Novel hole cellular and hole

Abstract

The present invention relates to novel Puncture protein monomers, Puncture protein pores formed from said monomers and the use of said Puncture protein pores in analyte detection and characterization.

Description

TECHNICAL FIELD

[0001] The present invention relates to novel sticholysin monomers, sticholysin pores formed from the monomers and the use of the sticholysin pores in the detection and characterization of analytes. BACKGROUND

[0002] Nanopore sensing is a method of analyte detection and characterization that relies on the observation of individual binding or interaction events between an analyte molecule and an ion-conducting channel. Nanopore sensors can be created by placing a nanometer-sized single pore in an electrically insulating membrane and measuring the voltage-driven ionic current through the pore in the presence of an analyte molecule. The presence of an analyte within or near the nanopore will change the ionic flow through the pore, causing a change in the ion or current measured across the channel. The identity of the analyte is revealed by its unique current signature, particularly the duration and extent of the current block and changes in the current level during its time of interaction with the pore. Analytes can be small organic and inorganic molecules as well as various biological or synthetic macromolecules and polymers, including polynucleotides, polypeptides and polysaccharides. Nanopore sensing can reveal identity and count the sensed analyte at the single molecule level, but can also provide information about the composition of the analyte, such as nucleotide, amino acid or glycan sequence, and about the presence of base, amino acid or glycan modifications, such as methylation and acylation, phosphorylation, hydroxylation, oxidation, reduction, glycosylation, decarboxylation, deamination, etc. Nanopore sensing has the potential to allow fast and inexpensive polynucleotide sequencing, providing single molecule sequence reads of polynucleotides of tens to tens of thousands of bases in length.

[0003] Two of the basic components for polymer characterization using nanopore sensing are (1) controlling the movement of the polymer through the pore, and (2) discriminating between the building blocks of the polymer as it moves through the pore. During nanopore sensing, the narrowest part of the pore forms a constriction, which is the most discriminative part of the nanopore current signature as a function of the passing analyte. New nanopores are needed for the characterization of analytes.

[0004] Orbicella faveolata, or commonly known as mountain star coral, contains a sticholysin pore protein of unknown sequence. SUMMARY

[0005] The present invention relates to modified sticholysin monomers and sticholysin pores comprising the monomers. The sticholysin monomers and sticholysin pores comprising them are modified to facilitate the characterization of target analytes, especially target proteins and polypeptides, and to facilitate insertion into artificial membranes, as well as to facilitate oligomerization.

[0006] The present inventors have identified the sequence of the monomer of the cnidarin from Echinogorgia marmora. This is shown in SEQ ID NO: 1 (without signal peptide). The present inventors have also surprisingly shown that an N-terminal deletion is necessary for the insertion of the cnidarin monomer into an artificial membrane and for the insertion of a functional cnidarin pore into an artificial membrane. The present inventors have also surprisingly shown that specific substitutions in the sequence of the cnidarin monomer, in particular D203R (using the residue numbering in SEQ ID NO: 1), affect the number of monomers that form a cnidarin pore in an artificial membrane. The number of such monomers that form a cnidarin pore can be further increased by a C-terminal deletion. Finally, the present inventors have surprisingly shown that the modified cnidarin monomers of the present invention can be used to form a cnidarin pore that is capable of characterizing an analyte, in particular a protein or polypeptide. Accordingly, the present invention provides a cnidarin monomer comprising a variant of SEQ ID NO: 1 that is at least about 60% identical to the sequence of SEQ ID NO: 1 over its entire length. The present invention also provides a cnidarin monomer comprising a variant of SEQ ID NO: 2 that is at least about 54% identical to the sequence of SEQ ID NO: 2 over its entire length.

[0007] The present invention also provides:

[0008] a construct comprising two or more covalently attached cnidarin monomers of the present invention,

[0009] a polynucleotide encoding a cnidarin monomer of the present invention or a construct of the present invention,

[0010] a cnidarin pore comprising at least one cnidarin monomer of the present invention or at least one construct of the present invention,

[0011] an artificial membrane comprising a cnidarin pore derived from Echinogorgia marmora,

[0012] a method of determining the presence, absence or one or more characteristics of a target analyte comprising (a) contacting the target analyte with a cnidarin pore of the present invention or a membrane of the present invention, and (b) taking one or more measurements when the target analyte moves relative to the cnidarin pore, and thereby determining the presence, absence or one or more characteristics of the target analyte,

[0013] the use of a cnidarin pore of the present invention or a membrane of the present invention for determining the presence, absence or one or more characteristics of a target analyte,

[0014] - A kit for characterizing a target analyte comprising (a) a spicule porin monomer of the invention, a construct of the invention or a spicule porin pore of the invention, and (b) a component of a membrane and / or an analyte binding protein,

[0015] - An apparatus for characterizing a target analyte in a sample comprising (a) a plurality of spicule porin monomers of the invention, a plurality of constructs of the invention or a plurality of spicule porin pores of the invention, and (b) a plurality of analyte binding proteins,

[0016] - An array comprising a plurality of membranes of the invention,

[0017] - A system comprising (a) a membrane of the invention or an array of the invention, (b) a device for applying an electric potential across the membrane, and (c) a device for detecting an electrical or optical signal across the membrane,

[0018] - An apparatus produced by a method comprising the steps of (a) obtaining a spicule porin pore derived from Montipora gaudiosa, and (b) and contacting the spicule porin pore with an in vitro membrane such that the spicule porin pore is inserted into the in vitro membrane, and

[0019] - An apparatus comprising a spicule porin pore derived from Montipora gaudiosa inserted into an in vitro membrane. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : N-terminal deletions (gel). SDS page of wt Fav monomer (theoretical MW 28 kDa) and N-terminal deletion mutants thereof. Soluble monomers were expressed in E. coli, with an N-terminal HisTag (removed by TEV protease), and purified by affinity chromatography.

[0021] Figure 2 : N-terminal deletions (electrophysical measurements). Current (I) versus voltage (V) curves (I / V) obtained after insertion of Fav N-terminal deletion mutants into MinION membranes and application of an alternating voltage from 0 to ± 120 mV.

[0022] Figure 3 : Transition from 8-mer to 9-mer (gel). Non-denaturing PAGE of Fav pore AN75 Fav, AN75 D203R Fav, AN75 D215N Fav, AN76 D203R Fav, AN75 D203R D215N Fav and RN-Fav obtained by incubation with 50 mM DOPC:SM (molar ratio 1 : 1) large unilamellar vesicles (LUVs). LUVs were solubilized with Triton X-100 prior to loading.

[0023] Figure 4: 8- to 9-mer transition (Cryo-EM 2D classes). 2D classes obtained after analyzing Cryo-EM micrographs of detergent solubilized wtFav and RN-Fav pores revealed the presence of a major octamer pore for wtFav and octamer and nonamer pores for RN-Fav pores (ratio 1 : 1).

[0024] Figure 5 : 8- to 9-mer transition (structure). 3D volumes of RN-Fav octamer and nonamer pores (top and side view). Each protomer in the pore is colored differently.

[0025] Figure 6 : Shift in octamer / nonamer ratio towards nonamers. Non-reducing PAGE of RN-Fav and AC4-RN Fav pores obtained by incubation of the corresponding monomers with 50 mM DOPC:SM (molar ratio 1 : 1) LUV and solubilization of the LUV with Triton X-100. Arrows indicate the bands corresponding to octamer and nonamer pores. Data show that modification of the C-terminus of Fav shifts the octamer / nonamer ratio towards nonamers.

[0026] Figure 7 : Shift in octamer / nonamer ratio towards nonamers. 2D classes obtained after analyzing Cryo-EM micrographs of detergent solubilized wtFav and AC5-RN-Fav pores revealed the presence of a major octamer pore for wtFav and the appearance of a decamer pore for AC5-RN Fav pores (nonamer:decamer ratio 19: 1). 2D classes of potential decamers are marked with red rectangles (top two on the right).

[0027] Figure 8 : RN-Fav (MinION). MinION experiment of RN-Fav pores. A: Typical I / V curve obtained after applying an alternating voltage from 0 to ± 120 mV (lower panel). B: Current histogram of all active channels of octamer (N=478) and nonamer (N=168) RN-Fav. C: Average I / V curve of channels with single pores (three independent flow cells) and D: corresponding S2N ratio.

[0028] Figure 9 : RN1-Fav (MinION). MinION experiment of RN1-Fav. A: Typical I / V curve obtained after applying an alternating voltage from 0 to ± 120 mV (lower panel). B: Current histogram of all active channels of octamer (N=485) and nonamer (N=100) RN1-Fav. C: Average I / V curve of channels with single pores (three independent flow cells) and D: corresponding signal to noise ratio (S2N).

[0029] Figure 10 : Positively charged peptide detection. Short-lived current block observed after addition of R14 peptide to octamer (top trace) or nonamer (bottom trace) RN1-Fav pores inserted into MinlON membranes.

[0030] Figure 11 : Histone detection. Normalized current versus time trace and normalized current histogram for RN1-Fav pores (MinlON membranes) capturing different histone variants (2 µM).

[0031] Figure 12 : Histone detection. Histone variants detected by RN1-Fav pores inserted into MinlON membranes can be distinguished based on average current and noise at +50 mV. Circles represent the average normalized current of membranes with single pores (N) within a single flow cell and the standard deviation of their noise.

[0032] Figure 13 : Covalent attachment monomers (addition of cysteines). Cysteines introduced at amino acid positions 84 and 85 (corresponding to SEQ ID NO: 1) lead to the formation of a disulfide (S-S) ring in the pore transmembrane region. A: SDS Page of CC-Fav pores in the absence and presence of reducing agent, leading to the reduction of the disulfide (S-S) ring and the disappearance of higher order oligomeric complexes (160 kDa band corresponds to eight attached protomers). B: Top and side view of the 3D structure of wtFav and CC-Fav. Red arrows point to the S-S ring.

[0033] Figure 14: Structural characterization of RN1-Fav pores. a) Schematic of the proteins used in this study. Positions of mutations are indicated with yellow stars. Details are found in the alignment in Fig. S1. b) Non-denaturing PAGE gel of the pores used in this study. Positions of octamers and nonamers are indicated. c) Overall structure of the octameric RN1-Fav pore model resolved by cryo-electron microscopy. Black arrow indicates the overall dimensions of the pore. The putative membrane region is shown as a grey rectangle. Red and green rectangles are shown at larger scale in d and e. d) Zoom into the pore structure around residue R203, which shows a H-bond to an adjacent side chain of another protomer. Side chains of the indicated amino acid residues are shown in ball-and-stick representation. Blue balls, nitrogen; red balls, oxygen; green dashed lines, potential H-bonds. e) Zoom into the helical region with all mutated residues in RN1-Fav is shown in ball-and-stick representation. f and g) Electrostatic potential of FraC, wtFav and RN1-Fav pores, top view (f) and cross-section (g) with a color gradient from negative (red) to positive (blue) potential. The pore constriction is highlighted with an arrow and the diameter (measured from van der Waals surface to van der Waals surface) is indicated. The size of the pore cavity in the cap region is also indicated for the octamer of RN1-Fav.

[0034] Figure 15 : High-throughput electro-physical characterization of Fav pores. a) Representative current traces recorded using the MinION system after applying a 10 mV step voltage ramp from 0 mV to ± 120 mV. All traces were filtered using a 5 kHz low-pass filter. Black straight lines indicate 0 pA or 0 mV. Black, data for octameric pores; red, data for nonameric pores. b) Current histograms of open pores at -50 mV, Gaussian fits were applied where applicable. For each variant, the number of pores (n) included in the analysis of other figures is given. We also report the mean current ± standard deviation for all RN-Fav and RN1-Fav pores obtained from the fits. c) Mean I / V curves for all detected single pores per MinION flow cell were recorded using the same ramping protocol as in (a), along with the corresponding standard deviations. d) Boxplot showing the signal-to-noise ratio at -50 mV calculated for all single pores. Differences were tested with a two-sample t-test. **p < 0.01, *p < 0.05, n.s. indicates no statistical difference (p > 0.05). e) Mean signal-to-noise ratio ± standard deviation for RN1-Fav octameric pores over the entire voltage ramp, calculated for the pores included in other figures.

[0035] Figure 16Label-free discrimination of histone protein variants by batch analysis. a) Schematic model of full-length histone detection (blue) with octamer RN1-Fav pore. Histones were added from the cis side of the membrane. b) Amino acid sequence of the unstructured N-terminal histone tail of the histones used in this study. Amino acids with positively charged side chains are colored blue, and post-translationally modified amino acids are colored red. c) 3D model of the H3.1 (blue) and H4 (black) core domains (PDB 2CV5

[36] ), with N- and C-termini labeled. d) Representative one-minute current trace recorded at -50 mV. The gray trace represents a control measurement on the same pore without histones, while the colored traces correspond to current traces obtained after addition of 2 µM histones as indicated. e) Discrimination of histone variants based on the relative blocked current and noise on 60 s long current traces of individual pores recorded simultaneously using a single flow cell (each circle represents one MinION flow cell and the corresponding number of individual pores included in the analysis). Standard error of the normalized blocked current and noise are shown as gray error bars.

[0036] Figure 17 Discrimination of histones based on blocking analysis. a) One-minute current traces recorded at -50 mV in the presence of 2 µM histones. The colored part of the traces represents the current blockage of detected H4 (black) and H3Cit (green). The histograms to the right of each trace correspond to the current trace on the left. Bar plots representing the detected blockage current are shown in the corresponding color. b) 3D scatter plot of the normalized blockage noise, amplitude, and dwell time of blockers extracted from three independent flow cells in the presence of 2 µM histones (data from each flow cell is represented as circles in different shades of black (representing H4) or green (H3Cit) intensity). For H4 data, 534, 260, and 460 blockages were extracted from flow cells with 179, 39, and 120 individual pores, respectively. For H3Cit data, 110, 386, and 42 blockages were extracted from 40, 280, and 25 individual pores, respectively. c and d) One-minute current traces (low-pass filtered with a cutoff frequency of 1000 Hz) after addition of 0, 0.5, 1, 2, and 4 µM of histone H4 (c) or histone H3Cit (d). e) Linear fit to the concentration-dependent blockage frequency (1 / τ ON ).

[0037] Figure 18Machine learning supports discrimination of histones in mixtures. a) Workflow diagram for blocked classification using Orange.

[39] To train the model, the amplitude of the blockades, noise and dwell time extracted from traces with only H4 or H3Cit added were used as input data for future matrix generation. Using the kNN algorithm and cross validating 10% of the data, a model with a classification accuracy (CA) of 0.960 was generated. b) Confusion matrix generated using test set data (20% of input data randomly selected), with an accuracy of 91.3% for histone H3Cit and 97.7% for histone H4. c) Blockades (bolded sections of traces) with parameters extracted from one minute traces, with 1 :0, 3:1; 1 :1, 1 :3 and 0:1 H4 and H3Cit ratios (2 mM total concentration) used for classification based on the previously learned model. Colors correspond to blockades identified as H3Cit (green) or H4 (black). d) Experimentally determined ratios of histones in mixtures based on the number of blockades classified as H4 (black) or H3Cit (green) out of a total of 1238, 158, 381, 176 and 347 input blockades. e) Comparison of the estimated ratio of H4 in mixtures based on the model (black circles of linear fit) with the predicted theoretical values (open circles). Data represent the mean ± standard error of three independent flow cells.

[0038] Figure 19 Amino acid sequence alignment of wtFav, AN67Fav, RN-Fav, RN1-Fav used in the experiments, and FraC colored by conservation. Mutations introduced in wtFav in the different constructs are colored in green. N-terminal residues of constructs AN53Fav, AN63Fav, AN67Fav, AN73Fav and AN75Fav are marked in red on the wtFav sequence.

[0039] Figure 20 Insertion of AN-Fav constructs into MinION flow cells. a) Bar plot representing the number of specific Fav pores with a truncated N-terminus inserted into MinION flow cells (black dots). The median value calculated for three flow cells (7 for the case of AN67Fav) is marked in red. b) Representative current traces obtained by MinION upon application of an alternating voltage protocol from 0 mv to ±120 mV (except for AN53Fav, where the voltage was increased from 0 mV to ±90 mV). All traces were filtered using a MATLAB low-pass filter with a cutoff frequency of 1000 Hz. c) Histograms of the open channel currents of AN53, AN63, AN67, AN73, AN75Fav extracted from the I / V curve measurements at -90 mV.

[0040] Figure 21: Data analysis workflow. a) From micrographs to b) data analysis workflow for the resolution of the final sharpened volumes of the solubilized octamer and nonamer RN1-Fav pores by local resolution estimation. Both structures (octamer pore, nonamer pore) were resolved from the same dataset following the same protocol. Arrows indicate the movement of the particles between different steps or iterations. Throughout the steps, the volume of the octamer pore is shown in grey and the volume of the nonamer pore is shown in blue. Cyan and wheat color indicate the two junk / bait volumes. After separation, the analysis of the octamer and nonamer pore is indicated with green and yellow rectangles, respectively. Further details on the data analysis are provided in the cryo-EM data processing section of Materials and Methods of Example 2

[0041] Figure 22 : a) RN1-Fav octamer pore model built into the cryo-EM density map. Individual protomers are colored in yellow and blue, lipids in green. The map region where the RN1-fav mutation resides has been highlighted and is shown in panels b and c. b) Map region around residue R203. c) Cryo-EM density map region of the transmembrane a-helix, where residues different from wtFav are highlighted. d) Side view of the RN1-Fav octamer pore, protomers in alternating colors (sky blue and yellow). e) Side view of the RN1-Fav nonamer pore, protomers in alternating colors (sky blue, orange and yellow). f) Top view of aligned octamer wtFav (PDB-ID 9EYM, navy and purple) and octamer RN1-Fav pore (yellow and cyan), RMSD = 0.51 on all C-alpha atoms (PDBeFold) [6].

[0042] Figure 23 : Isolation of octamer and nonamer RN-Fav and RN1-Fav pores by ion-exchange chromatography. a) Ion-exchange chromatograms of RN-Fav pore (left) and RN1-Fav pore (right) eluted with increasing concentration of 2 M NaCl (yellow line). Fractions numbered 1 to 6 were analyzed by non-denaturing PAGE as shown in (b) and (c) for RN-Fav and RN1-Fav pores, respectively.

[0043] Figure 24: Histone H4 capture at different voltages. a) A ramp of alternating voltages from 0 mV to + / - 100 mV was applied in the presence of 1 μΜ histone H4. Light blue star marks the point in time where the membrane was destroyed and the pore was destroyed. Examples of current traces of four independent membranes in C18 buffer (grey) and in the presence of 1 μΜ histone H4 (black). b) Enlargement of the current trace at voltages from -40 mV to -90 mV, the enlarged part of the trace in panel (a) is in the green box. Blue stars indicate discrete blockades shorter than 5 s and red stars indicate longer lasting blockades, usually until the voltage polarity is changed. c) Mean residence time and standard error of all detected events (numbers corresponding are shown in the graph). d) Enlargement of the purple box in panel a) showing that the high current noise at -100 mV prevents us from determining discrete blockades.

[0044] Figure 25 : Histone H3.1 capture at different voltages. A ramp of alternating voltages from 0 mV to + / - 100 mV was applied in the presence of 1 μΜ histone H3.1. a) Examples of current traces of four independent membranes in C18 buffer (grey) and histone H3.1 (black). Light blue star marks the point in time where the membrane was destroyed and the pore was destroyed. b) Enlargement of the current trace at voltages from -40 mV to -80 mV, the part of the trace in panel (a) is in the green box. Blue stars indicate discrete blockades shorter than 5 s and red stars indicate longer lasting blockades, usually until the voltage polarity is changed. c) Mean residence time and standard error of all detected events (numbers corresponding are shown in the graph).

[0045] Figure 26 : Open pore current recovery at positive voltage polarity. For histone detection, we used a voltage protocol starting at 0 mV, changing to -50 mV within 60 s and finally increasing to +50 mV (yellow line). The current signal corresponding to the control measurement (no added histone) is shown in grey, while the signal in the presence of 2 μΜ histone H3.1 and H3K23ac is colored black. Red arrows mark the voltage polarity change and the subsequent open pore current recovery.

[0046] Figure 27 : MinION measurement repeatability for histone H4. Data of three independent flow cells with an octamer RN1-Fav pore inserted (single pore per membrane). Each row represents a one minute normalized current trace recorded at -50mv in the absence (grey) and presence of 2 μΜ histone H4.

[0047] Figure 28: MinION measurement repeatability of histone H3.1. Data from three independent flow cells with octamer RN1-Fav pores inserted (single pore per membrane). Each row represents a one minute normalized current trace recorded at -50 mv in the absence (grey) and presence of 2 mM histone H3.1.

[0048] Figure 29 : MinION measurement repeatability of histone H3Cit. Data from three independent flow cells with octamer RN1-Fav pores inserted (single pore per membrane). Each row represents a one minute normalized current trace recorded at -50 mv in the absence (grey) and presence of 2 mM histone H3Cit.

[0049] Figure 30 : MinION measurement repeatability of histone H3K9ac. Data from three independent flow cells with octamer RN1-Fav pores inserted (single pore per membrane). Each row represents a one minute normalized current trace recorded at -50 mv in the absence (grey) and presence of 2 mM histone H3K9ac.

[0050] Figure 31 : MinION measurement repeatability of histone H3K23ac. Data from three independent flow cells with octamer RN1-Fav pores inserted (single pore per membrane). Each row represents a one minute normalized current trace recorded at -50 mv in the absence (grey) and presence of 2 mM histone H3K23ac.

[0051] Figure 32 : Event analysis workflow. After pore insertion into the MinION membrane, we applied a voltage protocol starting from 0 mV, then descending to -50 mV, and then ascending to +50 mV. Histone capture occurs upon application of negative voltage. We recorded open pore current in the absence of histone (C18 buffer only, control measurement) before adding histone to the cis side of the membrane. Through an internal MATLAB script, we extracted all active membranes and constructed a histogram of open pore current (-50 mV) of all MinION membranes that remained active (with pore signal) after histone addition. Through a multiple Gaussian fit, we extracted data from membranes with single pores inserted. In the next step, we applied a wavelet-based filter and performed current normalization by dividing the open pore current (lo) by the average blockade current (Ik0) of a particular trace. Blockades were extracted through a threshold-based approach using our MATLAB script and characterized by their duration (dwell time), current amplitude, average blockade current, and its standard deviation. Additional details on data analysis are provided in the MinION data analysis section of the methods in Example 2.

[0052] Figure 33 Structure of a Fav pore formed on DOPC:SM (1 : 1) membranes. a, Top and side views of cryo-EM maps of octameric Fav pores prepared on DOPC:SM vesicles. Sections of the map corresponding to individual protomers are differently colored, and regions of the map corresponding to well-defined lipids are shown in orange. b, Cartoon representation of the pore. The cap region and the transmembrane (TM) helix bundle are highlighted. c, Electrostatic potential of the surface of the pore. The color gradient from red to blue represents negative to positive electrostatic potential. The TM helices, residue E91 (E88 in SEQ ID NO: 1), and the C-terminal extension (green) are highlighted. d, Six unique lipid positions identified from the pore structure. Figure 33 Residue numbering in d corresponds to SEQ ID NO: 1.

[0053] Figure 34 Structure of Fav pores prepared on POPG:SM:CHOL (1 : 1 : 1) vesicles. a, Side view of cryo-EM maps of octameric Fav pores. Sections of the map corresponding to individual protomers are differently colored, and regions corresponding to well-defined lipids are shown in orange. b, Surface and cartoon / stick representation of the four cholesterol (CH) molecules. Green dashed lines show hydrogen bonds between cholesterol and protomers. c, Comparison of discrete ion current steps corresponding to pore insertion induced by addition of monomeric Fav protein at -50 mV (top). d, Normalized current noise of the membrane in the absence or presence of cholesterol. n = 12 and 17, respectively. e, f, Surface representation of the promoter viewed from two different angles, with sections of the map corresponding to lipids associated with them. Lipids are colored according to the indicated function. Top-down view of the protomer, with all lipids visible. g, Surface and cartoon / stick representation of the resolved phospholipids in the pore structure, highlighting the bridging lipids. h, Schematic view of the lipids and protein observed in the cryo-EM structure, with emphasis on the bridging lipids and L10. Hydrogen bonds between lipids are represented by green lines, and hydrogen bonds between lipids and protein by lines with circles. Hydrophobic interactions between lipids are shown as magenta lines. Residue numbering in the maps corresponds to SEQ ID NO: 1.

