x, xi} {t, vi, vii, viii, ix} {t, vi, vii , viii, x} {t, vi, vii, viii, xi} {t, vi, vii, ix, x} {t, vi, vii, ix, xi} {t, vi, vii, x, xi} { i, vi, viii, ix, x} {i, vi, viii, ix, xi} {t, vi, viii, x, xi}

{I, vi, ix, x, xi} {i, vii, viii, ix, x} {i, vii, viii, ix, xi} {i, vii, viii, x, xi} {i, vii, ix , x, xi} {i, viii, ix, x, xi} {ii, iii, iv, v, vi} {ii, iii, iv, v, vii} {ii, iii, iv, v, viii} { ii, iii, iv, v, ix} {ii, iii, iv, v, x} {ii, iii, iv, v, xi}

{Ii, iii, iv, vi, vii} {ii, iii, iv, vi, viii} {ii, iii, iv, vi, ix} {ii, iii, iv, vi, x} {ii, iii, iv , vi, xi} {ii, iii, iv, vii, viii} {ii, iii, iv, vii, ix} {ii, iii, iv, vii, x} {ii, iii, iv, vii, xi} { ii, iii, iv, viii, ix} {ii, iii, iv, viii, x} {ii, iii, iv, viii, xi} {ii, iii, iv, ix, x} {ii, iii, iv, ix, xi} {ii, iii, iv, x, xi} {ii, iii, v, vi, vii} {ii, iii, v, vi, viii} {ii, iii, v, vi, ix}

{Ii, iii, v, vi, x} {ii, iii, v, vi, xi} {ii, iii, v, vii, viii} {ii, iii, v, vii, ix} {ii, iii, v , vii, x} {ii, iii, v, vii, xi}

{Ii, iii, v, viii, ix} {ii, iii, v, viii, x} {ii, iii, v, viii, xi} {ii, iii, v, ix, x} {ii, iii, v , ix, xi} {ii, iii, v, x, xi}

{Ii, iii, vi, vii, viii} {ii, iii, vi, vii, ix} {ii, iii, vi, vii, x} {ii, iii, vi, vii, xi} {ii, iii, vi , viii, ix} {ii, iii, vi, viii, x} {ii, iii, vi, viii, xi} {ii, iii, vi, ix, x} {ii, iii, vi, ix, xi} { ii, iii, vi, x, xi} {ii, iii, vii, viii, ix} {ii, iii, vii, viii, x} {ii, iii, vii, viii, xi} {ii, iii, vii, ix, x} {ii, iii, vii, ix, xi} {ii, iii, vii, x, xi} {ii, iii, viii, ix, x} {ii, iii, viii, ix, xi} {ii , iii, viii, x, xi} {ii, iii, ix, x, xi} {ii, iv, v, vi, vii} {ii, iv, v, vi, viii} {ii, iv, v, vi , ix} {ii, iv, v, vi, x}

{Ii, iv, v, vi, xi} {ii, iv, v, vii, viii} {ii, iv, v, vii, ix} {ii, iv, v, vii, x} {ii, iv, v , vii, xi} {ii, iv, v, viii, ix} {ii, iv, v, viii, x} {ii, iv, v, viii, xi} {ii, iv, v, ix, x} { ii, iv, v, ix, xi} {ii, iv, v, x, xi} {ii, iv, vi, vii, viii}

{Ii, iv, vi, vii, ix} {ii, iv, vi, vii, x} {ii, iv, vi, vii, xi} {ii, iv, vi, viii, ix} {ii, iv, vi , viii, x} {ii, iv, vi, viii, xi} {ii, iv, vi, ix, x} {ii, iv, vi, ix, xi} {ii, iv, vi, x, xi} { ii, iv, vii, viii, ix} {ii, iv, vii, viii, x} {ii, iv, vii, viii, xi} {ii, iv, vii, ix, x} {ii, iv, vii, ix, xi} {ii, iv, vi, x, xi} {ii, iv, viii, ix, x} {ii, iv, viii, ix, xi} {ii, iv, viii, x, xi} {D , iii, ix, x, xi} {u, v, vi, vii, viii} {u, v, vi, vii, ix} {u, v, vi, vii, x} {u, v, vi, vii , xi} {u, v, vi, viii, ix} {u, v, vi, viii, x} {u, v, vi, viii, xi} {u, v, vi, ix, x} {D, v, vi, ix, xi} {u, v, w, x, xi} {u, v, vii, viii, ix}

{U, v, vii, viii, x} {u, v, vii, viii, xi} {u, v, vii, ix, x} {u, v, vii, ix, xi} {u, v, vii , x, xi} {u, v, viii, ix, x} {u, v, viii, ix, xi} {u, v, viii, x, xi} {u, v, ix, x, xi} { ii, vi, vii, viii, ix} {ii, vi, vii, viii, x} {ii, vi, vii, viii, xi} {ii, vi, vii, ix, x} {ii, vi, vii, ix, xi} {ii, vi, vii, x, xi} {ii, vi, viii, ix, x} {ii, vi, viii, ix, xi} {ii, vi, viii, x, xi}

{Ii, vi, ix, x, xi} {ii, vii, viii, ix, x} {ii, vii, viii, ix, xi} {ii, vii, viii, x, xi} {ii, vii, ix , x, xi} {ii, viii, ix, x, xi} {iii, iv, v, vi, vii} {iii, iv, v, vi, viii} {iii, iv, v, vi, ix} { iii, iv, v, vi, x} {iii, iv, v, vi, xi} {iii, iv, v, vii, viii} {iii, iv, v, vii, ix} {iii, iv, v, vii, x} {iii, iv, v, vii, xi} {iii, iv, v, viii, ix} {iii, iv, v, viii, x} {iii, iv, v, viii, xi} {iii , iv, v, ix, x} {iii, iv, v, ix, xi} {iii, iv, v, x, xi} {iii, iv, vi, vii, viii} {iii, iv, vi, vii , ix} {iii, iv, vi, vii, x} {iii, iv, vi, vii, xi} {iii, iv, vi, viii, ix} {iii, iv, vi, viii, x} {iii, iv, vi, viii, xi} {iii, iv, vi, ix, x} {iii, iv, vi, ix, xi} {iii, iv, vi, x, xi} {iii, iv, vii, viii, ix} {iii, iv, vii, viii, x} {iii, iv, vii, viii, xi} {iii, iv, vii, ix, x} {iii, iv, vii, ix, xi} {iii, iv , vii, x, xi} {iii, iv, viii, ix, x} {iii, iv, viii, ix, xi} {iii, iv, viii, x, xi} {iii, iv, ix, x, xi } {iii, v, vi, vii, viii} {iii, v, vi, vii, ix} {iii, v, vi, vii, x} {iii, v, vi, vii, xi} {iii, v, vi, viii, ix} {iii, v, vi, viii, x} {iii, v, vi, viii, xi} {iii, v, vi, ix, x} {iii, v, vi, ix, xi} {iii, v , vi, x, xi} {iii, v, vii, viii, ix} {iii, v, vii, viii, x} {iii, v, vii, viii, xi} {iii, v, vii, ix, x } {iii, v, vii, ix, xi} {iii, v, vii, x, xi} {iii, v, viii, ix, x} {iii, v, viii, ix, xi} {iii, v, viii, x, xi} {iii, v, ix, x, xi} {iii, vi, vii, viii, ix} {iii, vi, vii, viii, x} {iii, vi, vii, viii, xi} {iii, vi, vii, ix, x} {iii, vi, vii, ix, xi} {iii, vi, vii, x, xi} {iii, vi, viii, ix, x} {iii, vi, viii , ix, xi} {iii, vi, viii, x, xi} {iii, vi, ix, x, xi} {iii, vii, viii, ix, x} {iii, vii, viii, ix, xi} { iii, vii, viii, x, xi} {iii, vii, ix, x, xi} {iii, viii, ix, x, xi} {iv, v, vi, vii, viii} {iv, v, vi, vii, ix} {iv, v, vi, vii, x} {iv, v, vi, vii, xi} {iv, v, vi, viii, ix} {iv, v, vi, viii, x} {iv , v, vi, viii, xi} {iv, v, vi, ix, x} {iv, v, vi, ix, xi} {iv, v, vi, x, xi} {iv, v, vii, viii , ix} {iv, v, vii, viii, x} {iv, v, vii, viii, xi} {iv, v, vii, ix, x} {iv, v, vii, ix, xi} {iv, v, vi, x, xi} {iv, v, viii, ix, x} {iv, v, viii, ix, xi} {iv, v, viii, , Xi} {iv, v, ix, x, xi} {iv, vi, vii, viii, ix} {iv, vi, vii, viii, x} {iv, vi, vii, viii, xi} {iv, vi, vii, ix, x} {iv, vi, vii, ix, xi} {iv, vi, vii, x, xi} {iv, vi, viii, ix, x} {iv, vi, viii, ix, xi} {iv, vi, viii, x, xi} {iv, vi, ix, x, xi} {iv, vii, viii, ix, x} {iv, vii, viii, ix, xi} {iv, vii , viii, x, xi} {iv, vii, ix, x, xi} {iv, viii, ix, x, xi} {v, vi, vii, viii, ix} {v, vi, vii, viii, x } {v, vi, vii, viii, xi} {v, vi, vii, ix, x} {v, vi, vii, ix, xi} {v, vi, vii, x, xi} {v, vi, viii, ix, x} {v, vi, viii, ix, xi} {v, vi, viii, x, xi} {v, vi, ix, x, xi} {v, vii, viii, ix, x} {v, vii, viii, ix, xi} {v, vii, viii, x, xi} {v, vii, ix, x, xi} {v, viii, ix, x, xi} {vi, vii, viii , ix, x} {vi, vii, viii, ix, xi} {vi, vii, viii, x, xi} {vi, vii, ix, x, xi} {vi, viii, ix, x, xi}

{Vii, viii, ix, x, xi} {i, ii, iii, iv, v, vi} {i, ii, iii, iv, v, vii} {i, ii, iii, iv, v, viii} {i, ii, iii, iv, v, ix} {i, ii, iii, iv, v, x} {i, ii, iii, iv, v, xi} {i, ii, iii, iv, vi, vii} {i, ii, iii, iv, vi, viii} {i, ii, iii, iv, vi, ix} {i, ii, iii, iv, vi, x} {i, ii, iii, iv, vi, xi} {i, ii, iii, iv, vii, viii} {i, ii, iii, iv, vii, ix} {i, ii, iii, iv, vii, x} {i, ii, iii, iv, vii, xi} {i, ii, iii, iv, viii, ix}

{I, ii, iii, iv, viii, x} {i, ii, iii, iv, viii, xi} {i, ii, iii, iv, ix, x} {i, ii, iii, iv, ix, xi} {i, ii, iii, iv, x, xi} {i, ii, iii, v, vi, vii} {i, ii, iii, v, vi, viii} {i, ii, iii, v, vi, ix} {i, ii, iii, v, vi, x} {i, ii, iii, v, vi, xi} {i, ii, iii, v, vii, viii} {i, ii, iii, v, vii, ix} {i, ii, iii, v, vii, x} {i, ii, iii, v, vii, xi} {i, ii, iii, v, viii, ix} {i, ii, iii, v, viii, x} {i, ii, iii, v, viii, xi} {i, ii, iii, v, ix, x} {i, ii, iii, v, ix, xi} {i, ii, iii, v, x, xi} {i, ii, iii, vi, vii, viii} {i, ii, iii, vi, vii, ix} {i, ii, iii, vi, vii, x}

{I, ii, iii, vi, vii, xi} {i, ii, iii, vi, viii, ix} {i, ii, iii, vi, viii, x} {i, ii, iii, vi, viii, xi} {i, ii, iii, vi, ix, x}

{I, ii, iii, vi, ix, xi} {i, ii, iii, vi, x, xi} {i, ii, iii, vii, viii, ix} {i, ii, iii, vii, viii, {x} i, ii, iii, vii, viii, xi}

{I, ii, iii, vii, ix, x} {i, ii, iii, vii, ix, xi} {i, ii, iii, vii, x, xi} {i, ii, iii, viii, ix, {x} i, ii, iii, viii, ix, xi}

{I, ii, iii, viii, x, xi} {i, ii, iii, ix, x, xi} {i, ii, iv, v, vi, vii} {i, ii, iv, v, vi, viii} {i, ii, iv, v, vi, ix} {i, ii, iv, v, vi, x} {i, ii, iv, v, vi, xi} {i, ii, iv, v, vii, viii} {i, ii, iv, v, vii, ix} {i, ii, iv, v, vii, x} {i, ii, iv, v, vii, xi} {i, ii, iv, v, viii, ix} {i, ii, iv, v, viii, x} {i, ii, iv, v, viii, xi} {i, ii, iv, v, ix, x} {i, ii, iv, v, ix, xi} {i, ii, iv, v, x, xi} {i, ii, iv, vi, vii, viii}

{I, ii, iv, vi, vii, ix} {i, ii, iv, vi, vii, x} {i, ii, iv, vi, vii, xi} {i, ii, iv, vi, viii, ix} {i, ii, iv, vi, viii, x} {i, ii, iv, vi, viii, xi} {i, ii, iv, vi, ix, x} {i, ii, iv, vi, ix, xi} {i, ii, iv, vi, x, xi} {i, ii, iv, vii, viii, ix}

{I, ii, iv, vii, viii, x} {i, ii, iv, vii, viii, xi} {i, ii, iv, vii, ix, x} {i, ii, iv, vii, ix, xi} {i, ii, iv, vii, x, xi}

{I, ii, iv, viii, ix, x} {i, ii, iv, viii, ix, xi} {i, ii, iv, viii, x, xi} {i, ii, iv, ix, x, xi} {i, ii, v, vi, vii, viii}

{I, ii, v, vi, vii, ix} {i, ii, v, vi, vii, x} {i, ii, v, vi, vii, xi} {i, ii, v, vi, viii, ix} {i, ii, v, vi, vii, x} {i, ii, v, vi, viii, xi} {i, ii, v, vi, ix, x} {i, ii, v, vi, ix, xi} {i, ii, v, vi, x, xi} {i, ii, v, vii, viii, ix} {i, ii, v, vii, viii, x} {i, ii, v, vii, viii, xi} {i, ii, v, vii, ix, x} {i, ii, v, vii, ix, xi} {i, ii, v, vii, x, xi} {i, ii, v, viii, ix, x} {i, ii, v, viii, ix, xi} {i, ii, v, viii, x, xi} {i, ii, v, ix, x, xi} {i, ii, vi, vii, viii, ix} {i, ii, vi, vii, viii, x} {i, ii, vi, vii, viii, xi} {i, ii, vi, vii, ix, x}

{I, ii, vi, vii, ix, xi} {i, ii, vi, vii, x, xi} {i, ii, vi, viii, ix, x} {i, ii, vi, viii, ix, xi} {i, ii, vi, viii, x, xi}

{I, ii, vi, ix, x, xi} {i, ii, vii, viii, ix, x} {i, ii, vii, viii, ix, xi} {i, ii, vii, viii, x, xi} {i, ii, vii, ix, x, xi}

{I, ii, viii, ix, x, xi} {i, iii, iv, v, vi, vii} {i, iii, iv, v, vi, viii} {i, iii, iv, v, vi, ix} {i, iii, iv, v, vi, x}

{I, iii, iv, v, vi, xi} {i, iii, iv, v, vii, viii} {i, iii, iv, v, vii, ix} {i, iii, iv, v, vii, {x} i, iii, iv, v, vii, xi}

{I, iii, iv, v, viii, ix} {i, iii, iv, v, viii, x} {i, iii, iv, v, viii, xi} {i, iii, iv, v, ix, {x} i, iii, iv, v, ix, xi}

{I, iii, iv, v, x, xi} {i, iii, iv, vi, vii, viii} {i, iii, iv, vi, vii, ix} {i, iii, iv, vi, vii, {x} i, iii, iv, vi, vii, xi}

{I, iii, iv, vi, viii, ix} {i, iii, iv, vi, viii, x} {i, iii, iv, vi, viii, xi} {i, iii, iv, vi, ix, {x} i, iii, iv, vi, ix, xi}

{I, iii, iv, vi, x, xi} {i, iii, iv, vii, viii, ix} {i, iii, iv, vii, viii, x} {i, iii, iv, vii, viii, xi} {i, iii, iv, vii, ix, x} {i, iii, iv, vii, ix, xi} {i, iii, iv, vii, x, xi} {i, iii, iv, viii, ix, x} {i, iii, iv, viii, ix, xi} {i, iii, iv, viii, x, xi}

{I, iii, iv, ix, x, xi} {i, iii, v, vi, vii, viii} {i, iii, v, vi, vii, ix} {i, iii, v, vi, vii, {x} i, iii, v, vi, vii, xi}

{I, iii, v, vi, viii, ix} {i, iii, v, vi, vii, x} {i, iii, v, vi, viii, xi} {i, iii, v, vi, ix, {x} i, iii, v, vi, ix, xi}

{I, iii, v, vi, x, xi} {i, iii, v, vii, viii, ix} {i, iii, v, vii, viii, x} {i, iii, v, vii, viii, xi} {i, iii, v, vii, ix, x}

{I, iii, v, vii, ix, xi} {i, iii, v, vii, x, xi} {i, iii, v, viii, ix, x} {i, iii, v, viii, ix, xi} {i, iii, v, viii, x, xi}

{I, iii, v, ix, x, xi} {i, iii, vi, vii, viii, ix} {i, iii, vi, vii, viii, x} {i, iii, vi, vii, viii, xi} {i, iii, vi, vii, ix, x}

{I, iii, vi, vii, ix, xi} {i, iii, vi, vii, x, xi} {i, iii, vi, viii, ix, x} {i, iii, vi, viii, ix, xi} {i, iii, vi, viii, x, xi}

{I, iii, vi, ix, x, xi} {i, iii, vii, viii, ix, x} {i, iii, vii, viii, ix, xi} {i, iii, vii, viii, x, xi} {i, iii, vii, ix, x, xi}

{I, iii, viii, ix, x, xi} {i, iv, v, vi, vii, viii} {i, iv, v, vi, vii, ix} {i, iv, v, vi, vii, {x} i, iv, v, vi, vii, xi}

{I, iv, v, vi, viii, ix} {i, iv, v, vi, vii, x} {i, iv, v, vi, viii, xi} {i, iv, v, vi, ix, x} {i, iv, v, vi, ix, xi} {i, iv, v, vi, x, xi} {i, iv, v, vii, viii, ix} {i, iv, v, vii, viii, x} {i, iv, v, vii, viii, xi} {i, iv, v, vii, ix, x} {i, iv, v, vii, ix, xi}

{I, iv, v, vii, x, xi} {i, iv, v, viii, ix, x} {i, iv, v, viii, ix, xi} {i, iv, v, viii, x, xi} {i, iv, v, ix, x, xi}

{I, iv, vi, vii, viii, ix} {i, iv, vi, vii, viii, x} {i, iv, vi, vii, viii, xi} {i, iv, vi, vii, ix, x} {i, iv, vi, vii, ix, xi} {i, iv, vi, vii, x, xi} {i, iv, vi, viii, ix, x} {i, iv, vi, viii, ix, xi} {i, iv, vi, viii, x, xi} {i, iv, vi, ix, x, xi}

{I, iv, vii, viii, ix, x} {i, iv, vii, viii, ix, xi} {i, iv, vii, viii, x, xi} {i, iv, vii, ix, x, xi} {i, iv, viii, ix, x, xi}

{I, v, vi, vii, viii, ix} {i, v, vi, vii, viii, x} {i, v, vi, vii, viii, xi} {i, v, vi, vii, ix, {x} i, v, vi, vii, ix, xi}

{I, v, vi, vii, x, xi} {i, v, vi, viii, ix, x} {i, v, vi, viii, ix, xi} {i, v, vi, vii, x, xi} {i, v, vi, ix, x, xi}

{I, v, vii, viii, ix, x} {i, v, vii, viii, ix, xi} {i, v, vii, viii, x, xi} {i, v, vii, ix, x, xi} {i, v, viii, ix, x, xi}

{I, vi, vii, viii, ix, x} {i, vi, vii, viii, ix, xi} {i, vi, vii, viii, x, xi} {i, vi, vii, ix, x, xi} {i, vi, viii, ix, x, xi}

{I, vii, viii, ix, x, xi} {ii, iii, iv, v, vi, vii} {ii, iii, iv, v, vi, viii} {ii, iii, iv, v, vi, ix} {ii, iii, iv, v, vi, x} {ii, iii, iv, v, vi, xi} {ii, iii, iv, v, vii, viii} {ii, iii, iv, v, vii, ix} {ii, iii, iv, v, vii, x} {ii, iii, iv, v, vii, xi} {ii, iii, iv, v, viii, ix} {ii, iii, iv, v, viii, x} {ii, iii, iv, v, viii, xi} {ii, iii, iv, v, ix, x} {ii, iii, iv, v, ix, xi} {ii, iii, iv, v, x, xi} {ii, iii, iv, vi, vii, viii} {ii, iii, iv, vi, vii, ix} {ii, iii, iv, vi, vii, x} {ii. iii, iv, vi, vii, xi} {ii, iii, iv, vi, viii, ix} {ii, iii, iv, vi, viii, x} {ii, iii, iv, vi, viii, xi} { ii, iii, iv, vi, ix, x} {ii, iii, iv, vi, ix, xi} {ii, iii, iv, vi, x, xi} {ii, iii, iv, vii, viii, ix } {ii, iii, iv, vii, viii, x} {ii, iii, iv, vii, viii, xi} {ii, iii, iv, vii, ix, x} {ii, iii, iv, vii, ix Stacking} {ii, iii, iv, vii, x, xi} {ii, iii, iv, viii, ix, x} {ii, iii, iv, viii, ix, xi} {ii, iii, iv, viii , x, xi} {ii, iii, iv, ix, x, xi} {ii, iii, v, vi, vii, viii} {ii, iii, v, vi, vii, ix} {ii, iii, v , vi, vii, x} {ii, iii, v, vi, vii, xi} {ii, iii, v, vi, viii, ix} {ii, iii, v, vi, vii, x} {ii, iii , v, vi, viii, xi} {ii, iii, v, vi, ix, x} {ii, iii, v, vi, ix, xi} {ii, iii, v, vi, x, xi} {ii , iii, v, vii, viii, ix} {ii, iii, v, vii, viii, x} {ii, iii, v, vii, viii, xi} {ii, iii, v, vii, ix, x} {ii, iii, v, vii, ix, xi} {ii, iii, v, vii, x, xi} {ii, iii, v, viii, ix, x} {ii, iii, v, viii, ix, xi} {ii, iii, v, viii, x, xi} {ii, iii, v, ix, x, xi} {ii, iii, vi, vii, viii, ix} {ii, iii, vi, vii, viii, x} {ii. iii, vi, vii, viii, xi} {ii, iii, vi, vii, ix, x} {ii, iii, vi, vii, ix, xi} {ii, iii, vi, vii, x, xi} { ii, iii, vi, viii, ix, x} {ii, iii, vi, viii, ix, xi} {ii, iii, vi, viii, x, xi} {ii, iii, vi, ix, x, xi } {ii, iii, vii, viii, ix, x} {ii, iii, vii, viii, ix, xi} {ii, iii, vii, viii, x, xi} {ii, iii, vii, ix, x Stacking} {ii, iii, viii, ix, x, xi} {ii, iv, v, vi, vii, viii} {ii, iv, v, vi, vii, ix} {ii, iv, v, vi , vii, x} {ii, iv, v, vi, vii, xi} {ii, iv, v, vi, viii, ix} {ii, iv, v, vi, vii, x} {ii, iv, v , vi, viii, xi} {ii, iv, v, vi, ix, x} {ii, iv, v, vi, ix, xi}