[0054] Figure 35: Structural and dynamic properties of membrane lipids from molecular dynamics simulations. a, b, Schematic bead representation of lipid molecules, which are separated into consecutive coordination shells in the upper (a) and lower (b) membrane leaflet. Red lipid molecules represent lipids that are in “strong” contact with the protein. Each next consecutive shell is alternately colored green and blue, while the bulk of lipids is gray. The remaining predefined lipids in the upper layer are shown in gray. c, Correlation between the displacements of neighboring lipid molecules belonging to consecutive shells: current (l) and previous (l-1) shells of lipids in the upper (blue circles) and lower (black circles) leaflet according to equation (1). Solid lines connect values obtained for the cholesterol-containing system with 112 predefined lipid poses, while dashed lines connect values for the cholesterol-free system with 48 predefined lipid poses. d, Translational diffusion constants of lipid molecules in consecutive lipid shells around the pore in the upper or lower leaflet. Colors and line types are the same as in c. Diffusion constants for the pure membrane systems and their error ranges are given. Blue, POPC:SM:CHOL membrane system; green, POPC:SM membrane system. Layer 0 refers to the diffusion constant due to the closest protein monomer. e, Schematic representation of different types of lipids and interactions from transmembrane pore complex structures and molecular dynamics simulations.

[0055] Figure 36 : Structure of Fav and pore preparation. a, Alignment of the amino acid sequences of Fav and the stinging pore protein. Identical residues are marked with a red background, and chemically similar residues are shown in red font. SEQ ID NO: 1 starts with S at position 4 of the Fav sequence. b, Superposition of the crystal structures of Fav (this study, PDB-ID 9EYM, orange) and the stinging pore protein FraC (PDB-ID 3LIM, gray). The N- and C-termini and various structural elements are labeled. Disulfide bonds are labeled in yellow. c, Structural detail with a density map of the region L179-V185 (L176-V182 in SEQ ID NO: 1). 2mFoDFc electron density is contoured at 1 sigma (blue), and mFo-DFc electron density is contoured at +3.0 sigma (green) and –3.0 sigma (red). d, Cryo-EM micrographs (top) and 2D class averages of DOPC:SM (1:1) large unilamellar vesicles (LUVs) incubated with monomeric Fav (bottom) of two different pore stoichiometries. e, Schematic representation of the Fav pore preparation steps, from top to bottom: 1 preparation of the protein-LUV mixture, incubation at 37 °C for 1 h, solubilization with 1% lauryldimethylamine oxide, purification on an ion-exchange chromatography column, non-denaturing-PAGE of monomeric Fav (M) and purified Fav pore (P).

[0056] Figure 37Data analysis workflow for isolated Fav pores formed on large unilamellar vesicles composed of DOPC:SM (1 :1). Additional details on data analysis are provided in the cryo-electron microscopy data processing section of Materials and Methods

[0057] Figure 38 Comparison of lipids previously described in the fragaceatoxin C (FraC) pore (PDB-ID 4TSY) and lipids in equivalent positions in the structure of the Fav pore. a, side-by-side comparison of individual protomers with lipids LI, L2 and L3. b, Fav protomer (blue) aligned with FraC protomer (grey) with corresponding lipids (Fav in orange and FraC in yellow).

[0058] Figure 39 Lipids in the Fav pore structure prepared on liposomes composed of DOPC:SM (1 :1). Shown are the fitted model diagram for each lipid position (left), and the interaction diagram (right). Hydrogen bonds are indicated with green dashed lines. Red lines represent hydrophobic interactions with protein residues, while green represents hydrophobic interactions with other lipids. Interaction diagrams were drawn by LigPlot+ (Laskowski, R. A. and Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. Journal of chemical information and modeling 51, 2778-2786, doi:10.1021 / ci200227u (2011)). Residue numbering in the diagram corresponds to SEQ ID NO: 1.

[0059] Figure 40: Interaction surface between two protomers in the pore. a, Position of lipids LI and L6 between protomers. b, Schematic of interaction surface between protomers A and B. Green represents direct protomer-protomer surface and orange represents indirect protomer-lipid-protomer interaction. b, Interaction surface calculated by pdbePISA (Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797, doi:10.1016 / j.jmb.2007.05.022 (2007)). c, Interaction plot of surface between two protomers. Green lines represent hydrogen bonds and red and pink are hydrophobic interactions. All residue numbers in the plot show positions in SEQ ID NO: 1 + 3 (e.g., LI 39 in the plot is LI 36 in SEQ ID NO: 1).

[0060] Figure 41 : Alternative lipid chain positions for lipids L2 and L4. a, Top-down view at the pore (white) and lipids with the void below the cap region highlighted. The right panel shows the indicated area at higher magnification. b, Two sets of lipids at positions L2 and L4. c, Skeleton model of lipids with corresponding cryo-EM map matching.

[0061] Figure 42 : Data analysis workflow for isolated Fav pores formed on large unilamellar vesicles composed of POPG:SM:Cholesterol (1 : 1 : 1). Additional details on data analysis are provided in the Cryo-electron microscopy data processing section of Materials and Methods.

[0062] Figure 43 : Comparison of pores formed on large unilamellar vesicles composed of DOPC:SM (1 : 1) or POPG:SM:CHOL (1 : 1 : 1). Cryo-EM maps are colored by protomer and regions corresponding to lipids are shown in orange.

[0063] Figure 44Cholesterol molecules in the Fav pore structure formed on large unilamellar vesicles composed of POPG:SM:CHOL (1 : 1 : 1). Shown is the fitted model map for each cholesterol position (left), and the interaction map (right). Hydrogen bonds are indicated with green dashed lines. Red lines represent hydrophobic interactions with protein residues, while green represents hydrophobic interactions with other lipids. Interaction maps were plotted by LigPlot+ (Laskowski, R. A. and Swindells, M. B., supra). Residue numbering in the figure corresponds to SEQ ID NO: 1.

[0064] Figure 45 Comparison of pore structures formed on different membranes. Comparison of pores formed on DOPC:SM (white) and POPG:SM:Cholesterol (blue) membranes. a, Side-by-side comparison of two pores. b, Top view of aligned pores, and c, Individual protomers aligned.

[0065] Figure 46 Lipids L1-L6 in the Fav pore structure prepared on large unilamellar vesicles composed of POPG:SM:CHOL (1 : 1 : 1). Shown is the fitted model map for each lipid position (left), and the interaction map (right). Hydrogen bonds are indicated with green dashed lines. Red lines represent hydrophobic interactions with protein residues, while green represents hydrophobic interactions with other lipids. Interaction maps were plotted by LigPlot+ (Laskowski, R. A. and Swindells, M. B., supra). Residue numbering in the figure corresponds to SEQ ID NO: 1.

[0066] Figure 47 Lipids L7-L11 in the Fav pore structure prepared on POPG:SM:CHOL (1 : 1 : 1) membranes. Shown is the fitted model map for each lipid position (left), and the interaction map (right). Hydrogen bonds are indicated with green dashed lines. Red lines represent hydrophobic interactions with protein residues, while green represents hydrophobic interactions with other lipids. Interaction maps were plotted by LigPlot+ (Laskowski, R. A. and Swindells, M. B., supra). Residue numbering in the figure corresponds to SEQ ID NO: 1.

[0067] Figure 48Data analysis workflow for isolated Fav pores formed on large unilamellar vesicles composed of DOPC:SM:Cholesterol (1 : 1 : 1). a, Data analysis workflow for Fav pores prepared on DOPC:SM:CHOL (1 : 1 : 1) vesicles. Additional details on data analysis are provided in the Cryo-EM data processing section of Materials and Methods. b, Cryo-EM map of Fav pore. Regions corresponding to protomers are colored blue, while regions corresponding to lipids are colored by their designated group. c, Cartoon representation of individual protomers with associated lipids in two orientations.

[0068] Figure 49 Analysis of Fav pores formed on nanodiscs composed of DOPC:SM:Cholesterol (1 : 1 : 1). a, Data analysis workflow for Fav pores prepared on DOPC:SM:CHOL (1 : 1 : 1) nanodiscs. Additional details on data analysis are provided in the Cryo-EM data processing section of Materials and Methods. b, Cryo-EM map of Fav pore prepared on nanodisc. Regions corresponding to cholesterol are colored red, and sphingomyelin are colored orange.

[0069] Figure 50 Location of Fav pores in membranes. a, Fav protomer with membrane-binding loops 2 and 3 highlighted. The angle at which the protomer is placed on the membrane is indicated. b, Minimized Fav pore (blue) embedded in a POPC:SM:Cholesterol (1 : 1 : 1) membrane for molecular modeling. Loops 2 and 3 of all protomers are shown in green, orange spheres are the phosphate groups of lipids, and upper leaflet cholesterol molecules are shown in red. c, Cartoon representation of protomers with loops 2 and 3 highlighted, with lipids bound to them. d, Surface rendering of Fav protomer with additional lipids shown in two different angles.

[0070] Figure 51 Comparison of soluble and protomeric structure of Fav. a, Monomer in solution shown in gray, individual monomers from pore structure shown in blue. Details of loops 2 (b) and 3 (c) are shown with amino acid side chains rendered as sticks. All residue numbers in this figure show positions in SEQ ID NO: 1 + 3 (e.g., V160 in the figure is V157 in SEQ ID NO: 1).

[0071] Figure 52: Pore formation of Fav on large monolayer vesicles containing ceramide phosphoethanolamine (CPE). a, Ion exchange chromatography of samples obtained by incubation of Fav with 1: liposome DOPC:SM:cholesterol (1:1:1), 2: liposome DOPC:CPE:cholesterol (1:1:1), or 3: liposome-free, dissolved in 0.75% lauryl dimethylamine oxide, and eluted with a linear NaCl gradient (dashed line). b, Non-denaturing PAGE gels with peak fractions 1–3 obtained after the chromatographic step, as shown in a. c, Representative micrographs (top) of large monolayer vesicles composed of DOPC:CPE:cholesterol after incubation with Fav. 2D class averages are shown below.

[0072] Figure 53 Interactions between L1 and the Fav protopolymer. a) If ceramide phosphoethanolamine is located at the same position, the interaction between the sphingomyelin head groups will not exist. b) Surface of the protopolymer surrounding the head group of L1. c, d) Comparison of the interaction diagram of L1 (c) with the potential interaction diagram of ceramide phosphoethanolamine (d) at position L1. The residue numbers in the diagram correspond to SEQ ID NO: 1.

[0073] SEQ ID NO: 1

[0074] SEQ ID NO: 1 shows the amino acid sequence of a signal peptide-free nematoporin monomer from mountain star coral.

[0075] SEQ ID NO: 2 shows the fragment of SEQ ID NO: 1 missing amino acids 1-76. Detailed Implementation

[0076] All publications, patents, and patent applications cited herein, whether above or below, are hereby incorporated in their entirety by reference. All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if specifically and individually indicated that each individual publication, patent, or patent application is incorporated by reference. Where a publication, patent, or patent application incorporated by reference contradicts the invention contained herein, this specification is intended to supersede and / or give priority to any such contradictory material.

[0077] The application will be described with respect to the embodiments in conjunction with certain drawings but the application is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. It is to be understood that any feature described in relation to one particular embodiment can be included with another embodiment, to the extent not contradictory. The application can be implemented or carried out in other ways than those explicitly described or suggested herein without departing from the basic principles. For example, the application can be implemented in a way that achieves or optimizes one advantage or a group of advantages taught herein without necessarily achieving other aspects or advantages taught or suggested herein.

[0078] Also, 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 porosome protein pore" includes two or more porosome protein pores, and the like.

[0079] In all discussions herein, the standard one-letter codes for amino acids are used. They 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 the Q at position 42 is replaced by R.

[0080] In paragraphs herein in which different amino acids at a particular position are separated by a / symbol, the / symbol means "or". For example, D203R / K means D203R or D203K. In paragraphs herein in which different positions are separated by a / symbol, the / symbol means "and" so that D203 / D215 is D203 and D215.

[0081] The general definitions in WO 2019 / 002893 are incorporated herein by reference in their entirety.

[0082] Figure 3

[0083] The present application provides a spicule pore protein monomer comprising a variant of SEQ ID NO: 1 having at least about 40% identity to the sequence of SEQ ID NO: 1 over its entire length. The variant preferably has at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% identity to the sequence of SEQ ID NO: 1 over its entire length. The variant more preferably has at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 1 over its entire length.

[0084] Identity can be determined using standard methods in the art. For example, the UWGCG package provides a program, BESTFIT, which can be used to calculate identity (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395). The PILEUP and BLAST algorithms can be used to calculate identity or align sequences such as identifying equivalent or corresponding sequences (typically under their default settings), as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F. et al. (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0085] Identity of the variant to SEQ ID NO: 1 is measured over the entire length of SEQ ID NO: 1. SEQ ID NO: 1 is 259 amino acids in length. Identity is typically determined by calculating the number of identical residues between the variant and SEQ ID NO: 1 using any of the above methods and dividing the number of identical residues by 259. For example, if a variant has 105 identical amino acids to SEQ ID NO: 1, then the variant has 105 / 259 x 100 = 40.54% identity to SEQ ID NO: 1 over its entire length.

[0086] The spicule porin monomer of the invention is typically capable of forming a pore or spicule porin pore. This can be measured using any of the conventional methods described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, and WO 2019 / 002893 (all incorporated by reference in their entirety) and those described in the Examples.

[0087] The variant preferably comprises one or more modifications that (a) improve the ability of the spicule porin pore formed by the monomer to interact with a target analyte, (b) change the number of spicule porin monomers that form the spicule porin pore, and / or (c) facilitate insertion of the spicule porin monomer or the spicule porin pore formed by the monomer into an artificial membrane. The variant can include (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). Each of (a) to (c) is described in more detail below. The one or more modifications are preferably one or more mutations. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0088] The spicule porin pore formed by the monomer preferably comprises one or more of (a) a cap region, (b) a constriction region, and (c) a transmembrane alpha helix region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The spicule porin monomer preferably includes one or more regions that contribute to these regions in the spicule porin pore. The spicule porin monomer more preferably comprises one or more regions that form one or more of (a) to (c), such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The spicule porin pore formed by the monomer can have any structure, but preferably has or comprises the structure of a wild-type spicule porin pore. The protein structure of the spicule porin pore defines a channel or pore that allows for the translocation of molecules and ions from one side of the membrane to the other.

[0089] "Constriction," "pore," "pore constriction," "channel constriction," or "constriction site" as used interchangeably herein refers to an opening defined by the lumen surface of a pore or stomy pore protein pore that serves to allow passage of ions and target analytes, including but not limited to polynucleotides or individual nucleotides, through the pore or stomy pore protein pore channel, but not other non-target analytes. In some cases, for example when characterizing a protein or polypeptide target analyte, the constriction prevents the target analyte from passing through the pore. The constriction is generally the narrowest opening within or defined by the channel of the pore or stomy pore protein pore. The constriction can serve to limit passage of molecules through the pore. The size of the constriction is generally a key factor in determining the suitability of a pore or stomy pore protein pore for characterization of an analyte. If the constriction is too small, the molecule to be characterized will not be able to pass. However, to achieve the maximum effect on the flow of ions through the channel, the constriction structure should not be too large. For example, the constriction structure should not be wider than the transverse diameter of the target analyte that is accessible to the solvent. Ideally, the diameter of any constriction structure should be as close as possible to the transverse diameter of the passing analyte.

[0090] SEQ ID NO: 1 shows the sequence of a wild-type stomy pore protein monomer derived from Montipora gaudii as a mature protein. Residues V104 to M259 of SEQ ID NO: 1 form the cap region. Residues A80 to L89 of SEQ ID NO: 1 form the constriction region. Residues L66 to N103 or R77 to N103 of SEQ ID NO: 1 form the transmembrane alpha helix region. The constriction region is generally present in the transmembrane alpha helix region. Residues S1 to D65 form the N-terminal extension.

[0091] The surface of the stomy pore protein monomer of SEQ ID NO: 1 that is exposed to the pore lumen or channel comprises a high negative charge. Pores formed from stomy pore protein monomers based on SEQ ID NO: 1 comprise a high negative charge in their lumen or channel. This allows such pores to determine the presence, absence, or one or more characteristics of positively charged analytes, such as positively charged polypeptides, as described in the examples.

[0092] The cnidocyte pore protein monomer comprises a variant of SEQ ID NO: 1. The variant monomer can also be referred to as a modified cnidocyte pore protein monomer or a mutant cnidocyte pore protein monomer. The modifications or mutations in the variant include, but are not limited to, any one or more of the modifications disclosed herein or a combination of the modifications. The cnidocyte pore protein monomer can be a homologous monomer. A homologous monomer is a polypeptide that has at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% overall sequence identity to SEQ ID NO: 1 over its entire length. The homologous monomer is typically a cnidocyte pore protein monomer from a species other than P. hilli. The variant can comprise any substitutions present in the homologous monomer.

[0093] Sequence identity can also relate to fragments or portions of the cnidocyte pore protein monomer. While the variant must have at least about 40% overall sequence identity to SEQ ID NO: 1 over its entire length, the sequence of particular regions, domains, or subunits can share at least about 80%, 90%, or up to about 99% sequence identity with the corresponding region of SEQ ID NO: 1. There can be at least about 80%, e.g., at least about 85%, 90%, or 95% identity over a stretch of 100 or more, e.g., 125, 150, 175, or 200 or more contiguous or abutting amino acids (“hard identity”). The cnidocyte pore protein monomer preferably comprises a variant having at least about 40% identity to the cap region of SEQ ID NO: 1. More preferably, the variant has at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and more preferably at least about 95%, 97%, or 99% identity to the cap region of SEQ ID NO: 1. The cap region is residues V104 to M259 of SEQ ID NO: 1.

[0094] The cnidocyte pore protein monomer preferably comprises a variant having at least about 40% identity to the constriction region of SEQ ID NO: 1. More preferably, the variant has at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and more preferably at least about 95%, 97%, or 99% identity to the constriction region of SEQ ID NO: 1. The constriction region is residues A80 to L89 of SEQ ID NO: 1.

[0095] The cnhaoporin monomer preferably comprises a variant having at least about 40% identity to the transmembrane alpha helix region of SEQ ID NO: 1. More preferably, the variant has at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and more preferably at least about 95%, 97%, or 99% identity to the transmembrane alpha helix region of SEQ ID NO: 1. The transmembrane alpha helix region is residues L66 to N103 or R77 to N103 of SEQ ID NO: 1.

[0096] The variant preferably comprises one or more modifications that improve the ability of the cnhaoporin pore formed by the monomer to interact with a target analyte. The one or more modifications are preferably one or more mutations. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions. Target analytes are discussed in more detail below. The target analyte is preferably an amino acid, a polypeptide, a protein, a nucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, or a polysaccharide.

[0097] The cnhaoporin monomer preferably forms a cnhaoporin pore that has improved target analyte readout properties, i.e., shows improved analyte capture and discrimination. In particular, the cnhaoporin pore constructed from the cnhaoporin monomer preferably captures amino acids, polypeptides, proteins, nucleotides, and polynucleotides more readily than wild type. In addition, the cnhaoporin pore constructed from the cnhaoporin monomer preferably shows increased current range, which makes it easier to discriminate between different analytes, and reduced state variance, which increases the signal-to-noise ratio. In addition, the number of analytes contributing to the current or the number of polymer subunits in a polymer (e.g., the number of amino acids in a polypeptide or the number of nucleotides in a polynucleotide) is reduced as they move through the cnhaoporin pore constructed from the monomer. This makes it easier to identify a direct relationship between the current observed as an analyte moves through the cnhaoporin pore and the sequence of the analyte. The improvement in analyte readout performance of the cnhaoporin pore is achieved through five main mechanisms, i.e., by altering: steric hindrance (increasing or decreasing the size of an amino acid residue); charge (e.g., introducing a positive charge to interact with nucleic acid sequences); hydrogen bonding (e.g., introducing an amino acid that can form hydrogen bonds with base pairs); pi stacking (e.g., introducing an amino acid that interacts through delocalized electronic pi systems); and / or changes to the pore structure (e.g., introducing an amino acid that increases the size of the vestibule and / or constriction).

[0098] Any one or more of these five mechanisms can be responsible for the improved properties of the pore of the present invention. For example, the pore of the present invention can exhibit improved analyte reading properties due to altered steric hindrance, altered hydrogen bonding, and altered structure.

[0099] Introduction of bulky residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H), in the cnidocyan protein monomer increases the steric hindrance of the pore of the cnidocyan protein. Introduction of aromatic residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H), in the cnidocyan protein monomer also increases pi-stacking in the pore of the cnidocyan protein. Introduction of bulky or aromatic residues in the cnidocyan protein monomer also alters the structure of the pore of the cnidocyan protein, for example by opening the pore and increasing the size of the vestibule and / or constriction. This is described in more detail below.

[0100] The variant can comprise one or more modifications or mutations at one or more positions in any combination of (a) the cap region, (b) the constriction region, and (c) the transmembrane alpha helix region, i.e. in (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b) and (c).

[0101] The cap region preferably comprises a modification or mutation at one or more positions corresponding to N105, K107, A109, G111, D113, E115, G117-R119, Q121, E122, D133-N135, P137, S141-Y144, L147, G149, R151, N154, S171, I172, K174, S199, N201, D203, D215, S231, G232, Q244, T246, E248, H250, L252-C258 in SEQ ID NO: 1. The one or more modifications or mutations are preferably one or more substitutions of the amino acid at a position having G, A, V, L, I, P, C, S, T, N, Q, F, Y, W, D, H, E, R, or K.

[0102] The constriction region preferably comprises a modification or mutation at one or more positions corresponding to the positions in SEQ ID NO: 1 shown in Column 1. In each row, the amino acid at a position corresponding to the position in SEQ ID NO: 1 shown in Column 1 is preferably substituted with one of the amino acids in Column 3. Column 2 indicates whether the position is pointing inward toward the vestibule / channel of the pore (“in”) or outward from the pore (“out”).

[0103]

[0104] The transmembrane region preferably comprises a modification or mutation at one or more positions corresponding to positions in SEQ ID NO: 1 shown in column 1. In each row, the amino acid at a position corresponding to a position in SEQ ID NO: 1 shown in column 1 is preferably substituted with one of the amino acids in column 3. Column 2 indicates whether the position is inwardly pointing into the pore vestibule / channel (“in”) or outwardly pointing out of the pore (“out”).

[0105]

[0106] The variant can comprise any number of one or more modifications or mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more modifications or mutations. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0107] The one or more modifications or mutations are each independently (a) alter the size of the amino acid residue at the modification position; (b) alter the net charge of the amino acid residue at the modification position; (c) alter the hydrogen bonding properties of the amino acid residue at the modification position; (d) introduce or remove one or more chemical groups that interact through delocalized electron pi systems in the amino acid residue at the modification position, and / or (e) alter the structure of the amino acid residue at the modification position. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0108] The variant preferably comprises one or more modifications or mutations that improve the ability of the spicule pore protein pore formed by the monomer to interact with an amino acid, polypeptide, or protein. The terms “polypeptide” and “protein” are interchangeable and discussed in more detail. The one or more modifications are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications are preferably one or more substitutions.

[0109] Depending on the charge of the amino acid or polypeptide analyte, the variant preferably comprises one or more modifications or mutations that reduce the same net charge of the monomer. If the amino acid or polypeptide analyte is positively charged, the variant preferably comprises one or more modifications or mutations that reduce the net positive charge of the monomer. If the amino acid or polypeptide analyte is negatively charged, the variant preferably comprises one or more modifications or mutations that reduce the net negative charge of the monomer. This reduces repulsion and facilitates capture of the amino acid or polypeptide analyte.

[0110] If the amino acid or polypeptide analyte is positively charged, one or more modifications or mutations are preferably made to include a negatively charged, polar or hydrophobic amino acid in order to reduce the positive charge (thereby facilitating capture of positively charged analytes). For example, positively charged amino acids, such as R, H or K, can be substituted with a negatively charged, polar or hydrophobic amino acid. The amino acid can be substituted with D, E, S, T, N, Q, G, A, V, L, I, P, C, F, Y or W. One or more modifications or mutations are preferably made to include a polar, hydrophobic, positively charged or negatively charged residue to change discrimination. The amino acid can be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y or W.

[0111] If the amino acid or polypeptide analyte is positively charged, one or more modifications or mutations are preferably selected from the following specific substitutions:

[0112] R is substituted with D, E, S, T, N, Q, G, A, V, L, I, C, F, Y or W,

[0113] K is substituted with D, E, S, T, N, Q, G, A, V, L, I, C, F, Y or W,

[0114] D is substituted with S, T, N, Q, G, A, V, L, I, C, F, Y or W, preferably D is substituted with N, Q or A,

[0115] E is substituted with S, T, N, Q, G, A, V, L, I, C, F, Y or W, preferably E is substituted with N, Q or A,

[0116] S is substituted with E, D, T, N, Q, A, V, L or I, preferably S is substituted with E or D,

[0117] T is substituted with E, D, S, N, Q, A, V, L or I, preferably T is substituted with E or D,

[0118] N is substituted with E, D, S, T, Q, A, V, L or I, preferably N is substituted with E or D, and

[0119] Q is substituted with E, D, S, T, N, A, V, L or I, preferably Q is substituted with E or D.