{Ii, iv, v, vi, x, xi} {ii, iv, v, vii, viii, ix} {ii, iv, v, vii, viii, x} {ii, iv, v, vii, viii, xi} {ii, iv, v, vii, ix, x} {ii, iv, v, vii, ix, xi} {ii, iv, v, vii, x, xi} {ii, iv, v, viii, ix, x} {ii, iv, v, viii, ix, xi} {ii, iv, v, viii, x, xi} {ii, iv, v, ix, x, xi} {ii, iv, vi, vii, viii, ix} {ii, iv, vi, vii, viii, x} {ii, iv, vi, vii, viii, xi} {ii, iv, vi, vii, ix, x} {ii, iv, vi, vii, ix, xi} {ii, iv, vi, vii, x, xi} {ii, iv, vi, viii, ix, x} {ii, iv, vi, viii, ix, xi} {ii. iv, vi, viii, x, xi} {ii, iv, vi, ix, x, xi} {ii, iv, vii, viii, ix, x} {ii, iv, vii, viii, ix, xi} { ii, iv, vii, viii, x, xi} {ii, iv, vii, ix, x, xi} {ii, iv, viii, ix, x, xi} {ii, v, vi, vii, viii, ix } {ii, v, vi, vii, viii, x} {ii, v, vi, vii, viii, xi} {ii, v, vi, vii, ix, x} {ii, v, vi, vii, ix Stacking} {ii, v, vi, vii, x, xi} {ii, v, vi, viii, ix, x} {ii, v, vi, viii, ix, xi} {ii, v, vi, viii , x, xi} {ii, v, vi, ix, x, xi} {ii, v, vii, viii, ix, x} {ii, v, vii, viii, ix, xi} {ϋ, ν, νϋ , νίϋ, χ, χί} {ii, v, vii, ix, x, xi} {ii, v, viii, ix, x, xi} {ii, vi, vii, viii, ix, x} {ii, vi , vii, viii, ix, xi} {ii, vi, vii, viii, x, xi} {ii, vi, vii, ix, x, xi} {ii, vi, viii, ix, x, xi} {ii , vii, viii, ix, x, xi} {iii, iv, v, vi, vii, v iii} {iii, iv, v, vi, vii, ix} {iii, iv, v, vi, vii, x} {iii, iv, v, vi, vii, xi} {iii, iv, v, vi, viii, ix} {iii, iv, v, vi, vii, x} {iii, iv, v, vi, viii, xi} {iii, iv, v, vi, ix, x} {iii, iv, v, vi, ix, xi} {iii, iv, v, vi, x, xi} {iii, iv, v, vii, viii, ix} {iii, iv, v, vii, viii, x} {iii, iv, v, vii, viii, xi} {iii, iv, v, vii, ix, x} {iii, iv, v, vii, ix, xi} {iii, iv, v, vii, x, xi} {iii. iv, v, viii, ix, x} {iii, iv, v, viii, ix, xi} {iii, iv, v, viii, x, xi} {iii, iv, v, ix, x, xi} { iii, iv, vi, vii, viii, ix} {iii, iv, vi, vii, viii, x} {iii, iv, vi, vii, viii, xi} {iii, iv, vi, vii, ix, x } {iii, iv, vi, vii, ix, xi} {iii, iv, vi, vii, x, xi} {iii, iv, vi, viii, ix, x} {iii, iv, vi, viii, ix Stacking} {iii, iv, vi, viii, x, xi} {iii, iv, vi, ix, x, xi} {iii, iv, vii, viii, ix, x} {iii, iv, vii, viii , ix, xi} {iii, iv, vii, viii, x, xi} {iii, iv, vii, ix, x, xi} {iii, iv, viii, ix, x, xi} {iii, v, vi , vii, viii, ix} {iii, v, vi, vii, viii, x} {iii, v, vi, vii, viii, xi} {iii, v, vi, vii, ix, x} {iii, v , vi, vii, ix, xi} {iii, v, vi, vii, x, xi} {iii, v, vi, viii, ix, x} {iii, v, vi, viii, ix, xi} {iii , v, vi, viii, x, xi} {iii, v, vi, ix, x, xi} {iii, v, vii, viii, ix, x} {iii, v, vii, viii, ix, xi} {iii. v, vii, viii, x, xi} {iii, v, vii, ix, x, xi} {iii, v, viii, ix, x, xi} {iii, vi, vii, viii, ix, x} { iii, vi, vii, viii, ix, xi} {iii, vi, vii, viii, x, xi} {iii, vi, vii, ix, x, xi} {iii, vi, viii, ix, x, xi } {iii, vii, viii, ix, x, xi} {iv, v, vi, vii, viii, ix} {iv, v, vi, vii, viii, x} {iv, v, vi, vii, viii Stacking} {iv, v, vi, vii, ix, x} {iv, v, vi, vii, ix, xi} {iv, v, vi, vii, x, xi} {iv, v, vi, viii , ix, x} {iv, v, vi, viii, ix, xi} {iv, v, vi, viii, x, xi} {iv, v, vi, ix, x, xi} {iv, v, vii , viii, ix, x} {iv, v, vii, viii, ix, xi} {iv, v, vii, viii, x, xi} {iv, v, vii, ix, x, xi} {iv, v , viii, ix, x, xi} {iv, vi, vii, viii, ix, x} {iv, vi, vii, viii, ix, xi} {iv, vi, vii, viii, x, xi} {iv , vi, vii, ix, x, xi} {iv, vi, viii, ix, x, xi} {iv, vii, viii, ix, x, xi} {v, vi, vii, viii, ix, x} {v, vi, vii, viii, ix, xi} {v, vi, vii, viii, x, xi} {v, vi, vii, ix, x, xi} {v, vi, viii, ix, x, xi} {v, vii, viii, ix, x, xi} {vi, vii, viii, ix, x, xi} {i, ii, iii, iv, v, vi, vii} {i, ii, iii, iv, v, vi, viii} {i, ii, iii, iv, v, vi, ix} {i, ii, iii, iv, v, vi, x} {i, ii, iii, iv, v, vi Stacking} {i, ii, iii, iv, v, vii, viii} {i, ii, iii, iv, v, vii, ix} {i, ii, iii, iv, v, vii, x} {i , ii, iii, iv, v, vii, xi} {i, ii, iii, iv, v, viii, ix} {i, ii, iii, iv, v, viii, x} {i, ii, iii, iv, v, viii, xi} {i, ii, iii, iv, v, ix, x} { i, ii, iii, iv, v, ix, xi} {i, ii, iii, iv, v, x, xi} {i, ii, iii, iv, vi, vii, viii} {i, ii, iii , iv, vi, vii, ix} {i, ii, iii, iv, vi, vii, x} {i, ii, iii, iv, vi, vii, xi} {i, ii, iii, iv, vi, viii, ix} {i, ii, iii, iv, vi, viii, x} {i, ii, iii, iv, vi, viii, xi} {i, ii, iii, iv, vi, ix, x} { i, ii, iii, iv, vi, ix, xi} {i, ii, iii, iv, vi, x, xi} {i, ii, iii, iv, vii, viii, ix} {i, ii, iii , iv, vii, viii, x} {i, ii, iii, iv, vii, viii, xi}

{I, ii, iii, iv, vii, ix, x} {i, ii, iii, iv, vii, ix, xi} {i, ii, iii, iv, vii, x, xi} {i, ii, iii, iv, viii, ix, x} {i, ii, iii, iv, viii, ix, xi} {i, ii, iii, iv, viii, x, xi} {i, ii, iii, iv, ix , x, xi} {i, ii, iii, v, vi, vii, viii} {i, ii, iii, v, vi, vii, ix} {i, ii, iii, v, vi, vii, x} {i, ii, iii, v, vi, vii, xi} {i, ii, iii, v, vi, viii, ix} {i, ii, iii, v, vi, vii, x} {i, ii, iii, v, vi, viii, xi} {i, ii, iii, v, vi, ix, x} {i, ii, iii, v, vi, ix, xi} {i, ii, iii, v, vi , x, xi} {i, ii, iii, v, vii, viii, ix} {i, ii, iii, v, vii, viii, x} {i, ii, iii, v, vii, viii, xi} {i, ii, iii, v, vii, ix, x} {i, ii, iii, v, vii, ix, xi} {i, ii, iii, v, vii, x, xi} {i, ii, iii, v, viii, ix, x} {i, ii, iii, v, viii, ix, xi} {i, ii, iii, v, viii, x, xi} {i, ii, iii, v, ix , x, xi} {i, ii, iii, vi, vii, viii, ix} {i, ii, iii, vi, vii, viii, x} {i, ii, iii, vi, vii, viii, xi} {i, ii, iii, vi, vii, ix, x} {i, ii, iii, vi, vii, ix, xi} {i, ii, iii, vi, vii, x, xi} {i, ii, iii, vi, viii, ix, x} {i, ii, iii, vi, viii, ix, xi} {i, ii, iii, vi, viii, x, xi} {i, ii, iii, vi, ix , x, xi} {i, ii, iii, vii, viii, ix, x} {i, ii, iii, vii, viii, ix, xi}

{I, ii, iii, vii, viii, x, xi} {i, ii, iii, vii, ix, x, xi} {i, ii, iii, viii, ix, x, xi} {i, ii, iv, v, vi, vii, viii} {i, ii, iv, v, vi, vii, ix} {i, ii, iv, v, vi, vii, x} {i, ii, iv, v, vi , vii, xi} {i, ii, iv, v, vi, viii, ix} {i, ii, iv, v, vi, vii, x} {i, ii, iv, v, vi, viii, xi} {i, ii, iv, v, vi, ix, x} {i, ii, iv, v, vi, ix, xi} {i, ii, iv, v, vi, x, xi} {i, ii, iv, v, vii, viii, ix} {i, ii, iv, v, vii, viii, x} {i, ii, iv, v, vii, viii, xi} {i, ii, iv, v, vii , ix, x} {i, ii, iv, v, vii, ix, xi} {i, ii, iv, v, vii, x, xi} {i, ii, iv, v, viii, ix, x} {i, ii, iv, v, viii, ix, xi} {i, ii, iv, v, viii, x, xi} {i, ii, iv, v, ix, x, xi} {i, ii, iv, vi, vii, viii, ix} {i, ii, iv, vi, vii, viii, x} {i, ii, iv, vi, vii, viii, xi} {i, ii, iv, vi, vii , ix, x} {i, ii, iv, vi, vii, ix, xi} {i, ii, iv, vi, vii, x, xi} {i, ii, iv, vi, viii, ix, x} {i, ii, iv, vi, viii, ix, xi} {i, ii, iv, vi, viii, x, xi} {i, ii, iv, vi, ix, x, xi} {i, ii, iv, vii, viii, ix, x} {i, ii, iv, vii, viii, ix, xi} {i, ii, iv, vii, viii, x, xi} {i, ii, iv, vii, ix , x, xi} {i, ii, iv, viii, ix, x, xi} {i, ii, v, vi, vii, viii, ix} {i, ii, v, vi, vii, viii, x} {i, ii, v, vi, vii, viii, xi} {i, ii, v, vi, vii, ix, x} {i, ii, v, vi, vii, ix, xi} {i, ii, v, vi, vii, x, xi} {I, ii, v, vi, viii, ix, x} {i, ii, v, vi, viii, ix, xi} {i, ii, v, vi, viii, x, xi} {i, ii, v, vi, ix, x, xi} {i, ii, v, vii, viii, ix, x} {i, ii, v, vii, viii, ix, xi} {i, ii, v, vii, viii , x, xi} {i, ii, v, vii, ix, x, xi} {i, ii, v, viii, ix, x, xi} {i, ii, vi, vii, viii, ix, x} {i, ii, vi, vii, viii, ix, xi} {i, ii, vi, vii, viii, x, xi} {i, ii, vi, vii, ix, x, xi} {i, ii, vi, viii, ix, x, xi} {i, ii, vii, viii, ix, x, xi}