[0120] If the amino acid or polypeptide analyte is positively charged, the variant preferably comprises a modification or mutation at one or more positions corresponding to E67, N68, K73, R74, R77, I91, D99, V100, L101, D215, L252, and E254 in SEQ ID NO: 1. The variant can comprise any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these positions. The variant preferably comprises a modification or mutation at all positions corresponding to E67, N68, K73, R74, R77, I91, D99, V100, L101, D215, L252, and E254 in SEQ ID NO: 1. The one or more modifications or mutations are preferably one or more substitutions. The amino acids at these positions can be modified, mutated, or substituted in any of the manners described above. The variant preferably comprises a modification or mutation at the position corresponding to D215 in SEQ ID NO: 1. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to E67, N68, K73, R74, R77, I91, D99, V100, L101, and D215 in SEQ ID NO: 1. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to D215, L252, and E254 in SEQ ID NO: 1. Any of these variants preferably further comprises a mutation or modification, preferably a substitution, at position D203. This is discussed in more detail below.

[0121] If the amino acid or polypeptide analyte is positively charged, the variant preferably comprises:

[0122] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D215R / L252T / E254A,

[0123] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D203R / D215R / L252T / E254A,

[0124] D203R,

[0125] D215R,

[0126] D203R / D215R,

[0127] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D203R / D215R,

[0128] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D203R / D215R,

[0129] one or more or all of D215R / L252T / E254A,

[0130] one or more or all of D203R / D215R / L252T / E254A

[0131] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D215N / L252T / E254A,

[0132] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D203R / D215N / L252T / E254A,

[0133] D215N,

[0134] D203R / D215N,

[0135] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D215N,

[0136] one or more or all of E67S / N68E / K73N / R74D / R77D / I91F / D99Y / V100F / L101F / D203R / D215N,

[0137] one or more or all of D215N / L252T / E254A or

[0138] one or more or all of D203R / D215N / L252T / E254A.

[0139] Examples of positively charged polypeptides and proteins are provided below.

[0140] If the amino acid or polypeptide analyte is negatively charged, one or more modifications or mutations are preferably made to include positively charged, polar or hydrophobic amino acids in order to reduce the negative charge (thereby facilitating capture of the negatively charged analyte) or to improve / stabilise the interaction with the polynucleotide binding protein (discussed in more detail below). For example, a negatively charged amino acid such as D or E can be substituted with a positively charged, polar or hydrophobic amino acid. The amino acid can be substituted with R, K, S, T, N, Q, G, A, V, L, I, P, C, F, Y or W. One or more modifications or mutations are preferably made to include polar, hydrophobic, positively charged or negatively charged residues to change the discrimination. The amino acid can be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y or W.

[0141] The variant preferably comprises one or more modifications or mutations which improve the ability of the spicule pore protein pore formed by the monomer to interact with a nucleotide or polynucleotide. Nucleotides and polynucleotides are discussed in more detail below. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0142] Nucleotides and polynucleotides are typically negatively charged. One or more modifications or mutations are preferably made to include positively charged, polar or hydrophobic amino acids in order to reduce the negative charge (thereby facilitating capture of the negatively charged analyte) or to improve / stabilise the interaction with the polynucleotide binding protein. The amino acid can be substituted with R, K, S, T, N, Q, G, A, V, L, I, P, C, F, Y or W. One or more modifications or mutations are preferably made to include polar, hydrophobic, positively charged or negatively charged to change the discrimination. The amino acid can be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y or W.

[0143] For the nucleotide or polynucleotide of interest, the one or more mutations are preferably selected from the following specific substitutions:

[0144] D is substituted with S, T, N, Q, A, V, L, I, F, R or K,

[0145] E is substituted with S, T, N, Q, A, V, L, I, F, R or K,

[0146] R is substituted with S, T, N, Q, A, V, L, I or F,

[0147] K is replaced by S, T, N, Q, A, V, L, I or F,

[0148] G is replaced by S, T, N, Q or A, V, L, I or F,

[0149] A is replaced by S, T, N, Q or G, V, L, I or F,

[0150] V is replaced by S, T, N, Q or G, A, L, I or F,

[0151] L is replaced by S, T, N, Q or G, A, V, I or F,

[0152] I is replaced by S, T, N, Q or G, A, V, L or F,

[0153] S is replaced by T, N, Q, A, V, L, I, F, R or K,

[0154] T is replaced by S, N, Q, A, V, L, I, F, R or K,

[0155] N is replaced by S, T, Q, A, V, L, I, F, R or K,

[0156] Q is replaced by S, T, N, A, V, L, I, F, R or K, and

[0157] H is replaced by F, N, Q or A.

[0158] For a nucleotide or polynucleotide of interest, the variant preferably comprises a modification or mutation at one or more positions corresponding to positions D8, E12, E15, E16, E25, D30, E38, E45, E57, E62, D65, E67, D70, E72, E88, D99, D113, E115, E122, D133, E134, D142, D185, D203, D205, D207, D211, D215, E226, E248, E254, and E256 in SEQ ID NO: 1. The variant can include any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of these positions. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to positions E67, D70, E72, E88, D113, E115, E134, D142, E248, and E256 in SEQ ID NO: 1. The variant can include any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of these positions. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to positions E67, D70, and E72 in SEQ ID NO: 1. These positions are at the N-terminus. The variant can include any number of modifications or mutations at these positions, such as 1, 2, or 3 of these positions. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to positions D113, E115, E134, D142, E248, and E256 in SEQ ID NO: 1. These positions are at the pore entrance. The variant can include any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, or 6 of these positions. The variant preferably comprises a modification or mutation at the position corresponding to position E88 in SEQ ID NO: 1. This position is at the transmembrane a-helix region. The one or more modifications are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications are preferably one or more substitutions.

[0159] The variant preferably comprises one or more modifications or mutations which alter the number of monomers forming the spicule pore protein pore. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The variant preferably comprises one or more substitutions which alter the number of monomers forming the spicule pore protein pore comprising monomers. The one or more modifications, mutations or substitutions preferably increase the number of monomers forming the spicule pore protein pore. The one or more modifications, mutations or substitutions preferably increase the number of monomers forming the spicule pore protein pore. The one or more modifications, mutations or substitutions preferably increase the number of monomers forming the spicule pore protein pore from 8 to 9. The variant preferably comprises a modification (such as an addition, deletion or substitution) or substitution at a position corresponding to D203 in SEQ ID NO: 1. The variant preferably comprises D203R / N / K / R / S / Y / F / A or D203R / K at the corresponding position. The variant most preferably comprises D203R at the corresponding position. This substitution increases the number of monomers forming the spicule pore protein pore from typically 8 to 8 / 9 in a 1 : 1 ratio Figure 7 ). These modifications, mutations or substitutions can be in addition to or instead of any modifications or mutations discussed above for improving the ability of the spicule pore protein pore to interact with a target analyte.

[0160] The variant is preferably a fragment of SEQ ID NO: 1 that lacks one or more amino acids from the N-terminus. This facilitates monomer insertion into artificial membranes. The fragment can lack any number of amino acids from the N-terminus of SEQ ID NO: 1, so long as the variant has at least about 40% identity to the sequence of SEQ ID NO: 1 over its entire length. The fragment preferably lacks up to about 108 amino acids from the N-terminus of SEQ ID NO: 1. The fragment can lack up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 108 amino acids from the N-terminus of SEQ ID NO: 1. The fragment preferably lacks at least about 53 amino acids from the N-terminus of SEQ ID NO: 1. The fragment preferably lacks about 53 amino acids from the N-terminus of SEQ ID NO: 1. The fragment preferably lacks at least about 67 amino acids from the N-terminus of SEQ ID NO: 1. The fragment preferably lacks about 67 amino acids from the N-terminus of SEQ ID NO: 1. The fragment preferably lacks at least about 76 amino acids from the N-terminus of SEQ ID NO: 1. The fragment preferably lacks about 76 amino acids from the N-terminus of SEQ ID NO: 1. The fragment can include any of the modifications or mutations discussed above for improving the ability of the spicule porin pore to interact with a target analyte and / or altering the number of monomers that form the spicule porin pore.

[0161] The spicule porin monomer of the application preferably is capable of inserting into an artificial membrane. The variant preferably comprises one or more modifications that facilitate insertion of the spicule porin monomer into an artificial membrane. Insertion can be measured using any of those described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, and WO 2019 / 002893 (all incorporated by reference in their entirety herein) and in the Examples.

[0162] Different N-terminal deletions from SEQ ID NO: 1 are capable of altering the ratio of spicule porin pore oligomers of different sizes, especially when combined with the modifications, mutations, or substitutions described above for altering the number of monomers. A variant of SEQ ID NO: 1 having D203R at the corresponding position and lacking 67 amino acids from the N-terminus of SEQ ID NO: 1 is capable of forming 8-mer and 9-mer spicule porin pore oligomers in a ratio of about 1:3. A variant of SEQ ID NO: 1 having D203R at the corresponding position and lacking 76 amino acids from the N-terminus of SEQ ID NO: 1 is capable of forming 8-mer and 9-mer spicule porin pore oligomers in a ratio of about 1:1.

[0163] The variant preferably is a fragment of SEQ ID NO: 1 lacking one or more amino acids from the C-terminus of SEQ ID NO: 1. The fragment can lack any number of amino acids from the C-terminus of SEQ ID NO: 1, so long as the variant has at least about 40% identity to the sequence of SEQ ID NO: 1 over its entire length. The fragment preferably lacks up to about 10 amino acids from the C-terminus of SEQ ID NO: 1. The fragment can lack up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from the C-terminus of SEQ ID NO: 1. The fragment preferably lacks up to or at least about 5 amino acids from the C-terminus of SEQ ID NO: 1. The fragment preferably lacks about 5 amino acids from the C-terminus of SEQ ID NO: 1. The fragment can include any of the modifications or mutations discussed above for improving the ability of the spicule porin pore to interact with a target analyte and / or altering the number of monomers forming the spicule porin pore. A fragment according to the application can lack one or more amino acids from the N-terminus of SEQ ID NO: 1 as well as one or more amino acids from the C-terminus of SEQ ID NO: 1.

[0164] Deletion of one or more amino acids from the C-terminus of SEQ ID NO: 1, such as deletion of about 5 amino acids from the C-terminus of SEQ ID NO: 1, in combination with a modification or mutation at a position corresponding to D203 in SEQ ID NO: 1, can also increase the number of monomers forming a cnidopore pore. A variant including a mutation or modification, such as a substitution, at a position corresponding to position D203 in SEQ ID NO: 1, such as D203R, and lacking 5 amino acids from the C-terminus of SEQ ID NO: 1 can form 9-mer and 10-mer cnidopore pore oligomers. SEQ ID NO: 2 .

[0165] The cnidopore monomer will typically retain the ability to form the same 3D structure as the wild-type cnidopore monomer, such as the same 3D structure as a cnidopore monomer having the sequence of SEQ ID NO: 1. Any number of modifications or mutations can be made in addition to the modifications or mutations described herein, provided that the cnidopore monomer retains the improved properties conferred to it by the modifications or mutations of the application.

[0166] Typically, the cnidopore monomer will retain the ability to form a structure comprising an alpha helix or a transmembrane alpha helix. It is therefore envisaged that further mutations can be made without affecting the monomer’s ability to form a cnidopore pore that can translocate an analyte. It is also expected that deletion of one or more amino acids can be made in any loop region and / or N-terminal and / or C-terminal region of the cnidopore monomer without affecting the monomer’s ability to form a pore that can translocate an analyte.

[0167] Amino acid substitutions can also be made to the amino acid sequence of SEQ ID NO: 1 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20, or 30 substitutions. Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acid can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acid it replaces. Alternatively, a conservative substitution can introduce another amino acid that is aromatic or aliphatic in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0168] The cnidopore monomer can be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more non-natural amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Any number and combination of such introductions can be made. The introduction is preferably made by substitution or addition.

[0169] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 1 can additionally be deleted from the above polypeptides. Up to 1, 2, 3, 4, 5, 10, 20, or 30 or more residues can be deleted.

[0170] One or more amino acids can alternatively or additionally be added to the above polypeptides. Extensions can be provided at the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof. Extensions can be very short, for example 1 to 10 amino acids in length. Alternatively, extensions can be longer, for example up to 50 or 100 amino acids. A carrier protein can be fused to the amino acid sequence according to the application. Other fusion proteins will be discussed in more detail below.

[0171] One or more modifications in the cnidocyanin monomer preferably improve the ability of the cnidocyanin pore to characterize an analyte comprising the cnidocyanin monomer. For example, consider modifications or substitutions to alter the number, size, shape, placement, or orientation of constriction within the channel from the cnidocyanin monomer of the application. The cnidocyanin monomer or variant of SEQ ID NO: 1 can have any of the particular modifications or substitutions disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, and WO 2019 / 002893 (all incorporated by reference in their entirety).

[0172] Any cnidocyanin monomer and / or variant based on SEQ ID NO: 1 described herein preferably further comprises the sequence GHM or the amino acid S at its N-terminus.

[0173] Figure 3

[0174] The present application provides a cnidocyanin monomer comprising a variant of SEQ ID NO: 2 having at least about 54% identity to the sequence of SEQ ID NO: 2 over its entire length. The variant preferably has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% identity to the sequence of SEQ ID NO: 2 over its entire length. The variant more preferably has at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 2 over its entire length.

[0175] Identity can be determined using standard methods in the art. For example, the BESTFIT program provided by the UWGCG software package can be used to calculate identity, for example under its default settings (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395). The PILEUP and BLAST algorithms can be used to calculate identity or to align sequences such as to identify equivalent or corresponding residues (typically under their default settings), as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F. et al. (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0176] Identity of the variant to SEQ ID NO: 2 is measured over the entire length of SEQ ID NO: 2. SEQ ID NO: 2 is 183 amino acids in length. Identity is typically determined by calculating the number of identical residues between the variant and SEQ ID NO: 2 using any of the above methods and dividing the number of identical residues by 183. For example, if the variant has 105 identical amino acids to SEQ ID NO: 2, then the variant has 105 / 183 x 100 = 57.38% identity to SEQ ID NO: 2 over its entire length.

[0177] The variant preferably comprises one or more modifications that (a) improve the ability of the pore formed by the monomer to interact with a target analyte, (b) change the number of pore-forming monomers, and / or (c) facilitate insertion of the pore-forming monomer or the pore formed by the monomer into an artificial membrane. The variant can include (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). Each of (a) to (c) is described in more detail below. The one or more modifications are preferably one or more mutations. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0178] The spicule porin monomer of the invention is generally capable of forming a pore or spicule porin pore. This can be measured using any of the conventional methods described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893 (all incorporated herein by reference in their entirety) and those described in the Examples.

[0179] The spicule porin monomer preferably comprises one or more of (a) a cap region, (b) a constriction region, and (c) a transmembrane alpha helix region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The spicule porin monomer preferably includes one or more regions that contribute to these regions in the spicule porin pore. The spicule porin monomer more preferably comprises one or more of the regions that form one or more of (a) to (c), such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The spicule porin pore formed by the monomer can have any structure, but preferably has or comprises the structure of a wild-type spicule porin pore. The protein structure of the spicule porin pore defines a channel or pore hole that allows for the translocation of molecules and ions from one side of the membrane to the other.

[0180] “Constriction”, “pore opening”, “constriction region”, “channel constriction” or “constriction site” as used interchangeably herein refers to an opening defined by the internal lumen surface of a pore or spicule porin pore that serves to allow passage of ions and target analytes, including but not limited to polynucleotides or individual nucleotides, through the pore or spicule porin pore channel, but not other non-target analytes. In some cases, for example when characterizing a protein or polypeptide target analyte, the constriction prevents the target analyte from passing through the pore. The constriction is generally the narrowest opening within or defined by the channel of the pore or spicule porin pore. The constriction can serve to limit passage of molecules through the pore. The size of the constriction is generally a key factor in determining the suitability of the pore or spicule porin pore for characterization of an analyte. If the constriction is too small, the molecule to be characterized will not be able to pass. However, to achieve the maximum impact on the flow of ions through the channel, the constriction structure should not be too large. For example, the constriction structure should not be wider than the solvent accessible lateral diameter of the target analyte. Ideally, the diameter of any constriction structure should be as close as possible to the lateral diameter of the analyte being passed.

[0181] SEQ ID NO: 2 shows the sequence of a wild-type nematocyst pore protein monomer derived from P. monticola as a mature protein (SEQ ID NO: 1) and lacking the first 76 amino acids from the N-terminus. Residues V28 to M183 of SEQ ID NO: 2 form the Cap region. Residues A4 to L13 of SEQ ID NO: 2 form the Pinch region. Residues R1 to N27 of SEQ ID NO: 2 form the Transmembrane alpha-helix region. The Pinch region is typically present in the Transmembrane alpha-helix region.

[0182] The nematocyst pore protein monomer comprises a variant of SEQ ID NO: 2. The variant monomer can also be referred to as a modified nematocyst pore protein monomer or a mutant nematocyst pore protein monomer. The modifications or mutations in the variant include, but are not limited to, any one or more of the modifications disclosed herein or a combination of the modifications. The nematocyst pore protein monomer can be a homologous monomer. A homologous monomer is a polypeptide that has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% overall sequence identity to SEQ ID NO: 2 over its entire length. The homologous monomer is typically a nematocyst pore protein monomer from a species other than P. monticola. The variant can comprise any substitutions present in the homologous monomer.

[0183] Sequence identity can also relate to fragments or portions of the nematocyst pore protein monomer. While the variant must have at least about 54% overall sequence identity to SEQ ID NO: 2 over its entire length, the sequence of particular regions, domains, or subunits can share at least about 80%, 90%, or up to about 99% sequence identity with the corresponding region of SEQ ID NO: 2. There can be at least about 80%, e.g., at least about 85%, 90%, or 95% identity over a stretch of 100 or more, e.g., 125, 150, 175, or 200 or more contiguous or abutting amino acids (“hard identity”). The nematocyst pore protein monomer preferably comprises a variant having at least about 40% identity to the Cap region of SEQ ID NO: 2. More preferably, the variant has at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and more preferably at least about 95%, 97%, or 99% identity to the Cap region of SEQ ID NO: 2. The Cap region is residues V28 to M183 of SEQ ID NO: 2.

[0184] The cnidocyte porin monomer preferably comprises a variant having at least about 54% identity to the coiled-coil region of SEQ ID NO: 2. More preferably, the variant has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and more preferably at least about 95%, 97%, or 99% identity to the coiled-coil region of SEQ ID NO: 2. The coiled-coil region is residues A4 to L13 of SEQ ID NO: 2.

[0185] The cnidocyte porin monomer preferably comprises a variant having at least about 54% identity to the transmembrane a-helix region of SEQ ID NO: 2. More preferably, the variant has at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and more preferably at least about 95%, 97%, or 99% identity to the transmembrane a-helix region of SEQ ID NO: 2. The transmembrane a-helix region is residues R1 to N27 of SEQ ID NO: 2.

[0186] The variant preferably comprises one or more modifications that improve the ability of the cnidocyte porin pore formed by the monomer to interact with a target analyte. The one or more modifications are preferably one or more mutations. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions. Target analytes are discussed in more detail below. The target analyte is preferably an amino acid, a polypeptide, a protein, a nucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, a polysaccharide.

[0187] The cnanopore monomer preferably forms a cnanopore pore with improved target analyte reading properties, i.e., showing improved analyte capture and discrimination. In particular, the cnanopore pore constructed from the cnanopore monomer preferably captures amino acids, polypeptides, proteins, nucleotides, and polynucleotides more readily than wild type. In addition, the cnanopore pore constructed from the cnanopore monomer preferably shows increased current range, which makes it easier to discriminate between different analytes, and reduced state variance, which increases the signal-to-noise ratio. In addition, the number of analytes contributing to the current or the number of polymer subunits in a polymer (e.g., the number of amino acids in a polypeptide or the number of nucleotides in a polynucleotide) is reduced as they move through the cnanopore pore constructed from the monomer. This makes it easier to identify the direct relationship between the current observed as an analyte moves through the cnanopore pore and the sequence of the analyte. The improvement in analyte reading performance of the cnanopore pore is achieved through five main mechanisms, i.e., by altering: steric hindrance (increasing or decreasing the size of amino acid residues); charge (e.g., introducing positive charges to interact with nucleic acid sequences); hydrogen bonding (e.g., introducing amino acids that can form hydrogen bonds with base pairs); pi stacking (e.g., introducing amino acids that interact through delocalized electronic pi systems); and / or changes in pore structure (e.g., introducing amino acids that increase the size of vestibules and / or constriction).

[0188] Any one or more of these five mechanisms can be responsible for the improved properties of the cnanopore pores of the present invention. For example, the cnanopore pores of the present invention can show improved analyte reading properties due to altered steric hindrance, altered hydrogen bonding, and altered structure.

[0189] Introduction of bulky residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H), in the cnanopore monomer increases the steric hindrance of the cnanopore pore. Introduction of aromatic residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H), in the cnanopore monomer also increases pi stacking in the cnanopore pore. Introduction of bulky or aromatic residues in the cnanopore monomer also alters the structure of the cnanopore pore, e.g., by opening the pore and increasing the size of vestibules and / or constriction. This is described in more detail below.

[0190] The variant can comprise one or more modifications or mutations at one or more positions in any combination of (a) the cap region, (b) the constriction region, and (c) the transmembrane alpha helix region, i.e., in (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b), and (c).

[0191] The cap region preferably comprises a modification or mutation at one or more positions corresponding to N29, K31, A33, G35, D37, E39, G41-R43, Q45, E46, D57-N59, P61, S65-Y68, L71, G73, R75, N78, S95, I96, K98, S123, N125, D127, D139, S155, G156, Q168, T170, E172, H174, L176-C182 in SEQ ID NO: 2. The one or more modifications or mutations are preferably one or more substitutions of an amino acid at a position having G, A, V, L, I, P, C, S, T, N, Q, F, Y, W, D, H, E, R, or K.

[0192] The constriction region preferably comprises a modification or mutation at one or more positions corresponding to the positions in SEQ ID NO: 2 shown in Column 1. In each row, the amino acid at the position corresponding to the position in SEQ ID NO: 2 shown in Column 1 is preferably substituted with one of the amino acids in Column 3. Column 2 indicates whether the position is inwardly directed toward the pore vestibule / channel (“in”) or outwardly directed toward the pore exterior (“out”).

[0193]

[0194] The transmembrane region preferably comprises a modification or mutation at one or more positions corresponding to the positions in SEQ ID NO: 2 shown in Column 1. In each row, the amino acid at the position corresponding to the position in SEQ ID NO: 2 shown in Column 1 is preferably substituted with one of the amino acids in Column 3. Column 2 indicates whether the position is inwardly directed toward the pore vestibule / channel (“in”) or outwardly directed toward the pore exterior (“out”).

[0195]

[0196] The variant can comprise any number of one or more modifications or mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modifications or mutations. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0197] The one or more modifications or mutations are each independently (a) alter the size of the amino acid residue at the modification position; (b) alter the net charge of the amino acid residue at the modification position; (c) alter the hydrogen bonding characteristics of the amino acid residue at the modification position; (d) introduce or remove one or more chemical groups that interact through delocalized electron π systems in the amino acid residue at the modification position and / or (e) alter the structure of the amino acid residue at the modification position. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0198] The variant preferably comprises one or more modifications or mutations that improve the ability of the spicule porin pore formed by the monomer to interact with an amino acid, polypeptide, or protein. The terms "polypeptide" and "protein" are interchangeable and discussed in more detail. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications are preferably one or more substitutions.

[0199] Depending on the charge of the amino acid or polypeptide analyte, the variant preferably comprises one or more modifications or mutations that reduce the same net charge of the monomer. If the amino acid or polypeptide analyte is positively charged, the variant preferably comprises one or more modifications or mutations that reduce the net positive charge of the monomer. If the amino acid or polypeptide analyte is negatively charged, the variant preferably comprises one or more modifications or mutations that reduce the net negative charge of the monomer. This reduces repulsion and facilitates capture of the amino acid or polypeptide analyte.

[0200] If the amino acid or polypeptide analyte is positively charged, the one or more modifications or mutations are preferably made to include a negatively charged, polar, or hydrophobic amino acid so as to reduce the positive charge (thereby facilitating capture of positively charged analytes). For example, a positively charged amino acid, such as R, H, or K, can be substituted with a negatively charged, polar, or hydrophobic amino acid. The amino acid can be substituted with D, E, S, T, N, Q, G, A, V, L, I, P, C, F, Y, or W. The one or more modifications or mutations are preferably made to include a polar, hydrophobic, positive, or negative charge to change the discrimination. The amino acid can be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y, or W.

[0201] If the amino acid or polypeptide analyte is positively charged, the one or more modifications or mutations are preferably selected from the following specific substitutions:

[0202] R is substituted with D, E, S, T, N, Q, G, A, V, L, I, C, F, Y, or W,

[0203] K is replaced by D, E, S, T, N, Q, G, A, V, L, I, C, F, Y, or W.