{I, iii, iv, v, vi, vii, viii} {i, iii, iv, v, vi, vii, ix} {i, iii, iv, v, vi, vii, x} {i, iii, iv, v, vi, vii, xi} {i, iii, iv, v, vi, viii, ix} {i, iii, iv, v, vi, vii, x} {i, iii, iv, v, vi , viii, xi} {i, iii, iv, v, vi, ix, x} {i, iii, iv, v, vi, ix, xi} {i, iii, iv, v, vi, x, xi}

{I, iii, iv, v, vii, viii, ix} {i, iii, iv, v, vii, viii, x} {i, iii, iv, v, vii, viii, xi} {i, iii, iv, v, vii, ix, x}

{I, iii, iv, v, vii, ix, xi} {i, iii, iv, v, vii, x, xi} {i, iii, iv, v, viii, ix, x} {i, iii, iv, v, viii, ix, xi} {i, iii, iv, v, viii, x, xi} {i, iii, iv, v, ix, x, xi} {i, iii, iv, vi, vii , viii, ix} {i, iii, iv, vi, vii, viii, x} {i, iii, iv, vi, vii, viii, xi}

{I, iii, iv, vi, vii, ix, x} {i, iii, iv, vi, vii, ix, xi} {i, iii, iv, vi, vii, x, xi} {i, iii, iv, vi, viii, ix, x}

{I, iii, iv, vi, viii, ix, xi} {i, iii, iv, vi, viii, x, xi} {i, iii, iv, vi, ix, x, xi} {i, iii, iv, vii, viii, ix, x}

{I, iii, iv, vii, viii, ix, xi} {i, iii, iv, vii, viii, x, xi} {i, iii, iv, vii, ix, x, xi} {i, iii, iv, viii, ix, x, xi}

{I, iii, v, vi, vii, viii, ix} {i, iii, v, vi, vii, viii, x} {i, iii, v, vi, vii, viii, xi} {i, iii, v, vi, vii, ix, x}

{I, iii, v, vi, vii, ix, xi} {i, iii, v, vi, vii, x, xi} {i, iii, v, vi, viii, ix, x} {i, iii, v, vi, viii, ix, xi} {i, iii, v, vi, viii, x, xi} {i, iii, v, vi, ix, x, xi} {i, iii, v, vii, viii , ix, x} {i, iii, v, vii, viii, ix, xi} {i, iii, v, vii, viii, x, xi} {i, iii, v, vii, ix, x, xi} {i, iii, v, viii, ix, x, xi} {i, iii, vi, vii, viii, ix, x} {i, iii, vi, vii, viii, ix, xi} {i, iii, vi, vii, viii, x, xi}

{I, iii, vi, vii, ix, x, xi} {i, iii, vi, viii, ix, x, xi} {i, iii, vii, viii, ix, x, xi} {i, iv, v, vi, vii, viii, ix}

{I, iv, v, vi, vii, viii, x} {i, iv, v, vi, vii, viii, xi} {i, iv, v, vi, vii, ix, x} {i, iv, v, vi, vii, ix, xi} {i, iv, v, vi, vii, x, xi} {i, iv, v, vi, viii, ix, x} {i, iv, v, vi, viii , ix, xi} {i, iv, v, vi, viii, x, xi} {i, iv, v, vi, ix, x, xi} {i, iv, v, vii, viii, ix, x} {i, iv, v, vii, viii, ix, xi} {i, iv, v, vii, viii, x, xi} {i, iv, v, vii, ix, x, xi} {i, iv, v, viii, ix, x, xi}

{I, iv, vi, vii, viii, ix, x} {i, iv, vi, vii, viii, ix, xi} {i, iv, vi, vii, viii, x, xi} {i, iv, vi, vii, ix, x, xi}

{I, iv, vi, viii, ix, x, xi} {i, iv, vii, viii, ix, x, xi} {i, v, vi, vii, viii, ix, x} {i, v, vi, vii, viii, ix, xi}

{I, v, vi, vii, viii, x, xi} {i, v, vi, vii, ix, x, xi} {i, v, vi, viii, ix, x, xi} {i, v, vii, viii, ix, x, xi} {i, vi, vii, viii, ix, x, xi} {ii, iii, iv, v, vi, vii, viii} {ii, iii, iv, v, vi , vii, ix} {ii, iii, iv, v, vi, vii, x} {ii, iii, iv, v, vi, vii, xi}

{Ii, iii, iv, v, vi, viii, ix} {ii, iii, iv, v, vi, vii, x} {ii, iii, iv, v, vi, viii, xi} {ii, iii, iv, v, vi, ix, x}

{Ii, iii, iv, v, vi, ix, xi} {ii, iii, iv, v, vi, x, xi} {ii, iii, iv, v, vii, viii, ix} {ii, iii, iv, v, vii, viii, x}

{Ii, iii, iv, v, vii, viii, xi} {ii, iii, iv, v, vii, ix, x} {ii, iii, iv, v, vii, ix, xi} {ii, iii, iv, v, vii, x, xi}

{Ii, iii, iv, v, viii, ix, x} {ii, iii, iv, v, viii, ix, xi} {ii, iii, iv, v, viii, x, xi} {ii, iii, iv, v, ix, x, xi}

{Ii, iii, iv, vi, vii, viii, ix} {ii, iii, iv, vi, vii, viii, x} {ii, iii, iv, vi, vii, viii, xi} {ii, iii, iv, vi, vii, ix, x}

{Ii, iii, iv, vi, vii, ix, xi} {ii, iii, iv, vi, vii, x, xi} {ii, iii, iv, vi, viii, ix, x} {ii, iii, iv, vi, viii, ix, xi}

{Ii, iii, iv, vi, viii, x, xi} {ii, iii, iv, vi, ix, x, xi} {ii, iii, iv, vii, viii, ix, x} {ii, iii, iv, vii, viii, ix, xi}

{Ii, iii, iv, vii, viii, x, xi} {ii, iii, iv, vii, ix, x, xi} {ii, iii, iv, viii, ix, x, xi} {ii, iii, v, vi, vii, viii, ix}

{Ii, iii, v, vi, vii, viii, x} {ii, iii, v, vi, vii, viii, xi} {ii, iii, v, vi, vii, ix, x} {ii, iii, v, vi, vii, ix, xi}

{Ii, iii, v, vi, vii, x, xi} {ii, iii, v, vi, viii, ix, x} {ii, iii, v, vi, viii, ix, xi} {ii, iii, v, vi, viii, x, xi}

{Ii, iii, v, vi, ix, x, xi} {ii, iii, v, vii, viii, ix, x} {ii, iii, v, vii, viii, ix, xi} {ii, iii, v, vii, viii, x, xi}

{Ii, iii, v, vii, ix, x, xi} {ii, iii, v, viii, ix, x, xi} {ii, iii, vi, vii, viii, ix, x} {ii, iii, vi, vii, viii, ix, xi}

{Ii, iii, vi, vii, viii, x, xi} {ii, iii, vi, vii, ix, x, xi} {ii, iii, vi, viii, ix, x, xi} {ii, iii, vii, viii, ix, x, xi}

{Ii, iv, v, vi, vii, viii, ix} {ii, iv, v, vi, vii, viii, x} {ii, iv, v, vi, vii, viii, xi} {ii, iv, v, vi, vii, ix, x}

{Ii, iv, v, vi, vii, ix, xi} {ii, iv, v, vi, vii, x, xi} {ii, iv, v, vi, viii, ix, x} {ii, iv, v, vi, viii, ix, xi}

{Ii, iv, v, vi, viii, x, xi} {ii, iv, v, vi, ix, x, xi} {ii, iv, v, vii, viii, ix, x} {ii, iv, v, vii, viii, ix, xi}

{Ii, iv, v, vii, viii, x, xi} {ii, iv, v, vii, ix, x, xi} {ii, iv, v, viii, ix, x, xi} {ii, iv, vi, vii, viii, ix, x}