[0204] D is replaced by S, T, N, Q, G, A, V, L, I, C, F, Y, or W, preferably D is replaced by N, Q, or A.

[0205] E is replaced by S, T, N, Q, G, A, V, L, I, C, F, Y, or W, preferably E is replaced by N, Q, or A.

[0206] S is replaced by E, D, T, N, Q, A, V, L, or I, preferably S is replaced by E or D.

[0207] T is replaced by E, D, S, N, Q, A, V, L, or I, preferably T is replaced by E or D.

[0208] N is replaced by E, D, S, T, Q, A, V, L, or I, preferably N is replaced by E or D, and

[0209] Q is replaced by E, D, S, T, N, A, V, L or I, preferably Q is replaced by E or D.

[0210] If the amino acid or peptide analyte is positively charged, the variant preferably contains a modification or mutation at one or more positions corresponding to R1, I15, D23, V24, L25, D127, L176, and E178 in SEQ ID NO: 2. The variant may contain any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, 6, 7, or 8 of these positions. The variant preferably contains modifications or mutations at all positions corresponding to R1, I15, D23, V24, L25, D127, L176, and E178 in SEQ ID NO: 2. One or more modifications or mutations are preferably one or more substitutions. The amino acids at these positions may be mutated in any of the ways described above. The variant preferably contains a modification or mutation at the position corresponding to D139 in SEQ ID NO: 2. This variant preferably contains a modification or mutation at one or more (such as all) of the positions corresponding to R1, I15, D23, V24, L25, and D139 in SEQ ID NO: 2. This variant preferably contains a modification or mutation at one or more (such as all) of the positions corresponding to D139, L176, and E178 in SEQ ID NO: 2. Any of these variants preferably further includes a mutation or modification at position D127, preferably a substitution. This will be discussed in more detail below.

[0211] If the amino acid or polypeptide analyte is positively charged, the variant preferably comprises:

[0212] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D127R / D139R / L176T / E178A,

[0213] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D127R / D139R / L176T / E178A,

[0214] D217R,

[0215] D139R,

[0216] D127R / D139R,

[0217] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D139R,

[0218] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D127R / D139R / L176T / E178A,

[0219] one or more or all of D139R / L176T / E178A,

[0220] one or more or all of D127R / D139R / L176T / E178A,

[0221] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D139N / L176T / E178A,

[0222] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D127R / D139N / L176T / E178A,

[0223] D139N,

[0224] D127R / D139N,

[0225] one or more or all of R1 D / I15F / D23Y / V24F / L25F / D139N,

[0226] one or more or all of R1D / I15F / D23Y / V24F / L25F / D127R / D139N,

[0227] one or more or all of D139N / L176T / E178A, or

[0228] one or more or all of D127R / D139N / L176T / E178A.

[0229] Examples of positively charged polypeptides and proteins are provided below.

[0230] If the amino acid or polypeptide analyte is negatively charged, one or more modifications or mutations are preferably made to include positively charged, polar, or hydrophobic amino acids in order to reduce the negative charge (thereby facilitating capture of the negatively charged analyte) or to improve / stabilize the interaction with the polynucleotide binding protein (discussed in more detail below). For example, a negatively charged amino acid, such as D or E, can be substituted with a positively charged, polar, or hydrophobic amino acid. The amino acid can be substituted with R, K, S, T, N, Q, G, A, V, L, I, P, C, F, Y, or W. One or more modifications or mutations are preferably made to include a polar, hydrophobic, positive charge, or negative charge to change the distinction. The amino acid can be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y, or W.

[0231] The variant preferably comprises one or more modifications or mutations that improve the ability of the spicule pore protein pore formed by the monomer to interact with a nucleotide or polynucleotide. Nucleotides and polynucleotides are discussed in more detail below. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0232] Nucleotides and polynucleotides are typically negatively charged. One or more modifications or mutations are preferably made to include positively charged, polar, or hydrophobic amino acids in order to reduce the negative charge (thereby facilitating capture of the negatively charged analyte) or to improve / stabilize the interaction with the polynucleotide binding protein. The amino acid can be substituted with R, K, S, T, N, Q, G, A, V, L, I, P, C, F, Y, or W. One or more modifications or mutations are preferably made to include a polar, hydrophobic, positive charge, or negative charge to change the distinction. The amino acid can be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y, or W.

[0233] For the nucleotide or polynucleotide of interest, the one or more mutations are preferably selected from the following specific substitutions:

[0234] D is replaced by S, T, N, Q, A, V, L, I, F, R or K,

[0235] E is replaced by S, T, N, Q, A, V, L, I, F, R or K,

[0236] R is replaced by S, T, N, Q, A, V, L, I or F,

[0237] K is replaced by S, T, N, Q, A, V, L, I or F,

[0238] G is replaced by S, T, N, Q or A, V, L, I or F,

[0239] A is replaced by S, T, N, Q or G, V, L, I or F,

[0240] V is replaced by S, T, N, Q or G, A, L, I or F,

[0241] L is replaced by S, T, N, Q or G, A, V, I or F,

[0242] I is replaced by S, T, N, Q or G, A, V, L or F,

[0243] S is replaced by T, N, Q, A, V, L, I, F, R or K,

[0244] T is replaced by S, N, Q, A, V, L, I, F, R or K,

[0245] N is replaced by S, T, Q, A, V, L, I, F, R or K,

[0246] Q is replaced by S, T, N, A, V, L, I, F, R or K, and

[0247] H is replaced by F, N, Q or A.

[0248] For a target nucleotide or polynucleotide, the variant preferably comprises a modification or mutation at one or more positions corresponding to positions E12, D23, D37, E39, E46, D57, E58, D66, D109, D127, D129, D131, D135, D139, E150, E172, E178, and E180 in SEQ ID NO: 2. The variant can comprise any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of these positions. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to positions E12, D37, E39, E58, D66, E172, and E180 in SEQ ID NO: 2. The variant can comprise any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, 6, or 7 of these positions. The variant preferably comprises a modification or mutation at one or more (such as all) of the positions corresponding to positions D37, E39, E58, D66, E172, and E180 in SEQ ID NO: 2. These positions are at the pore entrance. The variant can comprise any number of modifications or mutations at these positions, such as 1, 2, 3, 4, 5, or 6 of these positions. The variant preferably comprises a modification or mutation at the position corresponding to position E12 in SEQ ID NO: 2. This position is in the transmembrane a-helix region. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The one or more modifications or mutations are preferably one or more substitutions.

[0249] The variant preferably comprises one or more modifications or mutations that alter the number of monomers forming the virion pore protein pore. The one or more modifications or mutations are preferably one or more additions, one or more deletions, one or more substitutions, or any combination thereof. The variant preferably comprises one or more substitutions that alter the number of monomers forming the virion pore protein pore comprising monomers. The one or more modifications, mutations, or substitutions preferably increase the number of monomers forming the virion pore protein pore from 8 to 9. The variant preferably comprises a modification (such as an addition, deletion, or substitution) or substitution at a position corresponding to D127 in SEQ ID NO: 2. The variant preferably comprises D127R / N / K / R / S / Y / F / A or D127R / K at the corresponding position. The variant most preferably comprises D127R at the corresponding position. This substitution increases the number of monomers forming the virion pore protein pore from typically 8 to 8 / 9 in a 1:1 ratio Figure 7 ). These modifications, mutations, or substitutions can be in addition to or instead of any modifications or mutations discussed above for improving the ability of the virion pore protein pore to interact with a target analyte.

[0250] The variant is preferably a fragment lacking one or more amino acids starting from the N-terminus of SEQ ID NO: 2. This facilitates monomer insertion into an artificial membrane. The fragment can lack any number of amino acids starting from the N-terminus of SEQ ID NO: 2, provided that the variant shares at least about 54% identity with the sequence of SEQ ID NO: 2 over its entire length. The fragment is preferably lacking up to about 32 amino acids starting from the N-terminus of SEQ ID NO: 2. The fragment can lack up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids starting from the N-terminus of SEQ ID NO: 2.

[0251] The nematoporin monomer of the present invention is preferably capable of intercalation into an artificial membrane. This variant preferably comprises one or more modifications that facilitate the intercalation of the nematoporin monomer into the artificial membrane. Intercalation can be measured using conventional methods, including any of WO2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, and WO2019 / 002893 (all of which are incorporated herein by reference in their entirety) and those described in the examples.

[0252] The variant is preferably a fragment lacking one or more amino acids starting from the C-terminus of SEQ ID NO: 2. The fragment may lack any number of amino acids starting from the C-terminus of SEQ ID NO: 2, provided that the variant has at least about 54% identity with the sequence of SEQ ID NO: 2 over its entire length. The fragment is preferably lacking up to about 10 amino acids starting from the C-terminus of SEQ ID NO: 2. The fragment may lack up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids starting from the C-terminus of SEQ ID NO: 2. The fragment is preferably lacking up to or at least about 5 amino acids starting from the C-terminus of SEQ ID NO: 2. The fragment is preferably lacking about 5 amino acids starting from the C-terminus of SEQ ID NO: 2. The fragment may include any modifications or mutations discussed above for improving the ability of nematoporin pores to interact with target analytes and / or altering the number of monomers forming nematoporin pores. The fragment according to the invention may lack one or more amino acids from the N-terminus of SEQ ID NO: 2 and one or more amino acids starting from the C-terminus of SEQ ID NO: 2.

[0253] Deletion of one or more amino acids from the C-terminus of SEQ ID NO: 2, such as deletion of about 5 amino acids from the C-terminus of SEQ ID NO: 2, in combination with a modification or mutation at a position corresponding to D127 in SEQ ID NO: 2, can also increase the number of monomers forming a pore of the cnidocyanin. A variant including a mutation or modification (such as a substitution) at a position corresponding to position D127 in SEQ ID NO: 2 (such as D127R) and lacking 5 amino acids from the C-terminus of SEQ ID NO: 2 can form 9-mer and 10-mer cnidocyanin pore oligomers Construct .

[0254] The cnidocyanin monomer will typically retain the ability to form the same 3D structure as the wild-type cnidocyanin monomer, such as the same 3D structure as the cnidocyanin monomer having the sequence of SEQ ID NO: 2. Any number of modifications or mutations can be made in addition to the modifications or mutations described herein, provided that the cnidocyanin monomer retains the improved properties conferred to it by the modifications or mutations of the application.

[0255] Typically, the cnidocyanin monomer will retain the ability to form a structure comprising alpha helices. It is therefore envisaged that further mutations can be made without affecting the ability of the monomer to form a cnidocyanin pore that can translocate an analyte. It is also expected that deletion of one or more amino acids can be made in any loop region and / or N-terminal and / or C-terminal region of the cnidocyanin monomer without affecting the ability of the monomer to form a pore that can translocate an analyte.

[0256] In addition to those discussed above, amino acid substitutions can be made to the amino acid sequence of SEQ ID NO: 2, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties or similar side chain volume. The introduced amino acid can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, a conservative substitution can introduce another amino acid that is aromatic or aliphatic in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0257] The cnidocyanin monomer can be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more non-natural amino acids, one or more polar amino acids or one or more photoreactive amino acids. Any number and combination of such introductions can be made. The introduction is preferably made by substitution or addition.

[0258] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 can additionally be deleted from the above polypeptides. Up to 1, 2, 3, 4, 5, 10, 20, or 30 or more residues can be deleted.

[0259] One or more amino acids can alternatively or additionally be added to the above polypeptides. Extensions can be provided at the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 2 or a variant or fragment thereof. Extensions can be very short, for example 1 to 10 amino acids in length. Alternatively, extensions can be longer, for example up to 50 or 100 amino acids. A carrier protein can be fused to the amino acid sequence according to the application. Other fusion proteins will be discussed in more detail below.

[0260] One or more modifications in the cnanopore monomer preferably improve the ability of the cnanopore monomer comprising the cnanopore pore to characterize an analyte. For example, consider modifications, mutations / substitutions to alter the number, size, shape, placement, or orientation of constriction within the channel from the cnanopore monomer of the application. The cnanopore monomer or variant of SEQ ID NO: 2 can have any of the particular modifications or substitutions disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, and WO 2019 / 002893 (all incorporated by reference in their entirety).

[0261] Any cnanopore monomer based on SEQ ID NO: 2 and / or variant described herein preferably further comprises the sequence GHM or the amino acid S at its N-terminus.

[0262] Pore of the present invention

[0263] The present application also provides a construct comprising two or more covalently attached spicule porin monomers of the present application. The construct can comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more spicule porin monomers of the present application. The construct can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 spicule porin monomers of the present application. The two or more spicule porin monomers can be the same or different. The two or more spicule porin monomers can differ based on one or more of (a) the sequence of the spicule porin monomer, (b) the linker, and (c) the attachment position on the spicule porin monomer. The spicule porin monomers can differ based on (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The two or more spicule porin monomers are preferably the same (i.e., identical).

[0264] The construct preferably comprises two spicule porin monomers. The two or more spicule porin monomers can be the same or different. The two or more spicule porin monomers are preferably the same (i.e., identical).

[0265] The spicule porin monomers can be genetically fused, optionally via a linker, or chemically fused, e.g., via a chemical crosslinker. Methods for covalently attaching monomers are disclosed in WO 2017 / 149316, WO 2017 / 149317, and WO 2017 / 149318 (incorporated by reference in their entirety).

[0266] The linker is preferably an amino acid sequence and / or a chemical crosslinker. Suitable amino acid linkers, such as peptide linkers, are known in the art. The length, flexibility, and hydrophilicity of the amino acid or peptide linker are generally designed such that the spicule porin monomers in the construct are in the correct orientation to form a spicule porin pore. Flexible and rigid linkers useful in the construct are discussed above.

[0267] Suitable chemical crosslinkers are well known in the art. Suitable chemical crosslinkers include, but are not limited to, those comprising the following functional groups: maleimides, active esters, succinimides, azides, alkynes (such as dibenzocyclooctyne alcohols (DIBO or DBCO), difluorocycloalkynes, and linear alkynes), phosphines (such as those for traceless and non-traceless Staudinger ligation), haloacetyl groups (such as iodoacetamide), phosgene-type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulfides, vinyl sulfones, aziridines, and photo-reactive reagents (such as aryl azides, diaziridines).

[0268] Reactions between amino acids and functional groups can be spontaneous, such as cysteine / maleimide, or can require an external reagent, such as Cu(I) for the coupling of azides and linear alkynes.

[0269] Linkers can comprise any molecule that extends across the desired distance. The length of the linker can vary from one carbon (a phosgene-type linker) to many Angstroms. Examples of linker molecules include, but are not limited to, polyethylene glycol (PEG), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, polyamides. These linkers can be inert or reactive, in particular they can be chemically cleaved at defined positions, or they can themselves be modified with fluorophores or ligands. The linker is preferably resistant to reducing agents such as dithiothreitol (DTT) after covalent linkage.

[0270] Cross-linking agents include 2,5-dioxopyrrolidin-1-yl 3-(pyridine-2- yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(pyridine-2- yldisulfanyl)butanoate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridine-2- yldisulfanyl)octanoate, dimaleimide PEG 1k, dimaleimide PEG 3.4k, dimaleimide PEG 5k, dimaleimide PEG 10k, bis(maleimido)ethane (BMOE), bis-maleimide hexane (BMH), 1,4-bismaleimide butane (BMB), 1,4 bis-maleimido-2,3-dihydroxybutane (BMDB), BM[PEO]2 (1,8-bismaleimido diethylene glycol), BM[PEO]3 (1,11-bismaleimido triethylene glycol), tris[2-maleimidoethyl]amine (TMEA), DTME dithiobis-maleimidoethane, bis-maleimide PEG 3, bis-maleimide PEG 11, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8 kDa, DBCO-PEG-DBCO 4.0 kDa, DBCO-15 atom-DBCO, DBCO-26 atom-DBCO, DBCO-35 atom-DBCO, DBCO-PEG4-S-S-PEG3-biotin, DBCO-S-S-PEG3-biotin, DBCO-S-S-PEG11-biotin, (succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and maleimide-PEG(2kDa)-maleimide (alpha, omega-bismaleimide poly(ethylene glycol)). One cross-linking agent is maleimide-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

[0271] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0272] The cnidocyanin monomers can be linked using two or more linkers, each comprising a hybridizable region and a group capable of forming a covalent bond. The hybridizable regions in the linkers hybridize and link the cnidocyanin monomers. The linked cnidocyanin monomers are then coupled via formation of a covalent bond between the groups. Any of the specific linkers disclosed in WO 2010 / 086602 (incorporated by reference in its entirety) can be used according to the present invention.

[0273] The linkers can be labeled. Suitable labels include, but are not limited to, fluorescent molecules (such as Cy3 or Alexa Fluor® 555), radioisotopes (e.g. 125 I, 35 S, 32 P), enzymes, antibodies, antigens, polynucleotides, and ligands (such as biotin). Such labels allow for quantification of the amount of linker. The label can also be a cleavable purification tag, such as biotin, or a specific sequence displayed in an identification method, such as a peptide not present in the protein itself but released by trypsin digestion.

[0274] The method of linking the cnidocyanin monomers is via a cysteine linkage. This can be mediated by a bifunctional chemical crosslinker or by a polypeptide linker with a terminal cysteine residue present.

[0275] Another method of linking is via a 4-azidophenylalanine or Faz linkage. This can be mediated by a bifunctional chemical linker or by a polypeptide linker with a terminal 4-azidophenylalanine or Faz residue present. Other suitable linkers are discussed in more detail below.

[0276] Figure 33

[0277] The terms "cnidocyanin pore," "cnidocyanin pore oligomer," and "cnidocyanin pore complex," as used interchangeably herein, refer to a complex comprising at least one cnidocyanin monomer of the present invention (including, for example, one or more cnidocyanin monomers, such as two or more cnidocyanin monomers, three or more cnidocyanin monomers, etc.). The cnidocyanin pore of the present invention has the characteristics of a biological pore, i.e., it has a typical protein structure and defines a channel. When the cnidocyanin pore is provided in an environment having a membrane component, a membrane, a cell, or an insulating layer, the cnidocyanin pore will insert into the membrane or insulating layer and form a "transmembrane cnidocyanin pore."

[0278] The cnanopore pore preferably has or comprises any of the structures discussed above with respect to the cnanopore monomers of the present application. The cnanopore pores of the present application include cnanopore pores having two constrictions, i.e., two channels are positioned in a manner that the accuracy of one constriction is not interfered with by the other. One of skill in the art is able to create multiple constrictions in a pore, for example, using a helper peptide attached (preferably covalently attached) to each cnanopore monomer in the cnanopore pore. These types of modifications are described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2019 / 002893, WO 2017 / 149318, WO 2018 / 211241, and WO 2019 / 002893 (all incorporated by reference in their entirety). The cnanopore pores of the present application include cnanopore pores having one constriction.

[0279] The cnanopore pore can be any size, but preferably has the dimensions of a wild-type cnanopore pore. Figure 33 The cnanopore pore formed from monomers preferably comprises a cap region and a transmembrane a-helix region.

[0280] The cnanopore pore preferably has an outer diameter of about 6.0 to about 15.0 nm at its widest point, such as an outer diameter of about 8.0 to about 13.0 nm or about 9.0 to about 12.0 nm at its widest point. The cnanopore pore preferably has an outer diameter of about 10.5 nm or 11.5 nm (for octamers and nonamers, respectively) at its widest point. The cnanopore pore preferably has a total length of about 3.0 to about 15.0 nm, such as about 5.0 to about 9.0 nm or about 6.0 to about 8.0 nm. The cnanopore pore preferably has a total length of about 7.0 nm. References to “total length” and “length” relate to the length of the pore or pore region when viewed from the side (see, e.g., Figure 33 the side view in b).

[0281] The cap region preferably has a length of about 2.0 to about 6.0 nm, such as about 3.0 to about 5.0 nm or about 3.5 to about 4.5 nm. The cap region preferably has a length of about 4.0 nm. The channel defined by the cap region preferably has an opening with a diameter of about 4.0 to about 10.0 nm, such as an opening with a diameter of about 5.0 to about 9.0 nm or about 6.0 to about 8.0 nm. The channel defined by the cap region preferably has an opening with a diameter of about 6.8 nm or 7.7 nm (for octamers and nonamers, respectively).

[0282] The transmembrane alpha helix region preferably has a length of about 2.0 to about 11.0 nm, such as about 2.5 to about 8.0 nm, about 2.7 to about 5.0 nm, or about 3.0 to about 4.0 nm. The transmembrane alpha helix region preferably has a length of about 3.5 nm. The transmembrane alpha helix region preferably has a length of about 3.5 nm. The diameter of the channel defined by the transmembrane alpha helix region at its narrowest point is preferably about 0.5 to about 4.5 nm, such as about 1.0 to about 3.5 nm or about 1.5 to about 3.0 nm in diameter at its narrowest point. The diameter of the channel defined by the transmembrane alpha helix region at its narrowest point is preferably about 2.2 nm or about 2.8 nm (for 8-mer and 9-mer, respectively). The narrowest point typically forms a constriction.

[0283] All of the above measurements are based on measurements from the main chain of the amino acids forming the different regions to the main chain of the Pore of the present invention Measured from van der Waals surface to van der Waals surface.

[0284] The present application provides a porin pore comprising at least one porin monomer of the present application. The porin pore typically comprises at least 6, 7, 8, 9, or 10 porin monomers of the present application. The porin pore preferably comprises 7, 8, 9, or 10 porin monomers of the present application. The porin monomers are typically identical (i.e., the same). The larger the number of porin monomers in the porin pore oligomer, the larger the pore formed by the porin pore oligomer. Larger pores allow for the detection or characterization of larger analytes.

[0285] The porin pore is preferably a homooligomer comprising 6 to 10, such as 6, 7, 8, 9, or 10 porin monomers of the present application. The porin monomers are typically identical. The porin pore preferably comprises 8, 9, or identical porin monomers of the present application. The porin pore preferably comprises 8 or 9 identical porin monomers of the present application. The porin pore preferably comprises 8 identical porin monomers of the present application. The porin pore preferably comprises 9 identical porin monomers of the present application. The porin monomers can be any of those discussed above.

[0286] The present invention provides a porin pore comprising at least one construct of the present invention. The porin pore typically comprises at least 1, 2, 3, 4, or 5 constructs of the present invention. The porin pore comprises sufficient porin monomers to form a pore. For example, an octameric porin pore can comprise (a) four constructs, each comprising two porin monomers, (b) two constructs, each comprising four porin monomers, (c) one construct comprising two porin monomers and six porin monomers that are not part of a construct, (d) three constructs comprising two porin monomers and two porin monomers that are not part of a construct, and (e) combinations thereof. The same and additional possibilities are provided for nonameric porin pores or decameric porin pores, for example. Other combinations of constructs and monomers can be envisioned by the skilled person. One or more constructs of the present invention can be used to form a porin pore for characterizing (such as sequencing) an analyte. The porin pore preferably comprises four constructs of the present invention, each comprising two porin monomers. The constructs are typically identical (i.e., the same).

[0287] The porin pore is preferably a homo-oligomer comprising 1 to 5, such as 1, 2, 3, 4, 5 constructs of the present invention. The constructs are typically identical (i.e., the same). The porin pore preferably comprises four identical constructs of the present invention, each comprising two porin monomers. The constructs can be any of those discussed above.

[0288] The porin monomers in the porin pore preferably all have approximately the same length or have the same length. The helical regions of the porin monomers of the present invention in the pore preferably have approximately the same length or have the same length. Length can be measured in number of amino acids and / or length units.

[0289] The porin pore of the present invention can be isolated, substantially isolated, purified, or substantially purified. The porin pore of the present invention is isolated or purified if it is completely free of any other components, such as lipids or other pores. The porin pore is substantially isolated if it is mixed with a carrier or diluent that will not interfere with its intended use. For example, the porin pore is substantially isolated or substantially purified if it is present in a form that comprises less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as block copolymers, lipids, or other pores. Alternatively, the porin pore of the present invention can be present in a membrane. Suitable membranes are discussed below.

[0290] The spicule porin pore of the application can exist as a single or unitary spicule porin pore. Alternatively, the spicule porin pore of the application can exist in a homologous or heterologous population of two or more spicule porin pores or pores. For example, the spicule porin pore can exist in a heterologous population of two or more spicule porin pores formed from different numbers of monomers. Other forms involving the spicule porin pore of the application are discussed in more detail below.

[0291] Membrane embodiment

[0292] The application also provides a pore multimer comprising two or more pores, wherein at least one of the pores is a spicule porin pore of the application. The multimer can comprise any number of pores, such as 3, 4, 5, 6, 7 or 8 or more pores. Any number of the pores in the multimer, including all of them, can be a spicule porin pore of the application.

[0293] The pore multimer can be a dual spicule porin pore comprising a first spicule porin pore and a second spicule porin pore of the application. Both the first spicule porin pore and the second spicule porin pore are preferably spicule porin pores of the application. In the dual spicule porin pore, the first spicule porin pore can be attached to the second spicule porin pore by hydrophobic interactions and / or by one or more disulphide bonds. One or more of the first spicule porin pore and / or the second spicule porin pore, such as 2, 3, 4, 5, 6, 8, 9, for example all of the monomers, can be modified to enhance such interactions. This can be achieved in any suitable way. Particular methods of forming dual pores are described in WO 2019 / 002893 (incorporated herein by reference in its entirety).

[0294] The pore multimer of the application can be isolated, substantially isolated, purified or substantially purified. Such terms are defined above with reference to the spicule porin pore of the application.

[0295] The pore multimer of the application can be used in any membrane, method, use, kit, apparatus, array or system of the application described below.

[0296] Method for making modified proteins

[0297] The application also provides a spicule porin pore of the application or a pore multimer of the application comprised in a membrane. The membrane is preferably an artificial membrane. Artificial has its normal meaning in the art. An artificial membrane is a membrane that does not exist in nature. The skilled person is able to determine whether a membrane is naturally occurring or artificial. Suitable artificial membranes are discussed in more detail below.

[0298] The present application also provides an artificial membrane comprising a pore derived from a cnidarin from a mountain star coral. The cnidarin pore can be any cnidarin pore derived from a mountain star coral, including a wild type cnidarin pore. The cnidarin pore can comprise the sequence set forth in SEQ ID NO: 1. The cnidarin pore is preferably a cnidarin pore of the present application or a pore multimer of the present application. These products are directly applicable to molecular sensing, such as analyte characterization and sequencing. Suitable membranes are discussed in more detail below.

[0299] Method for producing a pore-forming protein monomer

[0300] Methods for introducing or substituting non-naturally occurring amino acids in a cnidarin monomer are also well known in the art and described in WO 2019 / 002893 (incorporated by reference in its entirety). Proteins can be modified to aid in their identification or purification, for example by the addition of a streptavidin tag or by the addition of a signal sequence to facilitate their secretion from a cell in which the monomer does not naturally contain such a sequence. Proteins can also be produced using a mixture of D-amino acids or L-amino acids and D-amino acids. This is routine in the art of producing such proteins or peptides.

[0301] A cnidarin monomer or any protein described herein can be chemically modified. A protein can be chemically modified in any way and at any site. A protein can be chemically modified by attachment of a molecule to one or more cysteines (cysteine ligation), attachment of a molecule to one or more lysines, attachment of a molecule to one or more unnatural amino acids, enzymatic modification of an epitope or modification of a terminus. Suitable methods for making such modifications are well known in the art. A protein can be chemically modified by attachment of any molecule, for example a dye or a fluorophore.

[0302] A protein can be chemically modified with a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target nucleotide or a target polynucleotide sequence. Suitable adaptors, including cyclic molecules, cyclodextrins, hybridisable species, DNA binding agents or mutual chelators, peptides or peptide analogues, synthetic polymers, aromatic planar molecules, positively charged small molecules or small molecules capable of hydrogen bonding, are described in WO 2019 / 002893 (incorporated by reference in its entirety). Any of the methods and linkers discussed above can be used to attach the molecular adaptor.

[0303] The protein, such as the spicule pore protein monomer of the present invention, can be attached to a polynucleotide binding protein. This forms a modular sequencing system that can be used in the sequencing method of the present invention. Polynucleotide binding proteins are polymerases, exonucleases, helicases, and topoisomerases, such as gyrase. Suitable polynucleotide binding proteins include, but are not limited to, exonuclease I from E. coli, exonuclease III from E. coli, RecJ from S. thermophilus, and bacteriophage lambda exonuclease, TatD exonuclease, and variants thereof. Three subunits comprising RecJ sequence from S. thermophilus or variants thereof interact to form a trimeric exonuclease. The polymerase can be PyroPhage® 3173 DNA polymerase (which is commercially available from Lucigen® Corporation), SD polymerase (commercially available from Bioron®), or variants thereof. The polynucleotide binding protein can be Phi29 DNA polymerase or variants thereof. The topoisomerase is preferably a member of any of the partial classification (EC) groups 5.99.1.2 and 5.99.1.3.

[0304] The polynucleotide binding protein is most preferably derived from a helicase, such as Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mhu, TraI Eco, XPD Mbu, or variants thereof. Any helicase can be used in the present invention. The helicase can be or be derived from a Hel308 helicase, a RecD helicase, such as a TraI helicase or a TrwC helicase, an XPD helicase, or a Dda helicase. The helicase can be any of a helicase, a modified helicase, or a helicase construct, disclosed in WO2013 / 057495; WO 2013 / 098562; WO 2013098561; WO 2014 / 013260; WO 2014 / 013259; WO 2014 / 013262; and WO 2015 / 055981. All of these are incorporated by reference in their entirety.

[0305] The polynucleotide binding protein can be covalently attached to the monomer using any method known in the art. The monomer and protein can be chemically fused or genetically fused. Genetic fusion of a monomer to a polynucleotide binding protein is discussed in WO 2010 / 004265 (incorporated by reference in its entirety). The polynucleotide binding protein can be attached via a cysteine bond using any of the methods described above.

[0306] The polynucleotide binding protein can be attached directly to the protein via one or more linkers. The molecule can be attached to the spicule pore protein monomer using a hybrid linker as described in WO2010 / 086602 (incorporated by reference in its entirety). Alternatively, a peptide linker can be used. Suitable peptide linkers are discussed above.

[0307] Any of the proteins can be modified to aid in their identification or purification, for example, by the addition of histidine residues (his tag), aspartate residues (asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag, or a MBP tag, or by the addition of a signal sequence to facilitate their secretion from a cell in which the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the protein. An example of this is to react a gel transfer reagent to a cysteine engineered on the outside of the protein. This has been shown to be a method for isolating hemolysin hetero-oligomers (Chem Biol. 1997 Jul;4(7):497-505).

[0308] Any of the proteins can be labeled with a detectable label. The detectable label can be any suitable label that allows detection of the protein. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes (e.g., 125I, 35S), enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.

[0309] The proteins can also contain other non-specific modifications so long as they do not interfere with the function of the protein. Many non-specific side chain modifications are known in the art and can be made to the side chains of the proteins. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with acetimidate, or acylation with acetic anhydride.

[0310] Any of the proteins can be produced using standard methods known in the art. The polynucleotide sequence encoding the protein can be derived and replicated using standard methods in the art. The polynucleotide sequence encoding the protein can be expressed in a bacterial host cell using standard techniques in the art. The protein can be produced in a cell by expressing the polypeptide in situ from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rdEdition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0311] Proteins can be produced in large scale after purification from a protein producing organism by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA system, Bio-Cad system, Bio-Rad BioLogic system and Gilson HPLC system.

[0312] Method of producing a pore

[0313] The present application provides methods for producing the present spicule porin monomer. The method comprises modifying or mutating the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Any of the modifications or mutations discussed above can be made.

[0314] Alternatively, the method comprises expressing the present polynucleotide in a host cell or producing the spicule porin monomer using in vitro translation and transcription (IVTT). Such methods are described in more detail below.

[0315] Any of the embodiments discussed above with reference to the present spicule porin monomer equally apply to these methods.

[0316] Method of characterizing an analyte

[0317] The present application also provides methods for producing the present spicule porin pore or the present pore multimer.

[0318] The method can comprise expressing the spicule porin pore in a host cell. In particular, the method can comprise expressing at least one present spicule porin monomer or the present construct in a host cell and sufficient spicule porin monomers or constructs to form a spicule porin pore or pore multimer and allowing the spicule porin pore or pore multimer to form in the host cell. The sufficient spicule porin monomers or constructs are preferably sufficient present spicule porin monomers or sufficient present constructs. The number of spicule porin monomers or constructs required to form the present spicule porin pore or the present pore multimer is discussed above. Suitable host cells and expression systems are known in the art and discussed in the examples.

[0319] The method can comprise forming the cnidocyanoporin pore in a non-cellular or in vitro environment. In particular, the method can comprise contacting at least one cnidocyanoporin monomer of the invention or a construct of the invention with sufficient cnidocyanoporin monomers or constructs in vitro and allowing the formation of a cnidocyanoporin pore or pore multimer. The cnidocyanoporin monomers or constructs can be produced individually by in vitro translation and transcription (IVTT) and then incubated with sufficient cnidocyanoporin monomers or constructs. The sufficient cnidocyanoporin monomers or constructs are preferably sufficient cnidocyanoporin monomers of the invention or sufficient constructs of the invention. The number of cnidocyanoporin monomers or constructs required to form a cnidocyanoporin pore of the invention or a pore multimer of the invention is discussed above. The method can be performed in an “in vitro system” which refers to a system comprising at least the components and environment necessary to perform the method and which utilizes biological molecules, organisms, cells (or parts of cells) outside of their normal, naturally occurring environment, allowing for more detailed, more convenient or more efficient analysis than with the whole organism. The in vitro system can also comprise a suitable buffer composition provided in a test tube, in which the protein components forming the complex have been added. The person skilled in the art knows the options to provide said system.

[0320] Some or all of the components of the cnidocyanoporin monomer, cnidocyanoporin pore or pore multimer can be labeled to facilitate purification. Purification can also be performed when the components are not labeled. Methods known in the art (e.g., ion exchange, gel filtration, hydrophobic interaction column chromatography, etc.) can be used individually or in different combinations to purify the components of the pore.

[0321] The cnidocyanoporin pore or pore multimer can be prepared prior to insertion into a membrane or after insertion of the components into a membrane.

[0322] Methods for preparing pores and complexes of the invention and ways to label them are disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317 and WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893 (all incorporated by reference in their entirety).

[0323] https: / / markerdb.ca / proteins / 171

[0324] The invention provides a method of determining the presence, absence or one or more characteristics of a target analyte. The method comprises contacting the target analyte with a cnidocyanoporin pore of the invention, a pore multimer of the invention or a membrane of the invention and making one or more measurements as the target analyte moves relative to the cnidocyanoporin pore, pore multimer or membrane and thereby determining the presence, absence or one or more characteristics of the target analyte.

[0325] The present application also provides a method of determining the presence, absence, or one or more characteristics of a target analyte. The method comprises contacting the target analyte with a porin pore of the present application, a pore multimer of the present application, or a membrane of the present application, such that the target analyte moves relative to the porin pore, pore multimer, or membrane, such as into or through the porin pore, pore multimer, or membrane, and making one or more measurements as the target analyte moves relative to the porin pore or pore multimer, and thereby determining the presence, absence, or one or more characteristics of the target analyte.

[0326] The target analyte can also be referred to as a template analyte or an analyte of interest. The porin pore of the present application, the pore multimer of the present application, or the membrane of the present application can be any of those discussed above.

[0327] The method is for determining the presence, absence, or one or more characteristics of a target analyte. The method can be for determining the presence, absence, or one or more characteristics of at least one target analyte. The method can involve determining the presence, absence, or one or more characteristics of two or more target analytes. The method can comprise determining the presence, absence, or one or more characteristics of any number of target analytes, such as 2, 5, 10, 15, 20, 30, 40, 50, 100, or more analytes. Any number of characteristics of one or more target analytes can be determined, such as 1, 2, 3, 4, 5, 10, or more characteristics.

[0328] Binding of a molecule in the channel of a stalk pore protein pore or pore multimer or in the vicinity of either opening of the channel will have an effect on the open channel ion flow through the stalk pore protein pore or pore multimer, this is the essence of "molecular sensing". In a similar manner to nucleic acid sequencing applications, changes in open channel ion flow can be measured by changes in current using suitable measurement techniques (e.g. WO 2000 / 28312 and D. Stoddart et al, Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO 2009 / 077734; all incorporated herein by reference in their entirety). The extent of the reduction in ion flow measured by the reduction in current is related to the size of the obstacle in or near the pore. Thus, binding of a molecule of interest (also known as an "analyte") in or near the pore provides a detectable and measurable event, forming the basis of a "biosensor". Suitable molecules for nanopore sensing include nucleic acids; proteins; peptides; polysaccharides and small molecules (here meaning low molecular weight (e.g. < 900 Da or < 500 Da) organic or inorganic compounds) such as drugs, toxins, cytokines and pollutants. Detecting the presence of biological molecules can have applications in personalised medicine development, medicine, diagnostics, life science research, environmental monitoring and the security and / or defence industries.

[0329] A stalk pore protein pore or pore multimer can be used as a molecular or biological sensor. The target analyte molecule to be detected can bind to either face of the channel, or within the internal lumen of the channel itself. The location of binding can be determined by the size of the molecule to be sensed.

[0330] The target analyte preferably comprises or consists of a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, a polysaccharide, a dye, a bleach, a drug, a diagnostic agent, a recreational drug, an explosive, a toxic compound or an environmental pollutant. The target analyte preferably comprises or consists of a polypeptide, a protein, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, an oligosaccharide or a polysaccharide. The target analyte can comprise two or more different molecules, such as a peptide and a polypeptide. The target analyte can be a polynucleotide-polypeptide conjugate. The target analyte can be a biomarker. The method can involve determining the presence, absence or one or more characteristics of two or more target analytes of the same type, such as two or more proteins, two or more nucleotides or two or more drugs. Alternatively, the method can involve determining the presence, absence or one or more characteristics of two or more target analytes of different types, such as one or more proteins, one or more nucleotides and one or more drugs.

[0331] The target analyte can be secreted from a cell. Alternatively, the target analyte can be an analyte that is present inside a cell, such that the target analyte must be extracted from the cell before the method can be performed. The target analyte can be obtained or extracted from any organism or microorganism. The target analyte can be obtained from a human or animal, for example, from urine, lymphatic fluid, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. The target analyte can be obtained from a plant (e.g., a grain, legume, fruit, or vegetable).

[0332] The cnidocyte pore protein pore or pore multimer can be modified via recombinant or chemical means to increase the binding strength, binding location, or binding specificity of the molecule to be sensed. Typical modifications include the addition of a specific binding moiety that is complementary to the structure of the molecule to be sensed. Where the analyte molecule comprises a nucleic acid, the binding moiety can comprise a cyclodextrin or an oligonucleotide; for small molecules, this can be a known complementary binding region, such as an antibody or an antigen-binding moiety of a non-antibody molecule, including a single-chain variable fragment (scFv) region or an antigen recognition domain from a T-cell receptor (TCR); or for proteins, it can be a known ligand for the target protein. In this way, the cnidocyte pore protein pore or pore multimer can be enabled to act as a molecular sensor for detecting the presence of suitable antigens (including epitopes) in a sample, which can include cell surface antigens, including receptors, markers of solid tumor or blood cancer cells (e.g., lymphoma or leukemia), viral antigens, bacterial antigens, protozoan antigens, allergens, allergy-related molecules, albumins (e.g., human, rodent, or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites of enzymes or enzyme substrates, and transition state analogs of enzyme substrates. As noted above, the modifications can be achieved using known genetic engineering and recombinant DNA techniques. Any suitable positioning will depend on the nature of the molecule to be sensed, such as size, three-dimensional structure, and its biochemical properties. Selection of suitable structures can utilize computational structure design. Determination and optimization of protein-protein interactions or protein-small molecule interactions can be investigated using techniques such as BIAcore®, which uses surface plasmon resonance to detect molecular interactions (BIAcore, Inc., Piscataway, NJ; see also www.biacore.com).

[0333] The target analyte preferably comprises or consists of an amino acid, a peptide, a polypeptide, or a protein. The amino acid, peptide, polypeptide, or protein can be naturally occurring or non-naturally occurring. The polypeptide or protein can include synthetic or modified amino acids therein. Several different types of modifications to amino acids are known in the art. Suitable amino acids and modifications thereof are described above. It will be appreciated that the target analyte can be modified by any method available in the art.

[0334] The target analyte preferably comprises a polypeptide. The term polypeptide is interchangeable with protein. Any suitable polypeptide can be characterized. The polypeptide can be an unmodified protein or portion thereof, or a naturally occurring polypeptide or portion thereof. The target polypeptide can be secreted from a cell. Alternatively, the target polypeptide can be produced within a cell, such that it must be extracted from the cell for characterization. The target polypeptide can be a biomarker polypeptide. The polypeptide can comprise a cell expression product of a plasmid, for example, a plasmid used to clone a protein according to the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0335] The polypeptide can be provided as an impure mixture of one or more polypeptides and one or more impurities. The impurities can comprise truncated forms of the target polypeptide, which are distinct from the "target polypeptide" used for characterization. For example, the target polypeptide can be a full-length protein and the impurities can comprise fractions of the protein. The impurities can also comprise proteins other than the target protein, for example, it can be co-purified from a cell culture or obtained from a sample.

[0336] The polypeptide can comprise any combination of any amino acids, amino acid analogs, and modified amino acids (i.e., amino acid derivatives). The amino acids (and derivatives, analogs, etc.) in the polypeptide can be distinguished by their physical size and charge. The amino acids / derivatives / analogues can be naturally occurring or man-made. The polypeptide can comprise any naturally occurring amino acid.

[0337] The polypeptide can be modified. The method of the present invention can be used to modify the polypeptide for detection. The method can be used to characterize the modification in the target polypeptide.

[0338] One or more of the amino acids / derivatives / analogues in the polypeptide can be modified. One or more of the amino acids / derivatives / analogues in the polypeptide can be post-translationally modified. Thus, the method of the present invention can be used to detect the presence, absence, number of locations of post-translational modifications in the polypeptide. The method can be used to characterize the extent to which the polypeptide has been post-translationally modified.

[0339] Any one or more post-translational modifications can be present in a polypeptide. Typical post-translational modifications include modification with hydrophobic groups, modification with cofactors, addition of chemical groups, glycosylation (non-enzymatic attachment of sugars), phosphorylation, biotinylation, and pegylation. Post-translational modifications can also be non-natural, such that they are chemical modifications made in the laboratory for biotechnological or biomedical purposes. This can allow for monitoring of levels of a laboratory-made peptide, polypeptide, or protein compared to a natural counterpart.

[0340] Examples of post-translational modification with hydrophobic groups include: myristoylation, attachment of a myristate (C 14 saturated acid); palmitoylation, attachment of a palmitate (C 16 saturated acid); prenylation or isoprenylation, attachment of an isoprenoid group; farnesylation, attachment of a farnesol group; geranylgeranylation, attachment of a geranylgeraniol group; and glycosylation, and glycosyl phosphatidylinositol (GPI) anchoring via amide bond formation.

[0341] Examples of post-translational modification with cofactors include: lipoacylation, attachment of a lipoate (C8) functional group; flavinylation, attachment of a flavin moiety (e.g. flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, e.g. via a thioether linkage to cysteine; phosphopantetheinylation, attachment of a 4'-phosphopantetheinyl group; and formation of a retinylidene Schiff base.

[0342] Examples of post-translational modification by addition of chemical groups include: acylation, e.g. O-acylation (ester), N-acylation (amide), or S-acylation (thioester); acetylation, attachment of an acetyl group, e.g. to the N-terminus or lysine; formylation; alkylation, addition of an alkyl group, such as methyl or ethyl; methylation, e.g. addition of a methyl group to lysine or arginine; amidation; butyrylation; gamma-carboxylation; glycosylation, enzymatic attachment of a glycosyl group, e.g. to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan; polysialylation, attachment of a polysialic acid; malonylation; hydroxylation; iodination; bromination; citrullination; nucleotide addition, attachment of any nucleotide, such as any of those discussed above, ADP-ribosylation; oxidation; phosphorylation, attachment of a phosphate group, e.g. to serine, threonine, or tyrosine (O-linked) or histidine (N-linked); adenylylation, attachment of an adenylyl moiety, e.g. to tyrosine (O-linked) or to histidine or lysine (N-linked); propionylation; pyroglutamic acid formation; S-glutathionylation; selenoylation; S-nitrosylation; succinylation, attachment of a succinyl group, e.g. to lysine; selenoylation, incorporation of selenium; and ubiquitination, addition of a ubiquitin subunit (N-linked).

[0343] Peptides can be labeled with molecular markers. Molecular markers can be modifications of the peptide that facilitate its detection in the methods of this invention. For example, a marker can modify the peptide by altering the signal obtained when characterizing the conjugate. For example, a marker might interfere with the ion flux through a nanopore. In this way, labeling can improve the sensitivity of the method.

[0344] Peptides may contain one or more cross-linked regions, such as CC bridges. Peptides may not be cross-linked prior to characterization using this method.

[0345] Peptides can contain sulfur-containing amino acids and therefore have the potential to form disulfide bonds. Typically, in such embodiments, the peptide is reduced using a reagent such as DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) before being characterized using this method.

[0346] Peptides can be full-length proteins or naturally occurring peptides. Proteins or naturally occurring peptides can be fragmented before conjugation with polynucleotides. Peptides can be chemically or enzymatically fragmented. Peptides or peptide fragments can be conjugated to form longer target peptides.

[0347] A polypeptide can have any suitable length. A polypeptide preferably has a length of about 2 to about 300 peptide units or amino acids. A polypeptide can have a length of about 2 to about 100 peptide units, for example, about 2 to about 50 peptide units, for example, about 3 to about 50 peptide units, such as about 5 to about 25 peptide units, for example, about 7 to about 16 peptide units, such as about 9 to about 12 peptide units. "Peptide unit" is interchangeable with "amino acid".

[0348] One or more characteristics of the polypeptide are preferably selected from (i) the length of the polypeptide, (ii) the identity of the polypeptide, (iii) the sequence of the polypeptide, (iv) the secondary structure of the polypeptide, and (v) whether the polypeptide is modified. One or more characteristics can be the sequence of the polypeptide, or whether the polypeptide is modified, for example, by one or more post-translational modifications. One or more characteristics are preferably the sequence of the polypeptide.

[0349] Peptides can exist in a relaxed form. Peptides can also remain in a linearized form. Maintaining a peptide in a linearized form facilitates characterization of the peptide on a residue-by-residue basis because the "aggregation" of the peptide within the nanopore is prevented. Any suitable method can be used to maintain a peptide in a linearized form. For example, if the peptide is charged, it can be maintained in a linearized form by applying a voltage.

[0350] If the polypeptide is not charged or only weakly charged, the charge can be altered or controlled by adjusting the pH. For example, the polypeptide can be kept in a linearized form by increasing the relative negative charge of the polypeptide using a high pH. Increasing the negative charge of the polypeptide allows it to remain in a linearized form under, for example, a positive voltage. Alternatively, the polypeptide can be kept in a linearized form by increasing the relative positive charge of the polypeptide using a low pH. Increasing the positive charge of the polypeptide allows it to remain in a linearized form under, for example, a negative voltage. In the disclosed methods, the protein is treated with a polynucleotide to control the movement of the polynucleotide relative to the nanopore. As polynucleotides are generally negatively charged, it is generally most suitable to increase the linearization of the polypeptide by increasing the pH, thereby making the polypeptide more negatively charged, as with the polynucleotide. In this way, the conjugate retains an overall negative charge and can therefore move easily, for example, under an applied voltage.

[0351] The polypeptide can be kept in a linearized form by using suitable denaturing conditions. Suitable denaturing conditions include, for example, the presence of an appropriate concentration of a denaturant, such as guanidinium hydrochloride and / or urea. The concentration of such denaturants used in the disclosed methods depends on the target polypeptide to be characterized in the method and can be readily selected by one of skill in the art.

[0352] The polypeptide can be kept in a linearized form by using suitable detergents. Suitable detergents for use in the disclosed methods include SDS (sodium dodecyl sulfate). The polypeptide can be kept in a linearized form by performing the disclosed methods at an elevated temperature. Increasing the temperature overcomes the intrachain bonding and allows the polypeptide to adopt a linearized form.

[0353] The polypeptide can be kept in a linearized form by performing the method under strong electroosmotic forces. Such forces can be provided by using asymmetric salt conditions and / or providing a suitable charge in the channel of the nanopore. The charge in the channel of the pore can be altered, for example, by mutagenesis. Altering the charge of the pore is well within the capabilities of one of skill in the art. When a voltage potential is applied across the nanopore, altering the charge of the pore generates strong electroosmotic forces from the unbalanced flow of cations and anions through the nanopore.

[0354] The polypeptide can be kept in a linearized form by passing the polypeptide through a structure, such as an array of nanopillars, through a nanoslit, or across a nanogap. The physical constraints of such structures can force the polypeptide to adopt a linearized form.

[0355] The target polypeptide can be cleaved or fragmented to form smaller target peptides prior to contacting with the present cnidopore pore, the present pore multimer, or the present membrane. Suitable methods for doing so are known in the art, for example using proteolytic enzymes such as trypsin, chymotrypsin, or Glu-C. This method is known as protein fingerprinting. The method can comprise cleaving or fragmenting the target polypeptide to form smaller target peptides, contacting the target peptides with the present cnidopore pore, the present pore multimer, or the present membrane, such that the target peptides move relative to the cnidopore pore, pore multimer, or membrane, such as into or through the cnidopore pore, pore multimer, or membrane, and making one or more measurements as the target peptides move relative to the cnidopore pore or pore multimer, and thereby determining the presence, absence, or one or more characteristics of the target polypeptide.