ii, iv, vi, vii, viii, ix, xi} {ii, iv, vi, vii, viii, x, xi} {ii, iv, vi, vii, ix, x, xi} {ii, iv, vi , viii, ix, x, xi} i, iv, vii, viii, ix, x, xi} {ii, v, vi, vii, viii, ix, x} {ii, v, vi, vii, viii, ix Stacking} {ii, v, vi, vii, viii, x, xi} i, v, vi, vii, ix, x, xi} {ii, v, vi, viii, ix, x, xi} {ii. v, vii, viii, ix, x, xi} {ii, vi, vii, viii, ix, x, xi} ii, iv, v, vi, vii, viii, ix} {iii, iv, v, vi, vii, viii, x} {iii, iv, v, vi, vii, viii, xi} {iii, iv, v, vi, vii, ix, x} ii, iv, v, vi, vii, ix, xi} {iii, iv, v, vi, vii, x, xi} {iii, iv, v, vi, viii, ix, x} {iii, iv, v, vi, viii, ix, xi} ii, iv, v , vi, viii, x, xi} {iii, iv, v, vi, ix, x, xi} {iii, iv, v, vii, viii, ix, x} {iii, iv, v, vii, viii, ix, xi} ii, iv, v, vii, viii, x, xi} {iii, iv, v, vii, ix, x, xi} {iii, iv, v, viii, ix, x, xi} {iii , iv, vi, vii, viii, ix, x} ii, iv, vi, vii, viii, ix, xi} {iii, iv, vi, vii, viii, x, xi} {iii, iv, vi, vii , ix, x, xi} {iii, iv, vi, viii, ix, x, xi} ii, iv, vii, viii, ix, x, xi} {iii, v, vi, vii, viii, ix, x } {iii, v, vi, vii, viii, ix, xi} {iii, v, vi, vii, viii, x, xi} ii, v, vi, vii, ix, x, xi} {iii, v, vi, viii, ix, x, xi} {iii, v, vii, viii, ix, x, xi} {iii, vi, vii, viii, ix, x, xi} v, v, vi, vii, viii, ix, x} {iv, v, vi, vii , Viii, ix, xi} {iv, v, vi, vii, viii, x, xi} {iv, v, vi, vii, ix, x, xi} v, v, vi, viii, ix, x, xi } {iv, v, vii, viii, ix, x, xi} {iv, vi, vii, viii, ix, x, xi} {v, vi, vii, viii, ix, x, xi} ii, iii, iv, v, vi, vii, viii} {i, ii, iii, iv, v, vi, vii, ix} {i, ii, iii, iv, v, vi, vii, x} {i, ii, iii , iv, v, vi, vii, xi} ii, iii, iv, v, vi, viii, ix} {i, ii, iii, iv, v, vi, vii, x} {i, ii, iii, iv , v, vi, viii, xi} {i, ii, iii, iv, v, vi, ix, x} ii, iii, iv, v, vi, ix, xi} {i, ii, iii, iv, v , vi, x, xi} {i, ii, iii, iv, v, vii, viii, ix} {i, ii, iii, iv, v, vii, viii, x} ii, iii, iv, v, vii , viii, xi} {i, ii, iii, iv, v, vii, ix, x} {i, ii, iii, iv, v, vii, ix, xi} {i, ii, iii, iv, v, vii, x, xi} ii, iii, iv, v, viii, ix, x} {i, ii, iii, iv, v, viii, ix, xi} {i, ii, iii, iv, v, viii, x, xi} {i, ii, iii, iv, v, ix, x, xi} ii, iii, iv, vi, vii, viii, ix} {i, ii, iii, iv, vi, vii, viii, x} {i, ii, iii, iv, vi, vii, viii, xi} {i, ii, iii, iv, vi, vii, ix, x} ii, iii, iv, vi, vii, ix, xi} {i, ii, iii, iv, vi, vii, x, xi} {i, ii, iii, iv, vi, viii, ix, x} {i, ii, iii, iv, vi, viii, ix, xi } ii, iii, iv, vi, viii, x, xi} {i, ii, iii, iv, vi, ix, x, xi} {i, ii, iii, iv, vii, viii, ix, x} { i, ii, iii, iv, vii, viii, ix, xi} ii, iii, iv, vi i, viii, x, xi} {i, ii, iii, iv, vii, ix, x, xi} {i, ii, iii, iv, viii, ix, x, xi} {i, ii, iii, v , vi, vii, viii, ix} ii, iii, v, vi, vii, viii, x} {i, ii, iii, v, vi, vii, viii, xi} {i, ii, iii, v, vi , vii, ix, x} {i, ii, iii, v, vi, vii, ix, xi} ii, iii, v, vi, vii, x, xi} {i, ii, iii, v, vi, viii , ix, x} {i, ii, iii, v, vi, viii, ix, xi} {i, ii, iii, v, vi, viii, x, xi} ii, iii, v, vi, ix, x Stacking} {i, ii, iii, v, vii, viii, ix, x} {i, ii, iii, v, vii, viii, ix, xi} {i, ii, iii, v, vii, viii, x, xi} ii, iii, v, vii, ix, x, xi} {i, ii, iii, v, viii, ix, x, xi} {i, ii, iii, vi, vii, viii, ix, x} {i, ii, iii, vi, vii, viii, ix, xi} ii, iii, vi, vii, viii, x, xi} {i, ii, iii, vi, vii, ix, x, xi} {i, ii, iii, vi, viii, ix, x, xi} {i, ii, iii, vii, viii, ix, x, xi} ii, iv, v, vi, vii, viii, ix} {i , ii, iv, v, vi, vii, viii, x} {i, ii, iv, v, vi, vii, viii, xi} {i, ii, iv, v, vi, vii, ix, x} ii , iv, v, vi, vii, ix, xi} {i, ii, iv, v, vi, vii, x, xi} {i, ii, iv, v, vi, viii, ix, x} {i, ii, iv, v, vi, viii, ix, xi} ii, iv, v, vi, viii, x, xi} {i, ii, iv, v, vi, ix, x, xi} {i, ii, iv, v, vii, viii, ix, x} {i, ii, iv, v, vii, viii, ix, xi} ii, iv, v, vii, viii, x, xi} {i, ii, iv, v, vii, ix, x, xi} {i, ii, iv, v, viii, ix, x, xi} {i, ii, iv, vi, vii, viii, ix, x} ii, iv, vi, vii, viii, ix, xi} {i, ii, iv, vi, vii, viii, x, xi} {i, ii, iv, vi, vii, ix, x, xi} {i, ii, iv, vi, viii, ix, x, xi} ii, iv, vii, viii, ix, x, xi} {i, ii, v, vi, vii, viii, ix, x} {i, ii, v, vi, vii, viii, ix, xi} {i, ii, v, vi, vii, viii, x, xi} ii, v, vi, vii, ix, x, xi} {i, ii, v, vi, viii, ix, x, xi} {i, ii, v, vii, viii, ix, x, xi} {i, ii, vi, vii, viii, ix, x [xi} iii, iv, v, vi, vii, viii, ix} {i, iii, iv, v, vi, vii, viii, x} {i, iii, iv, v, vi, vii, viii, xi } {i, iii, iv, v, vi, vii, ix, x} iii, iv, v, vi, vii, ix, xi} {i, iii, iv, v, vi, vii, x, xi} { i, iii, iv, v, vi, viii, ix, x} {i, iii, iv, v, vi, viii, ix, xi}

{I, iii, iv, v, vi, viii, x, xi} {i, iii, iv, v, vi, ix, x, xi} {i, iii, iv, v, vii, viii, ix, x {} i, iii, iv, v, vii, viii, ix, xi}

{I, iii, iv, v, vii, viii, x, xi} {i, iii, iv, v, vii, ix, x, xi} {i, iii, iv, v, viii, ix, x, xi } {i, iii, iv, vi, vii, viii, ix, x} {i, iii, iv, vi, vii, viii, ix, xi} {i, iii, iv, vi, vii, viii, x, xi} {i, iii, iv, vi, vii, ix, x, xi} {i, iii, iv, vi, viii, ix, x, xi} {i, iii, iv, vii, viii, ix, x Stacking} {i, iii, v, vi, vii, viii, ix, x} {i, iii, v, vi, vii, viii, ix, xi} {i, iii, v, vi, vii, viii, x, xi} {i, iii, v, vi, vii, ix, x, xi} {i, iii, v, vi, viii, ix, x, xi} {i, iii, v, vii, viii, ix , x, xi} {i, iii, vi, vii, viii, ix, x, xi} {i, iv, v, vi, vii, viii, ix, x} {i, iv, v, vi, vii, viii, ix, xi} {i, iv, v, vi, vii, viii, x, xi} {i, iv, v, vi, vii, ix, x, xi} {i, iv, v, vi, viii , ix, x, xi} {i, iv, v, vii, viii, ix, x, xi} {i, iv, vi, vii, viii, ix, x, xi} {i, v, vi, vii, viii, ix, x, xi} {ii, iii, iv, v, vi, vii, viii, ix} {ii, iii, iv, v, vi, vii, viii, x} {ii, iii, iv, v , vi, vii, viii, xi} {ii, iii, iv, v, vi, vii, ix, x} {ii, iii, iv, v, vi, vii, ix, xi} {ii, iii, iv, v, vi, vii, x, xi} {ii, iii, iv, v, vi, viii, ix, x} {ii, iii, iv, v, vi, viii, ix, xi} {ii, iii, iv , v, vi, viii, x, xi} {ii, iii, iv, v, vi, ix, x, xi} {ii, iii, iv, v, vii, viii, ix, x} {ii, iii, iv, v, vii, viii, ix, xi} {ii, iii, iv, v, vii, viii, x, xi {} ii, iii, iv, v, vii, ix, x, xi} {ii, iii, iv, v, viii, ix, x, xi} {ii, iii, iv, vi, vii, viii, ix, x} {ii, iii, iv, vi, vii, viii, ix, xi} {ii, iii, iv, vi, vii, viii, x, xi} {ii, iii, iv, vi, vii, ix, x, xi } {ii, iii, iv, vi, viii, ix, x, xi} {ii, iii, iv, vii, viii, ix, x, xi} {ii, iii, v, vi, vii, viii, ix, x} {ii, iii, v, vi, vii, viii, ix, xi} {ii, iii, v, vi, vii, viii, x, xi} {ii, iii, v, vi, vii, ix, x Stacking} {ii, iii, v, vi, viii, ix, x, xi} {ii, iii, v, vii, viii, ix, x, xi} {ii, iii, vi, vii, viii, ix, x, xi} {ii, iv, v, vi, vii, viii, ix, x} {ii, iv, v, vi, vii, viii, ix, xi} {ii, iv, v, vi, vii, viii , x, xi} {ii, iv, v, vi, vii, ix, x, xi} {ii, iv, v, vi, viii, ix, x, xi} {ii, iv, v, vii, viii, ix, x, xi} {ii, iv, vi, vii, viii, ix, x, xi} {ii, v, vi, vii, viii, ix, x, xi} {iii, iv, v, vi, vii , viii, ix, x} {iii, iv, v, vi, vii, viii, ix, xi} {iii, iv, v, vi, vii, viii, x, xi} {iii, iv, v, vi, vii, ix, x, xi} {iii, iv, v, vi, viii, ix, x, xi} {iii, iv, v, vii, viii, ix, x, xi} {iii, iv, vi, vii , viii, ix, x, xi} {iii, v, vi, vii, viii, ix, x, xi} {iv, v, vi, vii, viii, ix, x, xi} {i, ii, iii, iv, v, vi, vii, viii, ix} {i, ii, iii, iv, v, vi, vii, viii, x}

{I, ii, iii, iv, v, vi, vii, viii, xi} {i, ii, iii, iv, v, vi, vii, ix, x} {i, ii, iii, iv, v, vi , vii, ix, xi} {i, ii, iii, iv, v, vi, vii, x, xi} {i, ii, iii, iv, v, vi, viii, ix, x} {i, ii, iii, iv, v, vi, viii, ix, xi} {i, ii, iii, iv, v, vi, viii, x, xi} {i, ii, iii, iv, v, vi, ix, x, xi} {i, ii, iii, iv, v, vii, viii, ix, x} {i, ii, iii, iv, v, vii, viii, ix, xi} {i, ii, iii, iv, v , vii, viii, x, xi} {i, ii, iii, iv, v, vii, ix, x, xi} {i, ii, iii, iv, v, viii, ix, x, xi} {i, ii, iii, iv, vi, vii, viii, ix, x} {i, ii, iii, iv, vi, vii, viii, ix, xi}

{I, ii, iii, iv, vi, vii, viii, x, xi} {i, ii, iii, iv, vi, vii, ix, x, xi} {i, ii, iii, iv, vi, viii , ix, x, xi}

{I, ii, iii, iv, vii, viii, ix, x, xi} {i, ii, iii, v, vi, vii, viii, ix, x} {i, ii, iii, v, vi, vii , viii, ix, xi}

{I, ii, iii, v, vi, vii, viii, x, xi} {i, ii, iii, v, vi, vii, ix, x, xi} {i, ii, iii, v, vi, viii , ix, x, xi} {i, ii, iii, v, vii, viii, ix, x, xi} {i, ii, iii, vi, vii, viii, ix, x, xi} {i, ii, iv, v, vi, vii, viii, ix, x} {i, ii, iv, v, vi, vii, viii, ix, xi}

{I, ii, iv, v, vi, vii, viii, x, xi} {i, ii, iv, v, vi, vii, ix, x, xi} {i, ii, iv, v, vi, viii , ix, x, xi} {i, ii, iv, v, vii, viii, ix, x, xi} {i, ii, iv, vi, vii, viii, ix, x, xi} {i, ii, v, vi, vii, viii, ix, x, xi} {i, iii, iv, v, vi, vii, viii, ix, x}

{I, iii, iv, v, vi, vii, viii, ix, xi} {i, iii, iv, v, vi, vii, viii, x, xi} {i, iii, iv, v, vi, vii , ix, x, xi}

{I, iii, iv, v, vi, viii, ix, x, xi} {i, iii, iv, v, vii, viii, ix, x, xi} {i, iii, iv, vi, vii, viii , ix, x, xi}

{I, iii, v, vi, vii, viii, ix, x, xi} {i, iv, v, vi, vii, viii, ix, x, xi} {ii, iii, iv, v, vi, vii , viii, ix, x}

{Ii, iii, iv, v, vi, vii, viii, ix, xi} {ii, iii, iv, v, vi, vii, viii, x, xi} {ii, iii, iv, v, vi, vii , ix, x, xi}

{Ii, iii, iv, v, vi, viii, ix, x, xi} {ii, iii, iv, v, vii, viii, ix, x, xi} {ii, iii, iv, vi, vii, viii , ix, x, xi}

{Ii, iii, v, vi, vii, viii, ix, x, xi} {ii, iv, v, vi, vii, viii, ix, x, xi} {iii, iv, v, vi, vii, viii , ix, x, xi}

{I, ii, iii, iv, v, vi, vii, viii, ix, x} {i, ii, iii, iv, v, vi, vii, viii, ix, xi} {i, ii, iii, iv , v, vi, vii, viii, x, xi} {i, ii, iii, iv, v, vi, vii, ix, x, xi} {i, ii, iii, iv, v, vi, viii, ix , x, xi} {i, ii, iii, iv, v, vii, viii, ix, x, xi}

{I, ii, iii, iv, vi, vii, viii, ix, x, xi} {i, ii, iii, v, vi, vii, viii, ix, x, xi} {i, ii, iv, v , vi, vii, viii, ix, x, xi}

{I, iii, iv, v, vi, vii, viii, ix, x, xi} {ii, iii, iv, v, vi, vii, viii, ix, x, xi} or {i, ii, iii, iv, v, vi, vii, viii, ix, x, xi}.