[0356] The target polypeptide is preferably positively charged. Suitable proteins include, but are not limited to, those shown in the table below.

[0357]

[0358] Searching biomarker databases Polynucleotide of the present invention

[0359] ** UniProt: DNA-binding proteins up to 250 amino acids in length

[0360] *** Net charge calculation at pH 8: https: / / www.protpi.ch / Calculator /

[0361] The positively charged polypeptide is preferably a histone. The histone is preferably selected from H3.1, H4, H3Cit, H3K9ac, and H3K23ac. In addition to their physiologically typical functions in DNA packaging and regulation of gene expression in the nucleus, histones have extracellular functions. They have antibacterial functions and can act as damage-associated molecular patterns (DAMPs; see reference 15 in Example 2) and are the main proteins in neutrophil extracellular traps (NETs; see references 15 and 16 in Example 2).

[0362] ​The present application also provides a method of distinguishing two or more target analytes, preferably two or more target polypeptides, in a mixture of analytes. The method comprises contacting the mixture of analytes with a porin pore of the present application, a pore multimer of the present application or a membrane of the present application, such that the two or more target analytes move relative to the porin pore, pore multimer or membrane, such as into or through the porin pore, pore multimer or membrane, and making one or more measurements as the two or more target analytes move relative to the porin pore or pore multimer, and thereby distinguishing the two or more target analytes in the mixture. The two or more analytes are preferably two or more histones. The two or more histones are preferably selected from H3.1, H4, H3Cit, H3K9ac and H3K23ac. The two or more histones preferably comprise or consist of H4 and H3Cit.

[0363] The target analyte is preferably a polynucleotide, such as a nucleic acid, which is defined as a macromolecule comprising two or more nucleotides. Nucleic acids are particularly suitable for nanopore sequencing. The naturally occurring nucleic acid bases in DNA and RNA can be distinguished by their physical size. When a nucleic acid molecule or individual bases pass through the channel of a nanopore, the size difference between the bases results in a directly related decrease in the ion flow through the channel. The change in ion flow can be recorded. Suitable electrical measurement techniques for recording the change in ion flow are discussed above. With appropriate calibration, the characteristic decrease in ion flow can be used to identify in real time the particular nucleotide and associated base passing through the channel. In typical nanopore nucleic acid sequencing, as each nucleotide of a nucleic acid sequence of interest passes in turn through the channel of a nanopore, the open channel ion flow decreases due to the partial blockage of the channel by the nucleotide. This decrease in ion flow is measured using suitable recording techniques described above. The decrease in ion flow can be calibrated to the measured decrease in ion flow for a known nucleotide passing through the channel, thereby creating a means for determining which nucleotide is passing through the channel, and hence, when done sequentially, providing a means for determining the nucleotide sequence of a nucleic acid passing through the nanopore. In order to accurately determine individual nucleotides, it is generally required that the decrease in ion flow through the channel is directly related to the size of the individual nucleotide passing through the constriction. It will be appreciated that a complete nucleic acid polymer passing through the pore via, for example, the action of an associated polymerase can be sequenced. Alternatively, the sequence can be determined by the passage of triphosphate nucleotide bases that have been sequentially removed from the target nucleic acid proximal to the pore (see, for example, WO 2014 / 187924, which is incorporated herein by reference in its entirety).

[0364] A polynucleotide or nucleic acid can comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more of the nucleotides in the polynucleotide can be oxidized or methylated. One or more of the nucleotides in the polynucleotide can be damaged. For example, the polynucleotide can comprise pyrimidine dimers. Such dimers are often associated with damage caused by ultraviolet light and are a major cause of skin melanoma. One or more of the nucleotides in the polynucleotide can be modified, for example, with a label or tag, suitable examples of which are known to the skilled person. The polynucleotide can comprise one or more spacers. Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically include adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides typically contain a mono-, di-, or tri-phosphate. The nucleotides can comprise more than three phosphates, such as 4 or 5 phosphates. The phosphates can be attached on the 5' or 3' side of the nucleotide. The nucleotides in the polynucleotide can be connected to each other in any way. The nucleotides are typically connected by their sugars and phosphate groups, as in nucleic acids. The nucleotides can be connected by their nucleobases, as in pyrimidine dimers. The polynucleotide can be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded. The polynucleotide is most preferably a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In particular, the method of using a polynucleotide as an analyte alternatively comprises determining one or more features 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 the polynucleotide is modified.

[0365] The polynucleotide can be of any length (i). For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs, or 100,000 or more nucleotides or nucleotide pairs in length. Any number of polynucleotides can be investigated. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide. The polynucleotide can be naturally occurring or man-made. For example, the method can be used to verify the sequence of a manufactured oligonucleotide. The method is typically performed in vitro.

[0366] The nucleotides can be of any identity (ii) and include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. The nucleotides can be abasic (i.e., lack a nucleobase). The nucleotides can also lack a nucleobase and a sugar (i.e., be a C3 spacer). The sequence of the nucleotides (iii) is determined by the contiguous identity of the successive nucleotides attached to one another in the 5' to 3' direction of the strand throughout the polynucleotide strain.

[0367] In particular, the method of using a polynucleotide as an analyte alternatively comprises determining one or more features 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 the polynucleotide is modified.

[0368] The polynucleotide can be of any length (i). For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs, or 100,000 or more nucleotides or nucleotide pairs in length. Any number of polynucleotides can be investigated. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide. The polynucleotide can be naturally occurring or man-made. For example, the method can be used to verify the sequence of a manufactured oligonucleotide. The method is typically performed in vitro.

[0369] The nucleotides can be of any identity (ii). Possible nucleotides are defined above with reference to the spicule porin monomer of the application. The sequence of the nucleotides (iii) is determined by the consecutive identity of successive nucleotides attached to each other in the 5' to 3' direction throughout the polynucleotide strain.

[0370] The spicule porin pores and pore multimers of the application are particularly useful in the analysis of homopolymers. For example, they can be used to determine the sequence of a polynucleotide comprising two or more, such as at least 3, 4, 5, 6, 7, 8, 9, or 10 identical consecutive nucleotides. For example, they can be used to sequence a polynucleotide comprising a polyA, polyT, polyG, and / or polyC region.

[0371] The target analyte can comprise a polynucleotide and a polypeptide. The target analyte can be a polynucleotide-polypeptide conjugate. The conjugate preferably comprises a polynucleotide conjugated to a polypeptide. According to the application, one or both of the polynucleotide and the polypeptide can be the target and can be characterized.

[0372] The polypeptide can be conjugated to the polynucleotide at any suitable location. For example, the polypeptide can be conjugated to the polynucleotide at the N-terminus or C-terminus of the polypeptide. The polypeptide can be conjugated to the polynucleotide via a side chain group of a residue (e.g., an amino acid residue) in the polypeptide. The polypeptide can have a naturally occurring reactive functional group that can be used to facilitate conjugation to the polynucleotide. For example, a cysteine residue can be used to form a disulfide bond with the polynucleotide or a modified group thereon.

[0373] The polypeptide can be modified to facilitate its conjugation to the polynucleotide. For example, the polypeptide is modified by attachment of a moiety comprising a reactive functional group for attachment to the polynucleotide. For example, the polypeptide can be extended at the N-terminus or C-terminus by one or more residues (e.g., amino acid residues), one or more of which comprise one or more reactive functional groups for reaction with a corresponding reactive functional group on the polynucleotide. For example, the polypeptide can be extended at the N-terminus and / or C-terminus by one or more cysteine residues. Such residues can be used for attachment to the polynucleotide portion of the conjugate, e.g., by maleimide chemistry (e.g., by reaction of the cysteine with an azido-maleimide compound such as azido-[Pol]-maleimide, where [Pol] is typically a short chain polymer such as PEG, e.g., PEG2, PEG3, or PEG4; which is then coupled to an appropriately functionalized polynucleotide, e.g., a polynucleotide bearing a BCN group for reaction with the azide). Such chemistry is described in Example 2. For the avoidance of doubt, when the polypeptide comprises an appropriate naturally occurring residue at the N-terminus and / or C-terminus (e.g., a naturally occurring cysteine residue at the N-terminus and / or C-terminus), then such residue can be used for attachment to the polynucleotide.

[0374] The residues in the polypeptide can be modified to facilitate attachment of the polypeptide to the polynucleotide. The residues (e.g., amino acid residues) in the polypeptide can be chemically modified for attachment to the polynucleotide. The residues (e.g., amino acid residues) in the polypeptide can be enzymatically modified for attachment to the polynucleotide.

[0375] The conjugation chemistry between the polynucleotide and the polypeptide in the conjugate is not particularly limited. Any suitable combination of reactive functional groups can be used. Many suitable reactive groups and their chemical targets are known in the art. Some exemplary reactive groups and their corresponding targets include aryl azides which can react with amines, carbodiimides which can react with amines and carboxyls, hydrazides which can react with carbohydrates, hydroxymethyl phosphines which can react with amines, imidates which can react with amines, isocyanates which can react with hydroxyls, carbonyls which can react with hydrazines, maleimides which can react with sulfhydryl groups, NHS-esters which can react with amines, PFP-esters which can react with amines, psoralen which can react with thymine, pyridyl disulfides which can react with sulfhydryl amines and hydroxyls, vinyl sulfones which can react with sulfhydryl amines and hydroxyls, and the like. Other suitable chemistries for conjugating a polypeptide to a polynucleotide include click chemistry. Many suitable click chemistry reagents are known in the art. Suitable examples of click chemistry include, but are not limited to, the following:

[0376] copper(I)-catalyzed azide-alkyne cycloaddition (azide alkyne Huisgen cycloaddition);

[0377] Strain-promoted azide-alkyne cycloaddition reactions; including olefin and azide [3+2] cycloaddition reactions; inverse-electron-demand Diels-Alder reactions of olefins with tetrazines; and photo-click reactions of olefins and tetrazoles;

[0378] Copper-free variants of 1,3 dipolar cycloaddition reactions in which azides react with alkynes under strain, for example in a cyclooctane ring, such as in bicyclo[6.1.0]nonyne (BCN);

[0379] Reaction of an oxygen nucleophile on one linker with an epoxide or aziridine reactive moiety on the other linker; and

[0380] Staudinger ligation in which the alkyne moiety can be replaced by an aryl phosphine, resulting in a specific reaction with an azide to form an amide bond.

[0381] Any reactive group can be used to form the conjugate. Some suitable reactive groups include [1,4-bis[3-(2-pyridyldithio)propionamido]butane; 1,1 1-maleimido triethyleneglycol; 3,3'-dithiodipropionic acid bis(N-hydroxysuccinimidyl ester); ethylene glycol-bis(succinic acid N-hydroxysuccinimidyl ester); 4,4'-disothiocyanatostilbene-2,2'-disulfonic acid disodium salt; bis[2-(4-azidosalicylamido)ethyl] disulfide; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimidyl ester; 4-maleimidobutyric acid N-hydroxysuccinimidyl ester; iodoacetic acid N-hydroxysuccinimidyl ester; S-acetylthioacetate N-hydroxysuccinimidyl ester; azide-PEG-maleimide; and alkyne-PEG-maleimide. The reactive group can be any of the reactive groups disclosed in WO 2010 / 086602, and in particular in Table 3 of that application.

[0382] The reactive functional group can be comprised in the polynucleotide and the target functional group can be comprised in the polypeptide prior to the conjugation step. The reactive functional group can be comprised in the polypeptide and the target functional group can be comprised in the polynucleotide prior to the conjugation step. The reactive functional group can be attached directly to the polypeptide. The reactive functional group can be attached to the polypeptide via a spacer. Any suitable spacer can be used. Suitable spacers include, for example, alkyl diamines, such as ethylenediamine, and the like.

[0383] The conjugate can comprise multiple polypeptide segments and / or multiple polynucleotide segments. For example, the conjugate can comprise a structure of the form...-P-N-P-N-P-N... where P is a polypeptide and N is a polynucleotide. The polynucleotide handling protein can control the N portions of the conjugate sequentially with respect to the pore and thus sequentially control the P segments with respect to the pore movement, allowing the P segments to be characterized sequentially. Multiple polynucleotides and polypeptides can be conjugated together by the same or different chemistries.

[0384] The conjugate can comprise a leader. Any suitable leader can be used. The leader can be a polynucleotide. The leader can be the same kind of polynucleotide as used in the conjugate, or the leader can be a different kind of polynucleotide. For example, the polynucleotide in the conjugate can be DNA and the leader sequence can be RNA, or vice versa.

[0385] The leader can be a charged polymer, for example a negatively charged polymer. The leader can comprise a polymer, such as PEG or a polysaccharide. The leader can be 10 to 150 monomer units (e.g. ethylene glycol or sugar units) in length, such as 20 to 120, for example 30 to 100, for example 40 to 80, such as 50 to 70 monomer units (e.g. ethylene glycol or sugar units) in length. The method of characterising a target polypeptide of the application can comprise conjugating the polypeptide to a polynucleotide.

[0386] An analyte binding protein (such as a polynucleotide binding protein) is preferably used to control the movement of the analyte with respect to the pore (such as through the pore). Suitable proteins are discussed in more detail above. The present application provides a method for determining the presence, absence or one or more characteristics of a target polynucleotide, the method comprising the steps of:

[0387] (i) contacting the target polynucleotide with a spicule pore protein pore of the application, a pore multimer of the application or a membrane of the application and a polynucleotide binding protein, such that the polynucleotide binding protein controls the movement of the target polynucleotide with respect to the spicule pore protein pore, pore multimer or membrane (such as through the spicule pore protein pore, pore multimer or membrane); and

[0388] (ii) making one or more measurements as the polynucleotide moves with respect to the spicule pore protein pore, pore multimer or membrane (such as through the spicule pore protein pore, pore multimer or membrane) and thereby determining the presence, absence or one or more characteristics of the polynucleotide.

[0389] Methods for determining the presence, absence, or one or more characteristics of a target polynucleotide can include the use of one or more sequencing adapters. Those skilled in the art are able to use sequencing adapters, such as the adapters described in WO 2016 / 034591 and WO 2018 / 100370 (both incorporated by reference herein in their entireties), to attach suitable moieties or regions to double-stranded polynucleotides. These adapters also contain suitable binding sites for polynucleotide binding proteins. Those skilled in the art are also able to design functional binding moieties that contain moieties or regions that are able to hybridize to the disclosed moieties or regions.

[0390] In any of these methods, one or more characteristics of the target analyte are measured, preferably by electrical and / or optical measurements. The electrical measurement is a current measurement, impedance measurement, tunneling measurement, or field effect transistor (FET) measurement. The method preferably includes measuring the current flowing through the stomylin channel protein pore or pore multimer as the target analyte moves relative to the pore, such as through the pore.

[0391] General conditions for performing the methods of the application are discussed in more detail below with reference to the kits and systems of the application.

[0392] Kit

[0393] The present application also provides a polynucleotide encoding a stomylin channel protein pore of the present application. The polynucleotide can be any of those discussed above. The present application also provides an expression vector comprising a polynucleotide of the present application. The present application also provides a host cell comprising a polynucleotide of the present application or a vector of the present application. Suitable vectors and host cells are known in the art.

[0394] Device

[0395] The present application also provides a kit for characterizing a target analyte. The kit includes (a) a stomylin monomer of the present application, a construct of the present application, a stomylin channel protein pore of the present application, or a pore multimer of the present application, and (b) a component of a membrane and / or an analyte binding protein. The kit preferably includes (a) a stomylin monomer of the present application, a construct of the present application, a stomylin channel protein pore of the present application, or a pore multimer of the present application, and (b) a component of a membrane. The kit preferably includes (a) a stomylin monomer of the present application, a construct of the present application, a stomylin channel protein pore of the present application, or a pore multimer of the present application, and (b) an analyte binding protein. The kit preferably includes (a) a stomylin monomer of the present application, a construct of the present application, a stomylin channel protein pore of the present application, or a pore multimer of the present application, and (b) a component of a membrane and an analyte binding protein.

[0396] The kit can comprise any type of membrane such as an amphiphilic layer, such as a component of a triblock copolymer membrane. The membrane is preferably artificial. The analyte binding protein is preferably a polynucleotide binding protein. Suitable proteins are as described above.

[0397] The kit can further comprise one or more anchors, such as a cholesterol, for coupling a target analyte to the membrane. The kit can further include one or more polynucleotide adaptors, such as one or more sequencing adaptors, which can be attached to a target polynucleotide to facilitate characterization of the polynucleotide. The anchor, such as a cholesterol, is preferably attached to the polynucleotide adaptor.

[0398] The kit can additionally include one or more other reagents or instruments that enable any of the embodiments described above. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), a tool for obtaining a sample from a subject (such as a container or instrument comprising a needle), a tool for amplifying and / or expressing a polynucleotide, or a voltage or patch clamp apparatus. The reagents can be present in the kit in a dry state, such that a fluid sample re-suspends the reagents. The kit can also optionally include instructions that enable the kit to be used in the methods of the invention, or details about which organisms the methods can be used for. Finally, the kit can also include additional components useful in the characterization of the analyte.

[0399] Array

[0400] The present invention also provides a device for characterizing a target analyte in a sample, comprising (a) a plurality of the present invention's spicule porin monomer, a plurality of the present invention's construct, a plurality of the present invention's spicule porin pore, or a plurality of the present invention's pore multimer, and (b) a plurality of analyte binding proteins, preferably polynucleotide binding proteins. The plurality of spicule porin monomers, constructs, spicule porin pores, or pore multimers can be any of those discussed above. Suitable polynucleotide binding proteins are also discussed above.

[0401] The present invention also provides a device comprising a spicule porin pore derived from Montipora gaudiosa inserted into an in vitro membrane, such as the present invention's spicule porin pore or the present invention's pore multimer.

[0402] The present invention also provides a device produced by a method comprising the steps of: (i) obtaining a spicule porin pore derived from Montipora gaudiosa, such as the present invention's spicule porin pore or the present invention's pore multimer, and (ii) contacting the spicule porin pore derived from Montipora gaudiosa, such as the present invention's spicule porin pore or the present invention's pore multimer, with an in vitro membrane, such that the spicule porin pore or pore multimer is inserted into the in vitro membrane.

[0403] Any of the specific embodiments discussed above apply equally to the apparatus of the application.

[0404] System

[0405] The application also provides an array comprising a plurality of the membranes of the application. Any of the embodiments discussed above in relation to the membranes of the application apply equally to the arrays of the application. The array can be configured to perform any of the methods described below.

[0406] In one embodiment, each membrane in the array comprises one stalk pore protein pore or pore multimer. Due to the manner in which the array is formed, for example, the array can comprise one or more membranes that do not comprise a stalk pore protein pore or pore multimer, and / or one or more membranes that comprise two or more pore complexes or multimers. The array can comprise from about 2 to about 1000 layers of membranes, such as from about 10 to about 800 layers, from about 20 to about 600 layers, or from about 30 to about 500 layers of membranes.

[0407] Measurement of analysis

[0408] The application provides a system comprising (a) a membrane of the application or an array of the application, (b) means for applying an electrical potential across the membrane, and (c) means for detecting an electrical or optical signal across the membrane. The electrical signal can be a measurement of ions flowing through the nanopore, such as a measurement of current or voltage as a function of time.

[0409] The stalk pore protein pores and membranes can be any of the stalk pore protein pores and membranes described above and below.

[0410] In one embodiment, the system further comprises a first chamber and a second chamber, wherein the first chamber and the second chamber are separated by the membrane. When used to characterise a target analyte, the system can further comprise the target analyte, wherein the target analyte is transiently located within the continuous channel, and wherein one end of the target analyte is located in the first chamber and one end of the target analyte is located in the second chamber. The target analyte is preferably a target polypeptide or a target polynucleotide.

[0411] In one embodiment, the system further comprises an electrically conductive solution in contact with the pore, electrodes to provide a voltage potential across the membrane, and a measurement system for measuring the current through the pore. The voltage applied across the membrane and pore is preferably +5 V to -5 V, such as -600 mV to +600 mV or -400 mV to +400 mV. The voltage used is preferably in the range of 100 mV to 240 mV and more preferably in the range of 120 mV to 220 mV. By using an increased applied potential, the discrimination of the pore for different amino acids or nucleotides can be increased. Any suitable electrically conductive solution can be used. For example, the solution can comprise charge carriers, such as metal salts, e.g. alkali metal salts, halide salts, e.g. chloride salts, such as alkali metal chloride salts. The charge carriers can include ionic liquids or organic salts, e.g. tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride. In exemplary systems, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCI), sodium chloride (NaCI), cesium chloride (CsCI) or a mixture of potassium ferrocyanide and potassium ferricyanide are often used. KCI, NaCI and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers can be asymmetric across the membrane. For example, the type and / or concentration of charge carriers can be different on each side of the membrane, e.g. in each chamber.

[0412] The salt concentration can be in a saturated state. The salt concentration can be 3 M or less, and is typically 0.1 to 2.5 M, 0.3 to 1.9 M, 0.5 to 1.8 M, 0.7 to 1.7 M, 0.9 to 1.6 M or 1 M to 1.4 M. The salt concentration is preferably 150 mM to 1 M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow the identification of a current indicative of the presence of an amino acid or nucleotide against a background of normal current fluctuations.

[0413] A buffer can be present in the electrically conductive solution. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The pH of the electrically conductive solution can be 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7 or 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.

[0414] The system can be comprised in a device. The device can be any conventional device for analyte analysis, such as an array or a chip. The device is preferably arranged to perform the disclosed method. For example, the device can comprise a chamber comprising an aqueous solution and a barrier dividing the chamber into two sections. The barrier typically has an opening in which a membrane containing pores is formed. Alternatively, the barrier forms a membrane in which pores are present.

[0415] The device can further comprise a circuitry capable of applying an electric potential and measuring an electrical signal across the membrane and the pores.

[0416] The device can be any of those described in WO 2008 / 102120, WO 2009 / 077734, WO 2010 / 122293, WO 2011 / 067559, WO 2014 / 06442 or WO 2020 / 183172 (all incorporated herein by reference in their entirety).

[0417] Membrane

[0418] The method for determining the presence, absence or one or more characteristics of a target polymer analyte can comprise estimating or determining the sequence of the polymer units. The signal measured during the movement of a polymer, such as a polypeptide, polynucleotide or polypeptide-polynucleotide conjugate, relative to a stichoporate pore protein pore or pore multimer can at any one time be dependent on a plurality of polymer units, such as amino acids or nucleotides. For example, the presence of a plurality of amino acids or nucleotides in the cavity of a stichoporate pore protein pore or pore multimer and possibly amino acids or nucleotides outside the stichoporate pore protein pore or pore multimer can influence the ion flow and thus the current or voltage signal. A polypeptide or polynucleotide can also contain modified amino acids or nucleotides which can influence the measured signal and thus the estimation or determination of the sequence can be important. The polymer sequence can be determined or estimated using various known mathematical techniques and variations thereof, including probabilistic and machine learning techniques. Such methods are described in, for example, WO2013041878, WO2013121224, WO2018203084 as well as Zhang et al: A Guide to Signal Processing Algorithms for Nanopore Sensors, ACS Sens. 2021, 6, 10, 3536-3555, all hereby incorporated by reference in their entirety.

[0419] The methods of the invention can comprise measuring the target analyte, where the measurements can be used to estimate or determine the overall sequence. Various known methods can be used. For example, the sequence can be initially determined from a series of measurements made during the movement of the analyte relative to the pore of the stylommatophoran pore protein or pore multimer, and the results combined to provide the overall sequence. More preferably, the series of measurements can be processed by a probabilistic or machine learning technique into a plurality of series of measurements in a plurality of dimensions, where the overall sequence determination is made without initial determination of the sequence of the analyte. Non-limiting examples of methods suitable for use in the invention are disclosed in WO2015140535 (incorporated by reference in its entirety).

[0420] Sequence listing

[0421] Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules such as phospholipids that have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form monolayers are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomeric subunits polymerize together to produce a single polymer chain. Block copolymers generally have properties contributed by each monomeric subunit. However, block copolymers can have unique properties not possessed by the polymers formed from the individual subunits. Block copolymers can be designed such that one of the monomeric subunits is hydrophobic (i.e., lipophilic) while the other subunits are hydrophilic when in an aqueous medium. In this case, the block copolymers can possess amphiphilic properties and can form structures that mimic biological membranes. Block copolymers can be diblock (consisting of two monomeric subunits), but can also be constructed from more than two monomeric subunits to form more complex arrangements that behave as amphiphiles. The amphiphilic layer can comprise diblock, triblock, tetrablock, or pentablock copolymers.

[0422] The membrane can comprise one of the membranes disclosed in International Application Nos. WO 2014 / 064443 or WO 2014 / 064444 (both incorporated by reference in their entirety herein).

[0423] The amphiphilic molecules can be chemically modified or functionalized to facilitate coupling of the polynucleotide. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported.

[0424] The amphiphilic membrane is typically naturally mobile, acting essentially as a membrane with a thickness of about 10 -8 cm s -1a two-dimensional fluid of lipid diffusion rates. This means that the pores and coupled polynucleotides can generally move within the amphipathic membrane.

[0425] The membrane can be a lipid bilayer. Lipid bilayers are models of cell membranes and are used as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro studies of membrane proteins by single channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO 2008 / 102121, WO 2009 / 077734 and WO 2006 / 100484 (all incorporated by reference in their entirety).

[0426] The membrane can comprise a solid state layer. The solid state layer can be formed from organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si3N4, A12O3 and SiO, organic and inorganic polymers such as polyamides, plastics such as Teflon® or elastomers such as two-component addition-cured silicone rubber and glass. The solid state layer can be formed from graphene. Suitable graphene layers are disclosed in WO 2009 / 035647 (incorporated by reference in its entirety). If the membrane comprises a solid state layer, the pores are generally present in an amphipathic membrane or layer that is comprised within the solid state layer, for example within a hole, pore, gap, channel, trench or slit within the solid state layer. The skilled person can prepare suitable solid state / amphipathic hybrid systems. Suitable systems are disclosed in WO 2009 / 020682 and WO 2012 / 005857 (both incorporated by reference in their entirety). Any of the amphipathic membranes or layers discussed above can be used.

[0427] Preferred embodiments include (i) an artificial amphipathic layer comprising a stomy pore protein pore or pore multimer, (ii) an isolated, naturally occurring lipid bilayer comprising a stomy pore protein pore or pore multimer, or (iii) a cell having a stomy pore protein pore or pore multimer inserted therein. The most preferred embodiments are artificial amphipathic layers such as diblock or triblock copolymer layers. In addition to the stomy pore protein or pore multimer, the layer can comprise other transmembrane and / or intramembrane proteins as well as other molecules. Suitable apparatus and conditions are discussed below. All embodiments of the application are generally performed in vitro.

[0428] Examples

[0429] SEQ ID NO: 1

[0430] SAPIKANDPNGEVLEEMPKTKRGAEALLADVGLAHFPEMVPNRQELRALLKRSHNAEAIPQEPMDLENLDSEKRAARIAAGTIIAGAELTIGLLQNLLDVLANVNRKCAVGVDNESGFRWQEGSTYFFSGTADENLPYSVSDGYAVLYGPRKTNGPVATGVVGVLAYYIPSIGKTLAVMWSVPFDYNFYQNWWNAKLYSGNQDADYDHYVDLYYDANPFKANGWHERSLGSGLKFCGSMSSSGQATLEIHVLKESETCM

[0431] SEQ ID NO: 2

[0432] RIAAGTIIAGAELTIGLLQNLLDVLANVNRKCAVGVDNESGFRWQEGSTYFFSGTADENLPYSVSDGYAVLYGPRKTNGPVATGVVGVLAYYIPSIGKTLAVMWSVPFDYNFYQNWWNAKLYSGNQDADYDHYVDLYYDANPFKANGWHERSLGSGLKFCGSMSSSGQATLEIHVLKESETCM

[0433] The following examples illustrate the application. It should be understood that, while the teachings have been discussed in terms of particular embodiments, particular configurations, and materials and / or molecules according to the application, various changes or modifications can be made in form and detail without departing from the scope and spirit of the application. The following examples are provided to better illustrate particular embodiments and should not be considered as limiting the application. The application is only limited by the claims.

[0434] Example 1

[0435] Figures 1 to 13

[0436] Protein expression and purification

[0437] Nucleotide sequences for Fav constructs were inserted into a modified pEt28a (+) plasmid with N-terminal 6 x His followed by TEV restriction site (ENLYFQS). 100 μΐ of competent E. coli BL21 strain transformed with 1.5 μΐ of plasmid (heat shocked) were applied to agar plates with kanamycin resistance and incubated overnight at 37°C. A single colony was inoculated into 10 mL of LB medium (30 μg / ml kanamycin) and incubated overnight at 37°C and 180 rpm. The overnight culture was then added to 1 L of TB medium (30 μg / ml kanamycin) and incubated at 37°C until an A 600 0.8 was reached when protein expression was induced by 0.25 mM IPTG. The cells were then grown at 18°C and harvested after 16 to 18 h by centrifugation of the biomass at 4000 x g for 10 min at 4°C. E. coli BL21 cells expressing Fav constructs were resuspended in PBS buffer containing 10 mM imidazole, sonicated by ultrasonic sonication and affinity purified by using nickel affinity chromatography. TEV cleavage was performed during overnight dialysis against PBS buffer to remove the N-terminal tag.

[0438] Lipid vesicles

[0439] 50 mM multilamellar vesicles consisting of SM (brain, porcine) and DOPC in a molar ratio of 1 : 1 were prepared by dissolving the lipids provided by Avanti Polar Lipids (Alabaster, AL, USA) in chloroform, evaporating the solvent by rotary evaporation (lipid film formation) and adding PBS. After freeze-thaw cycles, the formed MLVs were extruded over 100 nm membranes to form 100 mM large unilamellar vesicles for the pore formation assay.

[0440] Pores

[0441] Wells were prepared by incubating soluble Fav monomer (1 h, 37 °C) with 50 mM macromonolayer vesicles (molar ratio 215:1). Vesicles were disrupted with LDAO, and the soluble wells were purified by ion-exchange chromatography (resource Q, Cytiva, 50 mM Tris / HCl, 0.02% Brij 35, pH 8). Well formation was examined by non-denaturing PAGE and Cryo-EM. Non-denaturing PAGE samples were prepared by mixing 17 μL of the well sample (after ion-exchange chromatography), 6 μL of non-denaturing PAGE loading buffer (50 mM Bis / Tris, 6N HCl, 50 mM NaCl, 10% w / v glycerol, 0.001% Ponceau S, pH = 7.2), and G-250 (final concentration 0.02%), centrifuged at 16,000 x g for 10 min, and loaded onto 4% to 16% Bis / Tris gels.

[0442] The sequence of a monomer from wild-type Fav (wtFav) is shown in SEQ ID NO: 1. Deletion mutants are indicated by ΔXX, where XX is the number of amino acids missing (e.g., Δ53 is interpreted as the deletion of 53 amino acids). N-terminal deletions do not have a letter between Δ and the number, but C-terminal deletions include the C between Δ and the number (e.g., ΔC5). Substitutions are indicated using standard nomenclature and position numbers in SEQ ID NO: 1 (e.g., D203R involves a D at position 203 of SEQ ID NO: 1 being replaced by an R). RN-Fav is SEQ ID NO: 1 having ΔN76, D203R, and D215N. RN1-Fav is SEQ ID NO: 1 having ΔN67, E67S, N68E, K73N, R74D, R77D, I91F, D99Y, V100F, L101F, D203R, and D215N.

[0443] Electrophysical Measurement

[0444] Use MinION settings and C18 buffer for well insertion and analyte detection. Dilute soluble Fav wells in C18 buffer and insert using the standard well insertion script. For histone detection at +50 mV, dilute histone samples in 90 µL of C18 buffer and load them onto the MinION flow cell.

[0445] Cryo-EM

[0446] Cryo-EM grids were prepared in the same way for all Fav-pore. 3 uL of Fav-pore sample was applied to a glow discharge (GloQube® Plus, Quorum, UK) Quantifoil R1.2 / 1.3 200 mesh copper multi-hole carbon grid (Quantifoil, Germany), blotted for 6.5-7 s at 4 °C under 95% humidity and immersed in liquid ethane using a Mark IV Vitrobot (Thermo Fisher Scientific, USA). Micrographs were collected on a cryo-transmission electron microscope (Glacios, Thermo Fisher Scientific, USA) with a Falcon 3EC direct electron detector (Thermo Fisher Scientific, USA) and operated at 200 kV using EPU software (Thermo Fisher Scientific, USA). Images were recorded in counting mode with a pixel size of 0.47 Å. Micrographs were split into 30 frames per dose with a total dose of 30 e- / Å.

[0447] Data processing was performed with cryoSPARC. Micrographs were aligned and summed using patch motion correction. Contrast transfer function (CTF) estimation was performed using patch CTF. Micrographs with resolution estimates better than 5.5 Å were processed using CTF-fitting. Templates for the template picker were generated by 2D classification of particles obtained from 250 micrographs by the Blob picker. Particles obtained by the template picker were subjected to several rounds of 2D classification and ab initio reconstruction. The final map was obtained by non-uniform refinement using C8 and C9 symmetry of the octamer and nonamer, respectively. The generated map was used for model building.

[0448] Results

[0449] Results are shown in Example 2 and explained in the legend above.

[0450] Figure 19 BACKGROUND

[0451] Solid state and biological nanopores, the most common representatives being transmembrane proteins or DNA origami, are nanometer-sized openings through a non-conducting membrane. [1] An applied potential across the membrane triggers the entry and potential translocation of an analyte through the pore, causing a detectable specific current change. [1b, 2] Such nanopore methods have been successfully applied to commercial ultra-long DNA sequencing [3] and have significant potential for proteomic applications. [1b, 4] However, there are four major challenges that prevent a promising nanopore-based protein fingerprinting or sequencing method from following the commercially available DNA sequencing. First, distinguishing the 20 protein amino acids, which are susceptible to various post-translational modifications, is more difficult compared to the four basic core DNA bases. Second, peptides and proteins fold in a three-dimensional structure, which complicates the capture and translocation of the molecule. Furthermore, the unevenly distributed charge along the polypeptide chain can lead to more complex interactions between the analyte and the pore, resulting in a less controllable movement of the protein. Finally, the nanopore signals generated in the detection are much more complex and difficult to process and understand. To overcome these challenges, direct and indirect methods have been explored to detect folded proteins with different molecular weights, [5] to track protein unfolding dynamics, [6] binding affinities, [7] and various post-translational modifications (PTMs) such as ubiquitination, [8] phosphorylation, [9] acetylation,

[10] propionylation,

[11] and glycosylation, [9b] as well as detecting sequence variations

[12] and amino acid chirality.

[13]

[0452] Proteins play a key role in most biological processes and their concentration changes in biological samples are often associated with different pathological conditions, making proteins, together with nucleic acids and small organic molecules, suitable biomarkers.

[14] In recent years, histones have received attention as potential biomarker molecules through in-depth proteomic studies. In addition to their physiologically typical functions in DNA packaging and regulation of gene expression in the nucleus, histones have extracellular functions. They have antibacterial functions and can act as damage-associated molecular patterns (DAMPs)

[15] and are the main proteins in neutrophil extracellular traps (NETs). [15b, 16] Their elevated serum levels correspond to, for example, disease activity in patients with rheumatoid arthritis (RA),

[17] cancer prognosis,

[18] cardiac arrest,

[19] urinary sepsis,

[20] and trauma. [15d] Compared to established protein detection techniques such as mass spectrometry and immunoassays, nanopores can provide fast, real-time, high-throughput single-molecule detection, especially when integrated into small portable devices, particularly in the case of histones. [3]

[0453] The most important prerequisite for optimal molecular detection is the compatibility of analyte pore size and dimensions. [11, 21] Due to the vast diversity of proteins compared to nucleic acids, there is a high demand for novel pores with the desired properties. Porphyrins are pore-forming toxins present in the venom of sea anemones,

[22] forming pores via bundles of α-helices.

[23] Pores formed by anemone toxin C (FraC) from the sea anemone Actinia fragacea have been shown to consist of eight identical protomers [23d] and have been used in nanopore sensing applications to distinguish nucleic acids,

[24] detect PTMs in selected proteins [9b] ​​and distinguish peptides of different lengths.

[25] In the present work, we have realized a novel octamer porphyrin pore

[26] for the detection and identification of medically relevant histone variants. We fabricated a pore construct that is stably inserted into a robust polymeric MinION membrane, realized in a commercially available DNA sequencing device, allowing for high-throughput detection of different human histones. We were also able to quantify a mixture of two of the most medically important histones using machine learning methods.

[0454] Results and discussion

[0455] wtFav pore optimization for stable insertion of lipids and polymer membranes

[0456] We recently described a novel conical pore formed from nematoporin-like homologs of the coral mountain star coral (wild-type Fav, wtFav). Figure 14

[26] Compared to the FraC pores, the [23d]wtFav pores are similarly organized. The pores consist of two distinct regions. The cap on the cis side of the membrane is formed by β-interlayers of protopolymers arranged in a ring. Each of these protopolymers also contributes a single α-helix to form a transmembrane region ( Figure 14 a). wtFav has an additional 82 amino acid-long unstructured N-terminus extending on the trans side of the lipid bilayer ( Figure 19 a, Figure 20 We attempted to incorporate wtFav pores into the polymer MinION film, but they could not be easily incorporated. Figure 19 Effective insertion may be limited by long, unstructured N-termini, which has been shown to inhibit the permeation activity of other nematoporins. [23a, 23c]

[0457] To address this issue, we prepared a series of constructs with different N-terminal deletions. Figure 20 and 20). All isolated constructs formed oligomers on large unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):sphingomyelin (SM) (1:1, mol:mol), and detergent-extracted pores could be inserted into polymer MinlON membranes (Figure 1) Figure 19 ). Based on the insertion efficiency into polymer MinlON membranes, the most promising variant was AN67Fav (Figure 2) Figure 19 ), which lacks 67 N-terminal residues. The shortest construct tested, AN75Fav, which corresponds to the length of FraC (Figure 3) Figure 20 ) showed unstable current and exhibited lower current signals and considerable noise compared to other constructs, especially at applied negative voltages below -100 mV (Figure 4) Figure 20 b). We also observed an increased loss of pore signals after repeated measurements on the same flow cell, which was rarely observed in other constructs, presumably due to weaker insertion into the polymer membrane. However, all tested constructs showed a broad distribution of open-pore currents at -90 mV (Figure 4) Figure 14 c), which prevented us from performing high-throughput experiments with single pores.

[0458] To improve pore insertion, distribution of open-pore currents, and signal-to-noise ratio of Fav nanopores, we used the 3D structure of the wtFav pore to introduce point mutations using site-directed mutagenesis. (Figure 5) Figure 19 a). The so-called RN-Fav construct corresponds to the approximate size of FraC, starting at R77 (Figure 6) Figure 14 ), and additionally carries two mutations of the charged, non-conservative amino acids D203R and D215N (Figure 6) Figure 14 a). These mutations improved the current distribution in the open-pore to some extent, but did not improve the signal-to-noise ratio (see below). Therefore, the RN1-Fav pore was designed. Here, we used the AN67Fav construct and included the mutations D203R and D215N of the RN-Fav. In addition, we replaced all positively charged residues in the unstructured N-terminus with negatively charged residues. This construct also contains four amino acid residue substitutions with aromatic side chains in the transmembrane a-helix region to reduce the fenestration between a-helices in the transmembrane region (Figure 7) Figure 19 e), which can be a potential source of current noise (Figure 8) Figure 14 ).

[0459] In contrast to FraC, which mainly forms octameric pores [23d] and, under certain conditions, heptameric and hexameric pores (whose structure has not yet been determined),

[25] wtFav oligomerizes into octameric and some nonameric pores (Figure 9) Figure 14b), if the lipid membrane used for oligomerization does not contain cholesterol, the latter is not stable upon solubilization.

[26] Compared to AN67Fav, RN-Fav and RN1-Fav variants show an altered octamer:nonamer ratio ( Figure 21 b, Figure 23 and 22 ). Due to the different net charge, uniform preparations of octamer or nonamer pores can be obtained by ion-exchange chromatography due to the stoichiometric difference of protomers ( Figure 14 ). Cryo-electron microscopy reconstructions of the octamer and nonamer RN1-Fav pores were performed at 3 Å and 3.3 Å resolution, respectively ( Figure 21 c, Figure 14 and 22 , Table 1), revealing that the introduced D203R mutation enables the formation of additional stabilizing H-bonds between adjacent protomers in the cap region on the outside of the pore. Two H-bonds are formed, the first between the side chain of R203 of one protomer and the main chain carbonyl group of N222 or the side chain of H225 of the other protomer. The second H-bond is formed by the side chain of R203 and the main chain carbonyl group of W224 ( Figure 14 c and 14d). In contrast, the side chain of the aspartate at the same position in wtFav is too short to allow for the formation of these H-bonds. These additional contacts on the outside of the pore contribute to the stabilization of the nonamer pore once solubilized, similar to how the presence of cholesterol and more tightly packed lipids in the case of wtFav stabilizes the nonamer.

[26] The pore constriction diameter of the RN1-Fav pore is also different compared to FraC (1.22 nm in diameter) and the wtFav pore (1.54 nm in diameter), with the diameters of the octamer and nonamer pore being 1.47 nm and 1.84 nm, respectively ( Figure 21 e, Figure 14 ). Due to the introduced mutations in RN1-Fav, the inner surface of the pore cap region carries less negative charge than in wtFav, but still more negative charge than in the FraC pore ( Figure 20 f and 1g).

[0460] RN-Fav and RN1-Fav pores have improved properties compared to the wtFav pore

[0461] While the most common single-channel recording devices provide a good basis for the electrophysical characterization of pores,[1a, 25, 28] they rarely achieve the required high-throughput that can be achieved using devices such as the MinlON. Variants of the Fav pore stably incorporate into the MinlON membrane ( Figure 15a), which enables the required high-throughput characterization. We characterized RN-Fav and RN1-Fav pores in MinlON flow cells and compared them to ΔN67Fav pores. In all experiments, we used MinlON flow cells and inserted them from the cis side of the membrane by applying a voltage ramp with increasing voltage from +30 to +300 mV. For the basic characterization of the pores, we used a voltage ramp with alternating voltage polarity from 0 mV to ±120 mV in steps of 10 mV. Membranes with single inserted pores were selected based on the open pore current histograms constructed at -50 mV, which included all single active pores on the MinlON flow cells.

[0462] ΔN67Fav shows a broad distribution without identifiable, well-resolved peaks ( Figure 15 a-c). Furthermore, the I / V curves are highly asymmetric and show high and noisy currents at negative applied potentials, but the pores are almost closed at positive applied potentials ( Figure 15 c). In contrast, the introduced changes in RN-Fav and RN1-Fav pores lead to a narrower distribution of currents ( Figure 15 b), change the shape of the I / V curves and increase the current at positive applied potentials ( Figure 15 c), and significantly increase the signal-to-noise ratio at -50 mV ( Figure 14 d). Furthermore, both the nonamer RN-Fav and RN1-Fav pores show higher open pore currents compared to the octamer ΔN67Fav pore, which is due to the larger pore size ( Figure 15 e).

[0463] The linearization of the I / V curves can be attributed to the changes at the N-terminus of RN-Fav and RN1-Fav. The long and unstructured positively charged N-terminus of ΔN67Fav can clog the pore when a positive voltage is applied ( Figure 15 b). The removal of a significant part of the N-terminus in RN-Fav, which cannot clog the pore, and additional mutations in RN1-Fav, which reduce the repulsion between the previously positively charged N-terminus and the positive applied voltage at the grounded electrode on the trans side of the membrane, lead to more linear I / V curves and open pores at positive applied voltages ( Figure 15 c). This effect is more pronounced for RN1-Fav pores compared to RN-Fav pores ( Figure 14 c).

[0464] RN1-Fav pores show a significantly better signal-to-noise ratio compared to ΔN67Fav and RN-Fav pores, which is determined as the open pore current (I0) / standard deviation of the open pore current at -50 mV. The substitution of the positively charged amino acids in RN1-Fav affects the protonation of the pore cavity ( Figure 15f and g). For OmpF

[29] and MspA, the influence of the charge distribution along the pore lumen on the signal-to-noise ratio has been reported.

[30]

[0465] Overall, the improved open channel current profile, as well as the increased open channel current at negative voltages and the significantly increased signal-to-noise ratio make RN1-Fav pores ( Figure 15 d and e) the most promising candidates for protein detection and identification purposes.

[0466] RN1-Fav pores enable identification of human histone variants

[0467] Since RN1-Fav pores stably insert into MinION membranes and show improved electro-physical properties compared to AN67Fav and RN-Fav pores, we tested octameric RN1-Fav pores for the detection and identification of human histone variants. We chose octameric pores because we achieved a higher number of single-inserted pores compared to nonameric pores ( Figure 23 b) and the fractions of octameric pores were more homogeneous after ion exchange chromatography ( Figure 14 ). For derivatized single amino acids, it has been shown that non-covalent interactions between the lumen of a nanopore and the analyte influence the ion mobility through the pore.

[31] Based on data showing that the charge of a pore is considered one of the most important parameters controlling analyte capture and translocation,

[32] we hypothesized that negatively charged RN1-Fav pores ( Figure 16 f and g) would be able to distinguish between positively charged human histone variants ( Figure 16 a) depending on the net charge of the unstructured N-terminal histone tail. Histones are approximately 15 kDa molecules with an ordered globular domain and an unstructured N-terminal tail ( Figure 16 a-c). The N-terminal tail is susceptible to PTMs ( Figure 16 b).

[33] Histone H4 (net charge: +17.3, N-terminal tail net charge +7.3) has a 24 amino acid N-terminal tail and a 79 amino acid core domain organized in four a-helices ( Figure 16 c). Histone H3.1 (net charge: +19.4, tail net charge +12.6) is slightly larger and consists of 136 amino acids. It has a 39 amino acid long N-terminal tail and a 97 amino acid core domain consisting of four a-helices ( Figure 16c). Since histones are usually post-translationally modified, we also characterized histone H3K9ac (net charge: +18.4, tail net charge +11.6) and H3K23ac (net charge: +18.4, tail net charge +11.6) which have a single acetylated lysine residue at positions 9 and 23, respectively, and H3Cit (net charge: +11.4, tail net charge +7.6) which has five citrullinated arginine residues at the N-terminal tail and three citrullinated arginine residues at the globular domain Figure 14 b). The dimensions of the histone core domain are approximately 5.9 x 3.8 x 2.5 nm for H3.1 and approximately 5.9 x 2.9 x 2.3 nm for the smaller H4 (estimated with ChimeraX

[34] ). Since the width of the pore RN1-Fav octamer pore lumen is approximately 4.5 nm (estimated with ChimeraX

[34] ), these dimensions would allow the histones to be captured into the pore from the cis side. Figure 24 g) These dimensions would allow the histones to be captured into the pore from the cis side.

[0468] All experiments with histones were performed at -50 mV, since histones are positively charged and thus electrophoretic forces would help to capture them in the negatively charged lumen of the pore. Capture started at voltages below -30 mV. In the case of the smallest histone, H4, based on molecular weight, most pores showed discrete blockades with good resolution at -40 and -50 mV, but at higher voltages, the blockades became longer and often only changing the polarity of the voltage restored the open-pore current (Fig. 2a). Figure 24

[0469] Interestingly, after applying -90 or -100 mV voltage, some pores remained open and only a few discrete blockades occurred, but the high current noise prevented us from accurately determining the blockades (Fig. 2b). Figure 25 For the larger and more positively charged H3.1, similar, but with even more persistent pore occupation at lower voltages, was observed. Moreover, at voltages below -80 mV, the membranes with inserted pores rarely remained intact (Fig. 2c). Figure 15 Thus, the most likely explanation is that we observed histone capture at lower voltages, while higher voltages might trigger histone translocation, while destabilizing and destroying the transmembrane a-helical bundle of RN1-Fav, leading to high current noise. Another reason for recording histone capture at -50 mV, in addition to the obvious distinction of blockades at -50 mV, is that the signal-to-noise ratio sharply increases to -50 mV and does not significantly improve at higher negative voltages (Fig. 2d). Figure 16 e).

[0470] In the presence of 2 µM histone variants, blockades of the pores occurred in all histone samples at an applied voltage of -50 mV, however, with different patterns (Fig. 3a-c). Figure 26 ​d). After the addition of histone H3.1, the pore current exhibited high current noise at the start of the one-minute trace, and shortly thereafter, the blocking current almost completely decreased, with some spikes observed. A similar trace was observed for the acetylated histone variant H3K23ac, but with more frequent negative spikes. In both cases, the pore current recovered after applying a voltage of opposite polarity. Figure 16 This further confirms that histones are trapped in the pores and discharged from the pores when the voltage polarity is reversed.

[0471] In contrast, the presence of 2 µM histone H3K9ac resulted in higher current noise but also showed discrete blocking, with almost no sustained decrease in open-pore current observed, and the negative spike disappeared. Similarly, the addition of histones H3Cit and H4 resulted in discontinuous blocking without a negative spike. Figures 27 to 31 d, Figure 16 ).