If the variant comprises any one of (i) and (iii) to (xi), it may further comprise a mutation at one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

In (i), the variant may comprises mutations at any number and combination of N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192. In (i), the variant preferably comprises one or more mutations at at the following positions (i.e. mutations at one or more of the following positions) N40, D43, E44, S54, S57, Q62, E101, E131 and T150. In (i), the variant preferably comprises one or more mutations at the following positions (i.e. mutations at one or more of the following positions) N40, D43, E44, E101 and E131. In (i), the variant preferably comprises a mutation at S54 and/or S57. In (i), the variant more preferably comprises a mutation at (a) S54 and/or S57 and (b) one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56. If S54 and/or S57 are deleted in (xi), it/they cannot be mutated in (i) and vice versa. In (i), the variant preferably comprises a mutation at T150, such as T150I. Alternatively the variant preferably comprises a mutation at (a) T150 and (b) one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56. In (i), the variant preferably comprises a mutation at Q62, such as Q62R or Q62K. Alternatively the variant preferably comprises a mutation at (a) Q62 and (b) one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56. The variant may comprise a mutation at D43, E44, Q62 or any combination thereof, such as D43, E44, Q62, D43/E44, D43/Q62, E44/Q62 or D43/E44/Q62. Alternatively the variant preferably comprises a mutation at (a) D43, E44, Q62, D43/E44,

D43/Q62, E44/Q62 or D43/E44/Q62 and (b) one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

In (ii) and elsewhere in this application where different positions are separated by the / symbol, the / symbol means "and" such that Y51/N55 is Y51 and N55. In (ii), the variant preferably comprises mutations at Y51/N55. It has been proposed that the constriction in CsgG is composed of three stacked concentric rings formed by the side chains of residues Y51, N55 and F56 (Goyal et al, 2014, Nature, 516, 250-253). Mutation of these residues in (ii) may therefore decrease the number of nucleotides contributing to the current as the polynucleotide moves through the pore and thereby make it easier to identify a direct relationship between the observed current (as the polynucleotide moves through the pore) and the polynucleotide. F56

may be mutated in any of the ways discussed below with reference to variants and pores useful in the method of the invention.

In (v), the variant may comprise N102R, N102F, N102Y or N102W. The variant preferably comprises (a) N102R, N102F, N102Y or N102W and (b) a mutation at one or more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

WE CLAIM

1. A mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 2 which comprises R192D/Q/F/S/T.

2. A mutant CsgG monomer according to claim 1, wherein the variant comprises R192D.

3. A mutant CsgG monomer according to claim 1 or 2, wherein the variant comprises

- (a) one or more mutations at the following positions (i.e. mutations at one or more of the following positions) 141, R93, A98, Q100, G103, T104, A106, 1107, N108, LI 13, SI 15,

Tl 17, Y130, K135, E170, S208, D233, D238 and E244 and /or (b) one or more of D43S, E44S, F48S/N/Q/Y/W/I/V/H/R/K, Q87N/R/K, N91K/R, K94R/F/Y/W/L/S/N, R97F/Y/W/V/I/K/S/Q/H, E101I/L/A/H, N102K/Q/L/I/V/S/H, R110F/G/N, Q114R/K, R142Q/S, T150Y/A/V/L/S/Q/N and D248S/N/Q/K/R;

- (A) deletion of one or more positions F193, 1194, D195, Y196, Q197, R198, LI 99, L200 and E201 and /or (B) deletion of one or more of

V139/G140/D149/T150/V186/Q187/V204/G205,

G137/G138/Q151/Y152/Y184/E185/Y206/T207 and

A141/R142/G147/A148/A188/G189/G202/E203; and/or

- one or more of the following: (i) one or more mutations at the following positions N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142 and T150; (ii) mutations at Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56; (iii) Q42R or Q42K; (iv) K49R or K49Q; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; (xi) deletion of one or more of the following positions F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57; (xii) one or more mutations at the following positions L90, N91, 195, A99, Ql 14; and (xiii) R97W; R93W; R93Y and R97Y; F191T; deletion of V105, A106 and 1107; and/or deletion of F193, 1194, D195, Y196, Q197, R198 and L199 or deletion of D195, Y196, Q197, R198 and L199.

4. A mutant monomer according to any one of claims 1 to 3, wherein the the variant comprises a mutation at Y51 and/or Y56.

5. A mutant monomer according to claim 4, wherein the mutation at Y51 is Y51 A and/or the mutation at Y56 is F56Q.

6. A mutant monomer according to any one of the preceding claims, wherein

(a) the variant comprises one or more of the following substitutions N40R, N40K, D43N, D43Q, D43R, D43K, E44N, E44Q, E44R, E44K, S54P, S57P, Q62R, Q62K, R97N, R97G, R97L, E101N, E101Q, E101R, E101K, E101F, E101Y, E101W, E101T, E124N, E124Q, E124R, E124K, E124F, E124Y, E124W, E131D, R142E, R142N and T150I;

(b) the variant comprises F56N/N55Q, F56N/N55R, F56N/N55K, F56N/N55S,

F56N/N55G, F56N/N55A, F56N/N55T, F56Q/N55Q, F56Q/N55R, F56Q /N55K, F56Q/N55S, F56Q/N55G, F56Q/N55A, F56Q/N55T, F56R/N55Q, F56R/N55R, F56R/N55K, F56R/N55S, F56R/N55G, F56R/N55A, F56R/N55T, F56S/N55Q, F56S/N55R, F56S/N55K, F56S/N55S, F56S/N55G, F56S/N55A, F56S/N55T, F56G/N55Q, F56G/N55R, F56G/N55K, F56G/N55S, F56G/N55G, F56G/N55A, F56G/N55T, F56A/N55Q, F56A/N55R, F56A/N55K, F56A/N55S, F56A/N55G, F56A/N55A, F56A/N55T, F56K/N55Q, F56K/N55R,F56K/N55K, F56K/N55S, F56K/N55G, F56K/N55A, F56K/N55T, F56N/Y51L, F56N/Y51V, F56N/Y51A, F56N/Y51N, F56N/Y51Q, F56N/Y51 S, F56N/Y51G, F56Q/Y51L, F56Q/Y51V, F56Q/Y51A, F56Q/Y51N, F56Q/Y51Q, F56Q/Y51 S, F56Q/Y51G, F56R/Y51L, F56R/Y51V, F56R/Y51A, F56R/Y51N, F56R/Y51Q, F56R/Y51 S, F56R/Y51G, F56S/Y51L, F56S/Y51V, F56S/Y51A, F56S/Y51N, F56S/Y51Q, F56S/Y51 S, F56S/Y51G, F56G/Y51L, F56G/Y51V, F56G/Y51A, F56G/Y51N, F56G/Y51Q, F56G/Y51 S, F56G/Y51G, F56A/Y51L, F56A/Y51V, F56A/Y51A, F56A/Y51N, F56A/Y51Q, F56A/Y51 S, F56A/Y51G, F56K/Y51L, F56K/Y51V, F56K/Y51A, F56K/Y51N, F56K/Y51Q, F56K/Y51 S, F56K/Y51G, N55Q/Y51L, N55Q/Y51V, N55Q/Y51A, N55Q/Y51N, N55Q/Y51Q, N55Q/Y51 S, N55Q/Y51G, N55R/Y51L, N55R/Y51V, N55R/Y51A, N55R/Y51N, N55R/Y51Q, N55R/Y51 S, N55R/Y51G, N55K/Y51L, N55K/Y51V, N55K/Y51A, N55K/Y51N, N55K/Y51Q, N55K/Y51 S, N55K/Y51G, N55S/Y51L, N55S/Y51V, N55S/Y51A, N55S/Y51N, N55S/Y51Q, N55S/Y51 S, N55S/Y51G, N55G/Y51L, N55G/Y51V, N55G/Y51A, N55G/Y51N, N55G/Y51Q, N55G/Y51 S, N55G/Y51G, N55A/Y51L, N55A/Y51V, N55A/Y51A,

N55A/Y51N, N55A/Y51Q, N55A/Y51 S, N55A/Y51G, N55T/Y51L, N55T/Y51V, N55T/Y51A, N55T/Y51N, N55T/Y51Q, N55T/Y51 S, N55T/Y51G, F56N/N55Q/Y51L, F56N/N55Q/Y51V, F56N/N55Q/Y51A, F56N/N55Q/Y51N, F56N/N55Q/Y51Q, F56N/N55Q/Y51 S,

F56N/N55Q/Y51G, F56N/N55R/Y51L, F56N/N55R/Y51V, F56N/N55R/Y51A,

F56N/N55R/Y51N, F56N/N55R/Y51Q, F56N/N55R/Y51 S, F56N/N55R/Y51G,

F56N/N55K/Y51L, F56N/N55K/Y51V, F56N/N55K/Y51A, F56N/N55K/Y51N,

F56N/N55K/Y51Q, F56N/N55K/Y51 S, F56N/N55K/Y51G, F56N/N55S/Y51L,

F56N/N55S/Y51V, F56N/N55S/Y51A, F56N/N55S/Y51N, F56N/N55S/Y51Q, F56N/N55S/Y51 S, F56N/N55S/Y51G, F56N/N55G/Y51L, F56N/N55G/Y51V,

F56N/N55G/Y51A, F56N/N55G/Y51N, F56N/N55G/Y51Q, F56N/N55G/Y51 S,

F56N/N55G/Y51G, F56N/N55A/Y51L, F56N/N55A/Y51V, F56N/N55A/Y51A,

F56N/N55A/Y51N, F56N/N55A/Y51Q, F56N/N55A/Y51 S, F56N/N55A/Y51G,

F56N/N55T/Y51L, F56N/N55T/Y51V, F56N/N55T/Y51A, F56N/N55T/Y51N,

F56N/N55T/Y51Q, F56N/N55T/Y51 S, F56N/N55T/Y51G, F56Q/N55Q/Y51L,

F56Q/N55Q/Y51V, F56Q/N55Q/Y51A, F56Q/N55Q/Y51N, F56Q/N55Q/Y51Q,

F56Q/N55Q/Y51 S, F56Q/N55Q/Y51G, F56Q/N55R/Y51L, F56Q/N55R/Y51V,

F56Q/N55R/Y51A, F56Q/N55R/Y51N, F56Q/N55R/Y51Q, F56Q/N55R/Y51 S,

F56Q/N55R/Y51G, F56Q/N55K/Y51L, F56Q /N55K/Y51V, F56Q/N55K/Y51A, F56Q

/N55K/Y51N, F56Q/N55K/Y51Q, F56Q /N55K/Y51 S, F56Q /N55K/Y51G, F56Q/N55S/Y51L, F56Q/N55S/Y51V, F56Q/N55S/Y51A, F56Q/N55S/Y51N, F56Q/N55S/Y51Q,

F56Q/N55S/Y51 S, F56Q/N55S/Y51G, F56Q/N55G/Y51L, F56Q/N55G/Y51V,

F56Q/N55G/Y51A, F56Q/N55G/Y51N, F56Q/N55G/Y51Q, F56Q/N55G/Y51 S,

F56Q/N55G/Y51G, F56Q/N55A/Y51L, F56Q/N55A/Y51V, F56Q/N55A/Y51A,

F56Q/N55A/Y51N, F56Q/N55A/Y51Q, F56Q/N55A/Y51 S, F56Q/N55A/Y51G,

F56Q/N55T/Y51L, F56Q/N55T/Y51V, F56Q/N55T/Y51A, F56Q/N55T/Y51N,

F56Q/N55T/Y51Q, F56Q/N55T/Y51 S, F56Q/N55T/Y51G, F56R/N55Q/Y51L,

F56R/N55Q/Y51V, F56R/N55Q/Y51A, F56R/N55Q/Y51N, F56R/N55Q/Y51Q,

F56R/N55Q/Y51 S, F56R/N55Q/Y51G, F56R/N55R/Y51L, F56R/N55R/Y51V,

F56R/N55R/Y51A, F56R/N55R/Y51N, F56R/N55R/Y51Q, F56R/N55R/Y51 S,

F56R/N55R/Y51G, F56R/N55K/Y51L, F56R/N55K/Y51V, F56R/N55K/Y51A,

F56R/N55K/Y51N, F56R/N55K/Y51Q, F56R/N55K/Y51 S, F56R/N55K/Y51G,

F56R/N55S/Y51L, F56R/N55S/Y51V, F56R/N55S/Y51A, F56R/N55S/Y51N,

F56R/N55S/Y51Q, F56R/N55S/Y51 S, F56R/N55S/Y51G, F56R/N55G/Y51L,

F56R/N55G/Y51V, F56R/N55G/Y51A, F56R/N55G/Y51N, F56R/N55G/Y51Q,

F56R/N55G/Y51 S, F56R/N55G/Y51G, F56R/N55A/Y51L, F56R/N55A/Y51V,

F56R/N55A/Y51A, F56R/N55A/Y51N, F56R/N55A/Y51Q, F56R/N55A/Y51 S,

F56R/N55A/Y51G, F56R/N55T/Y51L, F56R/N55T/Y51V, F56R/N55T/Y51A,

F56R/N55T/Y51N, F56R/N55T/Y51Q, F56R/N55T/Y51 S, F56R/N55T/Y51G,

F56S/N55Q/Y51L, F56S/N55Q/Y51V, F56S/N55Q/Y51A, F56S/N55Q/Y51N,

F56S/N55Q/Y51Q, F56S/N55Q/Y51 S, F56S/N55Q/Y51G, F56S/N55R/Y51L,

F56S/N55R/Y51V, F56S/N55R/Y51A, F56S/N55R/Y51N, F56S/N55R/Y51Q,

F56S/N55R/Y51 S, F56S/N55R/Y51G, F56S/N55K/Y51L, F56S/N55K/Y51V,

F56S/N55K/Y51A, F56S/N55K/Y51N, F56S/N55K/Y51Q, F56S/N55K/Y51 S, F56S/N55K/Y51G, F56S/N55S/Y51L, F56S/N55S/Y51V, F56S/N55S/Y51A, F56S/N55S/Y51N, F56S/N55S/Y51Q, F56S/N55S/Y51 S, F56S/N55S/Y51G, F56S/N55G/Y51L, F56S/N55G/Y51V, F56S/N55G/Y51A, F56S/N55G/Y51N, F56S/N55G/Y51Q, F56S/N55G/Y51 S, F56S/N55G/Y51G, F56S/N55A/Y51L, F56S/N55A/Y51V, F56S/N55A/Y51A, F56S/N55A/Y51N, F56S/N55A/Y51Q, F56S/N55A/Y51 S, F56S/N55A/Y51G, F56S/N55T/Y51L, F56S/N55T/Y51V, F56S/N55T/Y51A, F56S/N55T/Y51N, F56S/N55T/Y51Q, F56S/N55T/Y51 S, F56S/N55T/Y51G, F56G/N55Q/Y51L, F56G/N55Q/Y51V, F56G/N55Q/Y51A, F56G/N55Q/Y51N, F56G/N55Q/Y51Q, F56G/N55Q/Y51 S, F56G/N55Q/Y51G, F56G/N55R/Y51L, F56G/N55R/Y51V, F56G/N55R/Y51A, F56G/N55R/Y51N, F56G/N55R/Y51Q, F56G/N55R/Y51 S, F56G/N55R/Y51G, F56G/N55K/Y51L, F56G/N55K/Y51V, F56G/N55K/Y51A, F56G/N55K/Y51N, F56G/N55K/Y51Q, F56G/N55K/Y51 S, F56G/N55K/Y51G, F56G/N55S/Y51L, F56G/N55S/Y51V, F56G/N55S/Y51A, F56G/N55S/Y51N, F56G/N55S/Y51Q, F56G/N55S/Y51 S, F56G/N55S/Y51G, F56G/N55G/Y51L, F56G/N55G/Y51V, F56G/N55G/Y51A, F56G/N55G/Y51N, F56G/N55G/Y51Q, F56G/N55G/Y51 S, F56G/N55G/Y51G, F56G/N55A/Y51L, F56G/N55A/Y51V, F56G/N55A/Y51A, F56G/N55A/Y51N, F56G/N55A/Y51Q, F56G/N55A/Y51 S, F56G/N55A/Y51G, F56G/N55T/Y51L, F56G/N55T/Y51V, F56G/N55T/Y51A, F56G/N55T/Y51N, F56G/N55T/Y51Q, F56G/N55T/Y51 S, F56G/N55T/Y51G, F56A/N55Q/Y51L, F56A/N55Q/Y51V, F56A/N55Q/Y51A, F56A/N55Q/Y51N, F56A/N55Q/Y51Q, F56A/N55Q/Y51 S, F56A/N55Q/Y51G, F56A/N55R/Y51L, F56A/N55R/Y51V, F56A/N55R/Y51A, F56A/N55R/Y51N, F56A/N55R/Y51Q, F56A/N55R/Y51 S, F56A/N55R/Y51G, F56A/N55K/Y51L, F56A/N55K/Y51V, F56A/N55K/Y51A, F56A/N55K/Y51N, F56A/N55K/Y51Q, F56A/N55K/Y51 S, F56A/N55K/Y51G, F56A/N55S/Y51L, F56A/N55S/Y51V, F56A/N55S/Y51A, F56A/N55S/Y51N, F56A/N55S/Y51Q, F56A/N55S/Y51 S, F56A/N55S/Y51G, F56A/N55G/Y51L, F56A/N55G/Y51V, F56A/N55G/Y51A, F56A/N55G/Y51N, F56A/N55G/Y51Q, F56A/N55G/Y51 S, F56A/N55G/Y51G, F56A/N55A/Y51L, F56A/N55A/Y51V, F56A/N55A/Y51A, F56A/N55A/Y51N, F56A/N55A/Y51Q, F56A/N55A/Y51 S, F56A/N55A/Y51G, F56A/N55T/Y51L, F56A/N55T/Y51V, F56A/N55T/Y51A, F56A/N55T/Y51N, F56A/N55T/Y51Q, F56A/N55T/Y51 S, F56A/N55T/Y51G, F56K/N55Q/Y51L, F56K/N55Q/Y51V, F56K/N55Q/Y51A, F56K/N55Q/Y51N, F56K/N55Q/Y51Q, F56K/N55Q/Y51 S, F56K/N55Q/Y51G, F56K/N55R/Y51L, F56K/N55R/Y51V, F56K/N55R/Y51A, F56K/N55R/Y51N, F56K/N55R/Y51Q, F56K/N55R/Y51 S, F56K/N55R/Y51G, F56K/N55K/Y51L, F56K/N55K/Y51V, F56K/N55K/Y51A,

F56K/N55K/Y51N, F56K/N55K/Y51Q, F56K/N55K/Y51 S, F56K/N55K/Y51G,

F56K/N55S/Y51L, F56K/N55S/Y51V, F56K/N55S/Y51A, F56K/N55S/Y51N,

F56K/N55S/Y51Q, F56K/N55S/Y51 S, F56K/N55S/Y51G, F56K/N55G/Y51L,

F56K/N55G/Y51V, F56K/N55G/Y51A, F56K/N55G/Y51N, F56K/N55G/Y51Q,

F56K/N55G/Y51 S, F56K/N55G/Y51G, F56K/N55A/Y51L, F56K/N55A/Y51V,

F56K/N55A/Y51A, F56K/N55A/Y51N, F56K/N55A/Y51Q, F56K/N55A/Y51 S,

F56K/N55A/Y51G, F56K/N55T/Y51L, F56K/N55T/Y51V, F56K/N55T/Y51A,

F56K/N55T/Y51N, F56K/N55T/Y51Q,F56K/N55T/Y51 S, F56K/N55T/Y51G, F56E/N55R, F56E/N55K, F56D/N55R, F56D/N55K, F56R/N55E, F56R/N55D, F56K/N55E or F56K/N55D; and/or

(c) the variant comprises deletion of Y51/P52, Y51/P52/A53, P50 to P52, P50 to A53, K49 to Y51, K49 to A53 and replacement with a single proline (P), K49 to S54 and replacement with a single P, Y51 to A53, Y51 to S54, N55/F56, N55 to S57, N55/F56 and replacement with a single P, N55/F56 and replacement with a single glycine (G), N55/F56 and replacement with a single alanine (A), N55/F56 and replacement with a single P and Y51N, N55/F56 and replacement with a single P and Y51Q, N55/F56 and replacement with a single P and Y51 S, N55/F56 and replacement with a single G and Y51N, N55/F56 and replacement with a single G and Y51Q, N55/F56 and replacement with a single G and Y51 S, N55/F56 and replacement with a single A and Y51N, N55/F56 and replacement with a single A/Y51Q or N55/F56 and replacement with a single A and Y51 S.

7. A mutant according to any one of the preceding claims, wherein the variant comprises D195N/E203N, D195Q/E203N, D195N/E203Q, D195Q/E203Q, E201N/E203N, E201Q/E203N, E201N/E203Q, E201Q/E203Q, E185N/E203Q, E185Q/E203Q, E185N/E203N, E185Q/E203N, D195N/E201N/E203N, D195Q/E201N/E203N, D195N/E201Q/E203N, D195N/E201N/E203Q, D195Q/E201Q/E203N, D195Q/E201N/E203Q, D195N/E201Q/E203Q, D195Q/E201Q/E203Q, D149N/E201N, D149Q/E201N, D149N/E201Q, D149Q/E201Q, D149N/E201N/D195N, D149Q/E201N/D195N, D149N/E201Q/D195N, D149N/E201N/D195Q,

D149Q/E201Q/D195N, D149Q/E201N/D195Q, D149N/E201Q/D195Q,

D149Q/E201Q/D195Q, D149N/E203N, D149Q/E203N, D149N/E203Q, D149Q/E203Q, D149N/E185N/E201N, D149Q/E185N/E201N, D149N/E185Q/E201N, D149N/E185N/E201Q, D149Q/E185Q/E201N, D149Q/E185N/E201Q, D149N/E185Q/E201Q, D149Q/E185Q/E201Q, D149N/E185N/E203N, D149Q/E185N/E203N, D149N/E185Q/E203N, D149N/E185N/E203Q,

D149Q/E185Q/E203N, D149Q/E185N/E203Q, D149N/E185Q/E203Q, D149Q/E185Q/E203Q,

D 149N/E185N/E201N/E203N, D 149Q/E185N/E201N/E203N, D 149N/E185Q/E201N/E203N,

D149N/E185N/E201Q/E203N, D149N/E185N/E201N/E203Q, D149Q/E185Q/E201N/E203N,

D149Q/E185N/E201Q/E203N, D149Q/E185N/E201N/E203Q, D149N/E185Q/E201Q/E203N,

D149N/E185Q/E201N/E203Q, D149N/E185N/E201Q/E203Q, D149Q/E185Q/E201Q/E203Q,

D149Q/E185Q/E201N/E203Q, D149Q/E185N/E201Q/E203Q, D149N/E185Q/E201Q/E203Q,

D 149Q/E 185Q/E201 Q/E203N, D 149N/E185N/D 195N/E201N/E203N,

D 149Q/E 185N/D 195N/E201N/E203N, D 149N/E185Q/D 195N/E201N/E203N,

D 149N/E 185N/D 195Q/E201N/E203N, D 149N/E185N/D 195N/E201 Q/E203N,

D 149N/E 185N/D 195N/E201N/E203 Q, D 149Q/E185Q/D 195N/E201N/E203N,

D 149Q/E 185N/D 195Q/E201N/E203N, D 149Q/E185N/D 195N/E201 Q/E203N,

D149Q/E185N/D195N/E201N/E203Q, D149N/E185Q/D195Q/E201N/E203N,

D 149N/E185Q/D 195N/E201 Q/E203N, D 149N/E185Q/D 195N/E201N/E203Q,

D 149N/E185N/D 195Q/E201 Q/E203N, D 149N/E185N/D 195Q/E201N/E203Q,

D 149N/E185N/D 195N/E201 Q/E203Q, D 149Q/E185Q/D 195Q/E201N/E203N,

D 149Q/E185Q/D 195N/E201 Q/E203N, D 149Q/E185Q/D 195N/E201N/E203Q,

D 149Q/E185N/D 195Q/E201 Q/E203N, D 149Q/E185N/D 195Q/E201N/E203Q,

D 149Q/E185N/D 195N/E201 Q/E203Q, D 149N/E185Q/D 195Q/E201 Q/E203N,

D149N/E185Q/D195Q/E201N/E203Q, D149N/E185Q/D195N/E201Q/E203Q,

D149N/E185N/D195Q/E201Q/E203Q, D149Q/E185Q/D195Q/E201Q/E203N,

D149Q/E185Q/D195Q/E201N/E203Q, D149Q/E185Q/D195N/E201Q/E203Q,

D149Q/E185N/D195Q/E201Q/E203Q, D149N/E185Q/D195Q/E201Q/E203Q,

D 149Q/E185Q/D 195Q/E201 Q/E203Q, D 149N/E185R/E201N/E203N,

D149Q/E185R/E201N/E203N, D149N/E185R/ E201Q/E203N, D149N/E185R/ E201N/E203Q, D149Q/E185R/ E201Q/E203N, D149Q/E185R/ E201N/E203Q, D149N/E185R/ E201Q/E203Q, D149Q/E185R/ E201Q/E203Q, D149R/E185N/ E201N/E203N, D149R/E185Q/ E201N/E203N, D149R/E185N/ E201Q/E203N, D149R/E185N/ E201N/E203Q, D149R/E185Q/ E201Q/E203N, D149R/E185Q/ E201N/E203Q, D149R/E185N/ E201Q/E203Q, D149R/E185Q/ E201Q/E203Q, D 149R/E 185N/D 195N/E201N/E203N, D 149R/E 185Q/D 195N/E201N/E203N,

D 149R/E 185N/D 195 Q/E201N/E203N, D 149R/E 185N/D 195N/E201 Q/E203N,

D 149R/E185Q/D 195N/E201N/E203Q, D 149R/E185Q/D 195Q/E201N/E203N,

D 149R/E 185 Q/D 195N/E201 Q/E203N, D 149R/E 185 Q/D 195N/E201N/E203 Q,

D 149R/E 185N/D 195 Q/E201 Q/E203N, D 149R/E 185N/D 195 Q/E201N/E203 Q,

D 149R/E 185N/D 195N/E201 Q/E203 Q, D 149R/E 185 Q/D 195 Q/E201 Q/E203N,

D 149R/E185Q/D 195Q/E201N/E203Q, D 149R/E185Q/D 195N/E201 Q/E203Q,

D149R/E185N/D195Q/E201Q/E203Q, D149R/E185Q/D195Q/E201Q/E203Q,

D 149N/E 185R/D 195N/E201N/E203N, D 149Q/E185R/D 195N/E201N/E203N,

D 149N/E 185R/D 195Q/E201N/E203N, D 149N/E185R/D 195N/E201 Q/E203N,

D 149N/E 185R/D 195N/E201N/E203 Q, D149Q/E185R/D195 Q/E201N/E203N,

D 149Q/E 185R/D 195N/E201 Q/E203N, D 149Q/E 185R/D 195N/E201N/E203 Q,

D149N/E185R/D195Q/E201Q/E203N, D149N/E185R/D195Q/E201N/E203Q,

D 149N/E 185R/D 195N/E201 Q/E203 Q, D149Q/E185R/D195 Q/E201 Q/E203N,

D 149Q/E 185R/D 195 Q/E201N/E203 Q, D149Q/E185R/D195N/E201 Q/E203 Q,

D149N/E185R/D195Q/E201Q/E203Q, D149Q/E185R/D195Q/E201Q/E203Q,

D 149N/E 185R/D 195N/E201 R/E203N, D 149Q/E 185R/D 195N/E201 R/E203N,

D149N/E185R/D195Q/E201R/E203N, D149N/E185R/D195N/E201R/E203Q,

D149Q/E185R/D195Q/E201R/E203N, D149Q/E185R/D195N/E201R/E203Q,

D149N/E185R/D195Q/E201R/E203Q, D149Q/E185R/D195Q/E201R/E203Q, E131D/K49R, E101N/N102F, E101N/N102Y, E101N/N102W, E101F/N102F, E101F/N102Y, E101F/N102W, E101Y/N102F, E101Y/N102Y, E101Y/N102W, E101W/N102F, E101W/N102Y,

E101W/N102W, E101N/N102R, E101F/N102R, E101 Y/N102R or E101W/N102F.

8. A mutant monomer according to any one of the preceding claims wherein the variant comprises a mutation at T150.

9. A construct comprising two or more covalently attached CsgG monomers, wherein at least one of the monomers is a mutant monomer according to any one of the preceding claims.

10. A construct according to claim 9, wherein the two or more mutant monomers are the same or different.

11. A construct according to claim 9 or 10, wherein the two or more mutant monomers are genetically fused.

12. A construct according to any one of claims 9 to 11, wherein the two or more mutant monomers are attached via one or more linkers.

13. A construct according to any one of claims 9 to 12, wherein the construct comprises two mutant monomers according to any one of claims 1 to 8.

14. A polynucleotide which encodes a mutant monomer according to any one of claims 1 to 8 or a construct according to claim 11.

15. A homo-oligomeric pore derived from CsgG comprising identical mutant monomers according to any one of claims 1 to 8 or identical constructs according to any one of claims 9 to 13.

16. A homo-oligomeric pore according to claim 15, wherein the pore comprises nine identical mutant monomers according to any one of claims 1 to 8.

17. A hetero-oligomeric pore derived from CsgG comprising at least one mutant monomer according to any one of claims 1 to 8 or at least one construct according any one of claims 9 to 13.

18. A hetero-oligomeric pore according to claim 17, wherein the pore comprises (a) nine mutant monomers according to any one of claims 1 to 8 and wherein at least one of them differs from the others or (b) one or more mutant monomers according to any one of claims 1 to 8 and sufficient additional monomers comprising SEQ ID NO: 2.

19. A method for determining the presence, absence or one or more characteristics of a target analyte, comprising:

(a) contacting the target analyte with a pore according to any one of claims 15 to 18 such that the target analyte moves with respect to the pore; and

(b) taking one or more measurements as the analyte moves with respect to the pore and thereby determining the presence, absence or one or more characteristics of the analyte.

20 A method according to claim 19, wherein the target analyte is a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an

oligonucleotide, a polynucleotide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive or an environmental pollutant.

21. A method according to claim 20, wherein the target analyte is a target polynucleotide.

22. A method according to claim 21, wherein the method is for characterising a target polynucleotide and the method comprises:

a) contacting the polynucleotide with the pore such that the polynucleotide moves with respect to the pore; and

b) taking one or more measurements as the polynucleotide moves with respect to the pore, wherein the measurements are indicative of one or more characteristics of the

polynucleotide, and thereby characterising the target polynucleotide.

23. A method according to claim 22, wherein the one or more characteristics are selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified.

24. A method according to claim 22 or 23, wherein the one or more characteristics of the polynucleotide are measured by electrical measurement and/or optical measurement.

25. A method according to claim 24, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.

26. A method according to any one of claims 22 to 25, wherein step a) further comprises contacting the polynucleotide with a polynucleotide binding protein such that the protein controls the movement of the polynucleotide through the pore.

27. A method according to claim 26, wherein the method comprises:

a) contacting the polynucleotide with the pore and the polynucleotide binding protein such that the protein controls the movement of the polynucleotide with respect to the pore; and b) measuring the current passing through the pore as the polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the polynucleotide and thereby characterising the target polynucleotide.

28. A method according to claim 26 or 27, wherein the polynucleotide binding protein is a helicase or is derived from a helicase.

29. A method according to claim 21, wherein the the method is for characterising a target polynucleotide and the method comprises:

a) contacting the polynucleotide with a pore according to any one of claims 15 to 18 and an exonucleoase such that the exonuclease digests individual nucleotides from one end of the target polynucleotide and the individual nucleotides move with respect to the pore; and

b) taking one or more measurements as the individual nucleotides move with respect to the pore, wherein the measurements are indicative of one or more characteristics of the individual nucleotides, and thereby characterising the target polynucleotide.

30. A method according to any one of claims 16 to 29, wherein the pore is in a membrane.

31. A method according to claim 30, wherein membrane is an amphiphilic layer or comprises a solid state layer.

32. A method according to claim 20 or 31, wherein the target analyte is coupled to the membrane before it is contacted with the pore.

33. A method according to any one of claims 30 to 32, wherein the target analyte is attached to a microparticle which delivers the analyte towards the membrane.

34. A method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore according to any one of claims 15 to 18 and a polynucleotide binding protein and thereby forming a sensor for characterising the target polynucleotide.

35. A method according to claim 34, wherein the complex is formed by (a) contacting the pore and the polynucleotide binding protein in the presence of the target polynucleotide and (a) applying a potential across the pore.

36. A method according to claim 35, wherein the potential is a voltage potential or a chemical potential.

37. A method according to claim 34, wherein the complex is formed by covalently attaching the pore to the protein.

38. A sensor for characterising a target polynucleotide, comprising a complex between a pore according to any one of claims 15 to 18 and a polynucleotide binding protein.

39. Use of a pore according to any one of claims 15 to 18 to determine the presence, absence or one or more characteristics of a target analyte.

40. A kit for characterising a target analyte comprising (a) a pore according to any one of claims 15 to 18 and (b) the components of a membrane.

41. An apparatus for characterising target analytes in a sample, comprising (a) a plurality of pores according to any one of claims 15 to 18 and (b) a plurality of membranes.

42. An apparatus according to claim 41, wherein the apparatus comprises:

a sensor device that is capable of supporting the plurality of pores and membranes being operable to perform analyte characterisation using the pores and membranes; and

at least one port for delivery of the material for performing the characterisation.

43. An apparatus according to claim 42, wherein the apparatus comprises:

a sensor device that is capable of supporting the plurality of pores and membranes being operable to perform analyte characterisation using the pores and membranes; and

at least one reservoir for holding material for performing the characterisation.

44. An apparatus according to claim 42 or 43, wherein the apparatus further comprises:

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.

45. A method of characterising a target polynucleotide, comprising:

a) contacting the polynucleotide with a pore according to any one of claims 15 to 18, a polymerase and labelled nucleotides such that phosphate labelled species are sequentially added to the target polynucleotide by the polymerase, wherein the phosphate species contain a label specific for each nucleotide; and

b) detecting the phosphate labelled species using the pore and thereby characterising the polynucleotide.

46. A method of producing a mutant monomer according to any one of claims 1 to 8 or a construct according to claim 11, comprising expressing a polynucleotide according to claim 14 in a suitable host cell and thereby producing a mutant monomer according to any one of claims 1 to 8 or a construct according to claim 11.