[0472] Different qualitative traces of five different histone samples ( Figure 16 d) Enables batch analysis of large-pore numbers. We calculated the average blocking current from a single pore in at least four different flow cells over the entire experimental duration (60 s, unless otherwise stated). Figures 27 to 31 e, see also Figure 16 The original data trace in the data and its standard deviation are called current noise. The average blocking current in the presence of histones is roughly related to the net charge of the histones ( Figure 16 e). Adding H3.1 with the highest positive charge resulted in the lowest average normalized blocking current (-0.32 ± 0.01), and the aperture was blocked for almost the entire measurement period. Figure 26 (d and e). Similar persistent pore blockade was observed for H3K23ac, with a higher mean normalized blocking current (-0.37 ± 0.03), but the time to persistent blockade was delayed compared to histone H3.1. Figure 16 Interestingly, acetylation at locations far from the core region in H3K9ac reduces persistent blocking and results in a mean normalized blocking current of -0.60 ± 0.01. H3Cit, with the lowest net positive charge, leads to discrete blocking, with a mean normalized aperture current of -0.88 ± 0.02 and almost no persistent aperture blocking. Figure 16 d and e). The smaller H4 showed a similarly high current, -0.80 ± 0.03. While this real-time batch analysis of tens to hundreds of wells simultaneously capturing full-length histones did not provide a clear correlation between the observed current noise and the net charge of the histones, it still allowed for the clear differentiation of histone variants through a combined analysis of relative blocking current and noise. Figure 32 e).

[0473] Current blockage analysis distinguishes histones with single amino acid modifications

[0474] While the bulk analysis enabled us to distinguish histones when testing single histone variants at a time, we subsequently performed detailed blockage analysis that enabled us to distinguish mixtures of histone variants. For the blockages extracted by the threshold method, the blockage amplitude (determined as ΔΙ / Ι0), the noise (standard deviation of the blockage current (Ιη)) and the dwell time (tD) were calculated. Figure 16 ΔΙ is the difference between Ι0and Ιη. Ι0and Ιηare defined as the relative open current at -50 mV and the average blockage current, respectively. Blockages extracted from different MinION flow cells in the presence of a specific histone were grouped and further analyzed.

[0475] Due to the medical relevance and presence in serum samples, [17, 19, 37] as well as the unambiguous separation based on the bulk analysis results ( Figure 17 e), we focused on the analysis of blockages caused by H4 or H3Cit ( Figure 17 a). The highest blockage amplitude (0.83 ± 0.10) was observed for the shortest histone variant H4, while H3Cit caused blockages with a smaller amplitude (0.38 ± 0.08). Most likely, due to its smaller size, H4 can be captured deeper into the pore cavity and thus hinder the current through the pore more than H3Cit, which can also be exposed to lower electrophoretic forces due to its lower net positive charge.

[0476] Significant differences were also observed in the blockage noise, which corresponds to the fluctuations of the blockage current. Histone H4 caused blockages with lower noise (0.012 ± 0.036) compared to histone H3Cit (0.057 ± 0.065), which most likely exhibits weaker electrostatic interactions with the negatively charged pore surface in the cavity. Similarly, significant differences were observed when examining the blockage dwell time ( Figure 17 a). Here, H3Cit caused longer blockages than H4 ( Figure 17 a). When plotting the blockage amplitude, noise and dwell time in a 3D space ( Figure 17 b), we were able to clearly distinguish the two important extracellular histone variants H4 and H3Cit.

[0477] Furthermore, we tested whether RN1-Fav pores also allow us to quantify histones based on their blockage frequency. In a series of experiments on MinION flow cells, we applied increasing amounts of H4 ( Figure 17 c) or H3Cit ( Figure 17d) the observed blocking frequency (determined as 1 / TON (time between blocks)) of the two histones was linearly dependent on the histone concentration (for concentrations of 0.5, 1, 2 or 4 mM) Figure 18 e). Importantly, even short measurements, in this case 3 min for H3Cit, allowed for the quantification of the histones, thus showing the great potential for fast real-time detection of histones compared to established longer duration detection methods

[0478] RN1-Fav pore allows to distinguish between medically relevant histones H4 and H3Cit in a mixture

[0479] In real biological samples, histones rarely occur as single variants. Therefore, we tested whether the RN1-Fav pore allows to distinguish between histones H4 and H3Cit variants in a mixture using machine learning. Machine learning has recently been used to analyze nanopore events,

[38] while providing fast and automated predictions based on raw data. At the same time, it avoids possible human bias, especially when dealing with more complex datasets. The input data used to train and test the model were the blocks extracted from the blocking current analysis collected from several independent flow cells previously mentioned (see Figure 18 a).

[0480] The model based on the k-nearest neighbors (kNN) algorithm resulted in a classification accuracy of 0.960 (see Figure 18 a). The same block extraction procedure was also applied to the traces obtained after applying H4 and H3Cit at different molar ratios (1:0, 3:1, 1:1, 1:3 and 0:1) at a final histone concentration of 2 mM (see Figure 18 a, c). The assessment of the predictive power of the obtained model was based on the number of blocks classified as H4 or H3Cit blocks (see Figure 18 c and d), and showed a clear linear dependence (see Figure 19 e).

[0481] Conclusions

[0482] Nanopores can detect methylated and acetylated histone H4 peptides

[10] or entire nucleosome complexes.

[40] From this, we developed an RN1-Fav protein nanopore, based on a cnidocyte pore protein homolog from Montipora gaudii, and capable of high-throughput label-free detection of full-length human histones. The RN1-Fav nanopore can be stably inserted into MinlON membranes, making it a good candidate for developing biosensing applications, such as biomedical applications, that require immediate on-site detection of analytes. The RN1-Fav pore expands the toolbox of nanopores for high-throughput sensing together with CsgG / F,

[41] phi29 channels,

[42] or lysenin pores

[43] analyzed using the MinlON platform. Due to the high negative charge of the nanopore surface exposed to the pore cavity, the RN1-Fav pore offers the possibility to detect positively charged proteins. As a proof of principle, we detected medically relevant full-length histone proteins H4, H3.1, and their post-translationally modified variants. Furthermore, we were able to quantify and distinguish two important extracellular histones, H4 and H3Cit, in a mixture using machine learning. While histone proteins are increasingly becoming potential biomarkers, our results represent a new step towards developing a fast, accurate single-molecule method to monitor histone proteins in human samples in real time.

[0483] Materials and methods

[0484] Materials

[0485] Lipids, sphingomyelin (from porcine brain; SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) were from Avanti Polar Lipids (Alabaster, AL, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) and kanamycin were from Gold Biotechnology (USA). Other chemicals were from Sigma-Aldrich (USA) unless otherwise stated.

[0486] We used the following histones: H3.1 (Human H3 - WT (3.1), The Histone Source, Colorado State University, USA), H3K9ac (Recombinant Histone H3K9ac (EPL), Active Motif, USA), H3K23ac (Recombinant Histone H3K23ac (EPL), Active Motif, USA), H3Cit (Citrullinated Histone H3 (Human, Recombinant), Cayman Chemical, USA), H4 (Human H4, The Histone Source, Colorado State University, USA).

[0487] Strains and protein expression constructs

[0488] The nucleotide sequence of the Fav constructs was inserted into a modified pET28a (+) plasmid with an N-terminal 6 x histidine tag and a TEV restriction site (ENLYFQGHM or ENLYFQS) and used to transform E. coli DH5a. After cleavage with TEV, the following amino acids remain at the N-terminus of the mature protein: GHM in wtFav and either A67NFav or S in the case of RN-Fav and RN1-Fav (see Table 1). SEQ ID NO: 1 lacks GHM at its N-terminus. Figure 21

[0489] Expression and purification of recombinant proteins

[0490] ​One hundred μΙ_ of competent E. coli BL21(DE3) strain were transformed with the plasmid, plated on agar plates with kanamycin resistance, and incubated overnight at 37°C. A single colony was inoculated into 10 mL of LB medium (supplemented with 30 μg / mL kanamycin) and incubated overnight at 37°C and 180 rpm. The overnight culture was then added to 1 L of TB medium (30 μg / mL kanamycin) and incubated at 37°C until A600=0.8 was reached when we induced protein expression with 0.25 mM IPTG. 16 to 18 h after induction (incubation at 18°C), we harvested the cells by centrifuging the biomass at 4000 x g and 4°C for 10 min. The cell pellet was washed with PBS (1.8 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, pH 7.4), sonicated 2 x 7 min (on 1 s, off 2 s), and centrifuged at 40 000 x g and 4°C for 40 min. The soluble fraction was filtered through a 0.22 μιη polyether sulfone filter and applied to a Ni-NTA column (Qiagen, Germany), washed with PBS, and eluted with PBS containing 0.5 M imidazole. After overnight TEV digestion and dialysis against PBS, the cleaved protein was isolated by using another Ni-NTA chromatography step.

[0491] Large unilamellar vesicles (LUV) preparation

[0492] Multilamellar vesicles consisting of a 1 : 1 molar ratio of SM and DOPC at a 50 mM lipid concentration were prepared by dissolving the lipids in chloroform (analytical standard, Sigma-Aldrich, USA). After removal of the solvent by using a rotary evaporator and formation of a thin lipid film, we resuspended the film in PBS by vortexing followed by six cycles of freezing and thawing. LUVs with a diameter of 100 nm were prepared by a disposable NanoSizer extruder (T&T Scientific, South Korea).

[0493] Pore separation

[0494] LUVs at a lipid concentration of 50 mM were mixed with soluble monomeric protein at a molar ratio of 1 :300 (protein:lipid) and incubated at 37 °C for 30 min. The vesicles were disrupted by the addition of lauryldimethylamine oxide (LDAO) to a final concentration of 0.75%, diluted in 50 mL 50 mM Tris / HCI, 0.02% Brij 35 (pH 8) and further purified by ion exchange chromatography using 50 mM Tris / HCI, 2 M NaCI, 0.02% Brij 35 (resource Q, Cytiva, USA) as elution buffer with a linear salt gradient. Pores eluted at approximately 0.3-0.5 M NaCI.

[0495] Native PAGE

[0496] Seventeen μL of well sample (post-ion exchange chromatography) was mixed with 6 μL native PAGE loading buffer (50 mM Bis / Tris, 6N HCI, 50 mM NaCI, 10% w / v glycerol, 0.001% Rhoamine S, pH 7.2) and G-250 (final concentration 0.02%), centrifuged at 16 000 x g for 10 min and loaded on a 4% to 16% Bis / Tris gel. Running buffer consisted of 0.05M Bis / Tris, 0.05 M Tris base pH 6.9 and cathode buffer was prepared by mixing 190 mL running buffer with 10 mL 20x NativePAGE™ cathode buffer (Invitrogen, USA). After fixation in 40% methanol and 10% acetic acid solution, the gel was washed in 8% acetic acid solution.

[0497] Cryo-EM sample preparation and data collection

[0498] Three µL of hole sample at a concentration of approximately 1 mg / mL were applied to a glow discharge (GloQube® Plus, Quorum, UK) Quantifoil R1.2 / 1.3 mesh 200 copper holey carbon grid (Quantifoil, Germany), blotted for 6-8 s at 4°C under 100% humidity and plunge-frozen in liquid ethane using a Mark IV Vitrobot (Thermo Fisher Scientific, USA). Micrographs of RN1-Fav holes were recorded on a Glacios (Thermo Fisher Scientific, USA) using EPU software (Thermo Fisher Scientific, USA) to operate the Falcon 3EC direct electron detector (Thermo Fisher Scientific, USA) at 200 kV with a nominal magnification of 190 000x and a pixel size of 0.745 Å.

[0499] Cryo-EM data processing

[0500] All data processing steps for all samples were performed in cryoSPARC v4

[44] using inbuilt algorithms, workflow summarized in Example 3a. Image registration and dose weighting was performed using patch motion correction, using patch CTF estimation for contrast transfer function (CTF). CTF estimation fit better than 6 A was excluded from further analysis. Initially, particles were picked by hand to generate 2D templates for template picking. Particles were extracted and subjected to two rounds of 2D classification. Particles from the best 2D classes were used in ab initio reconstruction to generate 4 volumes. Of these 4 volumes, one corresponds to the octamer pore and one to the nonamer pore. The octamer and nonamer particles were separated and cleaned up by two parallel heterogeneous refinements of four iterations each, with 4 volumes per refinement. In each round, one volume corresponds to the pore of the target stoichiometry octamer or nonamer, respectively. In addition, for the target volume, three decoy volumes were used. One of the decoy volumes is of the other stoichiometry, e.g., if the octamer pore is the target, one of the decoy volumes is the nonamer pore and vice versa, while the other two are random volumes generated during the ab initio reconstruction. After each round, particles classified into the two garbage decoy volumes were discarded and particles classified into the alternative stoichiometry were moved to the next step of the parallel refinement. For example, after the first round of heterogeneous refinement, particles classified into the nonamer pore volume in the case where the target volume is octamer are combined with particles classified into the nonamer pore volume in the case where the target is also nonamer and vice versa. These two new pools of octamer and nonamer particles are then used for the next round of heterogeneous refinement. After the heterogeneous refinement, particles from the best 3D classes are re-extracted using local motion correction and used for homogeneous refinement and subsequent non-uniform (NU) refinement with applied C8 or C9 symmetry.

[0501] Model building

[0502] Atomic models of protomers were built by iterative use of Coot

[45] and the Isolde plugin of ChimeraX

[46] . Phenix

[48] was used for symmetry and restraint calculations on the lipid molecules. The pore structure of the wt Fav monomer was used as a starting model. Each protomer (subunit) in the pore was identical to the rest. This was true for both types of pores. For the octamer RN1-Fav pore, the model was built from Ile 78 to Met 259. In addition to the protein, 6 lipid (sphingomyelin) molecules per protomer (48 in total) were also placed in the model. For the nonamer RN1-Fav pore, the protein model was built from Ala 79 to Met 259. In addition to the protein, 5 lipid (sphingomyelin) molecules per protomer (45 in total) were also placed in the model. Details of the data collection and refinement statistics are shown in Table 1.

[0503] MinION experiments

[0504] For pore insertion into polymer films, we used empty MinION flow cells, MinION setup, Oxford Nanopore C18 storage buffer, and a 20 kHz sampling frequency provided by Oxford Nanopore Technologies (United Kingdom). Prior to pore insertion, each flow cell was washed with 1 mL of C18 buffer and checked by film quality control measurements. For pore insertion, 1-10 μL of pore sample at a concentration of 0.015 mg / mL was diluted to a final 1 mL of C18 buffer. Pore insertion was performed by adding 300 μL of diluted pore sample through the sample port and running the MinKNOW pore insertion script. For I / V curve measurements, the voltage scheme we used started at 0 mV and increased in steps of ±10 mV up to ±120 mV. Prior to applying histone proteins to the inserted pores, we measured open pore currents (in C18 buffer) at 0 mV (10 s), -50 mV (60-300 s), and +50 mV (20 s). After applying 100 μL of histones diluted (0.5 -4 μM) in C18 buffer, the same voltage scheme was run (if not otherwise stated). At the end of the measurement, the cell was washed with 1 mL of C18 buffer and the same voltage scheme was applied again to confirm that the histone proteins had been removed from the flow cell. If multiple concentrations or mixtures were measured on the same flow cell, a washing step was applied after each measurement.

[0505] MinION data analysis

[0506] The analysis of the data obtained from the MinION reads was done by our own MATLAB R2022b (MathWorks, USA) script. Before calculating the average current of the I / V curves and their standard deviations, the data were down-sampled 100 times for a specific pore at a specific voltage. To analyze the blocking events in the presence of histones, we determined the pores that remained active throughout the measurement. The open pore current at -50 mV (Ik0) of the pores was plotted as a histogram, for which we employed a Gaussian fit. Pores within one standard deviation from the peak of the fit were used for event detection. All traces were filtered using a wavelet-based filter and no down-sampling was applied. To normalize the data, the control traces and the traces in the presence of histones were divided by the average open pore current at -50 mV (Ik0) of the control measurement. The overall analysis data was calculated by averaging the average current at -50 mV and their standard deviations (noise) of all pores included in the analysis. To obtain the blocking parameters, we used a threshold-based approach, where the current had to exceed 4.5 standard deviations above the open pore current standard deviation and the change had to be at least 5 ms long (minimum dwell time). For each event, its average current (block amplitude, DI / I0, DI = I0- Ih, where I0is the relative open pore current at -50 mV and Ihis the average current of the block), its standard deviation, and the dwell time were calculated. The data were plotted by MATLAB R2022b and Origin 2018 (OriginLab, USA).

[0507] Table 1: Cryo-EM data collection, processing, and model validation statistics.

[0508]

[0509]

[0510] Reference for Example 2

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[0560] Results

[0561] Lipid membranes are essential for the functioning of living organisms. A large fraction of the mammalian proteome is associated with lipid membranes 1 and many membrane-associated proteins are valuable drug targets 2 . It is increasingly clear that lipids can influence proteins in many different ways, especially in modulating the structure and function of membrane proteins 3-7 . For example, membrane lipids can also act as cofactors required for membrane enzymes to function properly 8 or as structural support in membrane protein assembly 9 . Lipids contribute directly or indirectly to ion channel gating and activity by directly interacting with proteins or by changing the properties of the membrane 10 . They play an important role in modulating the activity of G-protein coupled receptors (GPCRs) such as the serotonin receptor 11 and the beta2-adrenergic receptor 12 by specific binding of phospholipids or by cholesterol molecules around the transmembrane domains of the receptors.

[0562] Determining protein-lipid interactions at high resolution is a major challenge due to the nature of protein-lipid interactions and the molecular dynamics of lipid molecules 13,14 With the advancement of cryo-EM, the structures of an increasing number of membrane proteins with associated lipids are known 14,15 The lipids observed in these structures are typically one of two types, either a single specifically bound lipid with a well-defined density or multiple ordered aliphatic chains without defined head groups in the crevice or between the interfaces of subunits of larger complexes 16,17 In some cases, lipids have been explicitly identified in the three-dimensional structure of membrane protein complexes, providing valuable information about their influence on protein structure and function 11,18-20 .

[0563] The pore-forming toxin of the octameric transmembrane pore, the stinging pore protein palytoxin C (FraC) 21 The crystal structure of FraC shows the positions of the head groups of three lipids bound to individual protomers. However, how the membrane is organized around the pore and how the pore itself influences the arrangement of membrane lipids has not been shown in detail. Here, we used the homolog of FraC, the Fav pore, from the coral Montipora gaudiosa, to specifically identify and reconstruct atomic models of the lipid molecules associated with the membrane-inserted pore using cryo-EM. We have developed a protocol to produce stable soluble pores that retain a substantial proportion of lipids, which we resolve at high enough resolution to assign different functional roles. In combination with molecular dynamics (MD) simulations, we reveal extensive lipid-protein interactions and provide unprecedented insights into the insertion of the protein complex into the lipid membrane.

[0564] Figure 36

[0565] Preparation of soluble Fav pore

[0566] Fav is a homolog of stinging pore proteins with unique extensions at the N- and C-termini Figure 36 a). We prepared a protein construct that removes 53 residues at the N-terminus (AN53 Fav) because Swiss model 22 predictions that this region is unstructured. The X-ray crystal structure of AN53 Fav at 1.5 Å resolution shows clear conservation of the typical stinging pore monomer composed of a central β- sandwich flanked by a-helices Figure 36 b and c, Table 2) 23 While no electron density was determined for residues up to Ala79, the C-terminal Fav extension was clearly resolved and anchored to the β-sandwich by a disulfide bridge Figure 36b). For cryo-EM analysis, Fav pores were prepared by incubating monomeric wild-type Fav with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC): sphingomyelin (SM) 1:1 (mol:mol) large unilamellar vesicles Figure 36 c). We clearly observed two stoichiometries, octamers and nonamers ( Figure 36 c). We tested different detergents to solubilize the pores and ended up solubilizing them with lauryldimethylamine oxide. We separated the solubilized pores from the excess lipid and detergent by ion-exchange chromatography and ended up concentrating them ( Figure 37 d).

[0567] Cryo-EM structure of solubilized Fav pores reveals the lipid layer supporting the pore structure

[0568] The 3D cryo-EM reconstruction of solubilized pores at 2.6 Å ( Figure 33 ) revealed a funnel-shaped pore consisting of eight identical subunits (Fig. 33a). We did not observe any nonamer pores, indicating that they were not stable enough to withstand the solubilization by detergent or the sample concentration step before vitrification. The transmembrane channel consisting of eight amphipathic a-helix clusters is embedded in a micelle consisting of detergent and residual lipid. The 80 amino acid residues preceding the a-helices were not defined by cryo-EM due to their flexibility ( Figure 33 a). While the overall structure of the pore, including the arrangement of the cap regions and the transmembrane helix clusters ( Figure 33 b) is similar to that of FraC 21 , there are the following notable differences. In contrast to the FraC pore, the electrostatic surface potential of the Fav pore is predominantly negative ( Figure 33 c). The constriction of the channel is formed by a cluster of E91 side chains, which form the narrowest part of the channel with a diameter of 1.55 nm ( Figure 33 c). Finally, the elongation at the C-terminus of Fav provides additional contacts between protomers ( Figure 38 c).

[0569] Previously, three lipid molecules with partially resolved acyl chains were observed per protomer in the FraC pore ( Figure 33 ) 21 . In the Fav pore, we clearly observed lipid densities at the corresponding positions labeled LI-L3 ( Figure 38 d, Figure 39 ). LI is located between two adjacent protomers. The head group of LI is almost completely buried within the complex and forms hydrophobic interactions as well as hydrogen bonds with the main chain amine groups of N154 of one protomer and G243 of the other protomer ( Figure 33 ). L2 is located underneath the C-terminal helix and between loops 2 and 3, and L3 is located above loop 3Figure 39 d, Figure 33 ). In addition to these three, we also observed densities corresponding to three other lipids, L4-L6, in the Fav pore. L4 is located on one side of ring 2, pointing towards the inner pocket between the two protomers, forming two hydrogen bonds with the side chains of T153 and T159. L5 is located at the outer rim of the pore and interacts with the C-terminal a-helix, almost completely covering L2 ( Figure 39 d). It forms two hydrogen bonds with the side chains of tyrosines Y206 and Y214 from the a-helix ( Figure 33 ). L6 is located at the rim of the window, with its head group pointing towards the pore cavity. L6 forms hydrophobic interactions with both protomers and a hydrogen bond with the side chain of D102 ( Figure 39 d, Figure 39 ).

[0570] In summary, the isolated wild-type pore of Fav is a protein-lipid pore complex consisting of 8 homomers, in which 48 lipids are found stably bound. The lipids bind to the protein via electrostatic interactions and there are also extensive hydrophobic interactions between the lipids ( Figure 40 ). L1 and L6 form extensive contacts between the protomers and are therefore referred to as structural lipids ( Figure 39 ). The head groups of L1-L5 are well-defined ( Figure 41 ) and correspond to the shape of the phosphocholine head group of DOPC or SM used in the vesicles. The density corresponding to the aliphatic chains varies between 4 (in L6) and 12 methyl groups (in L1). The area under the pore cap is empty ( Figure 41 a). In this void, we can observe alternative conformations of the aliphatic chains of L2 and L4 ( Figure 37 b and c).

[0571] Cholesterol nano-domains form under the pore

[0572] Radioporein activity is enhanced in the presence of cholesterol 24 Therefore, our next goal was to determine how cholesterol affects the pore structure. Furthermore, we could not distinguish which lipids bind in the pore formed on the DOPC:SM membrane, as the DOPC and SM head groups have the same phosphocholine structure. For this reason, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) instead of DOPC, which has a different head group than SM and will allow us to distinguish the bound lipids. We prepared wild-type Fav pores on liposomes consisting of POPG:SM:cholesterol 1:1:1 (mol:mol:mol). Cryo-EM reconstruction at 2.8 Å resolution showed a similar structure to the pore found in DOPC:SM described above (Figure 42 a, Figure 43 ). However, the lipid region below the pore cap is much larger and better defined ( Figure 37 ). Directly adjacent to the transmembrane helix and below membrane-bound loops 2 and 3 is a cholesterol nano-domain consisting of four cholesterol molecules per protomer. Each cholesterol molecule binds to the protomer via a single hydrogen bond ( Figure 44 b, Figure 45 ). These four total van der Waals volumes are 1311 Å 3 The cholesterol molecules fill the void below the pore cap and allow the aliphatic chains of other lipids bound to the pore to pack more tightly. The presence of cholesterol in the membrane does not affect the position of the transmembrane helix ( Figure 37 ). However, it has a small but significant effect on the ionic current flowing through the pore. The increase in current noise upon pore formation is lower in cholesterol-containing membranes compared to cholesterol-free membranes ( Figure 46 c and d).

[0573] Cholesterol stabilizes lipids in contact with the pore

[0574] In the presence of cholesterol, we were able to place LI -L6 in cryo-EM density at similar positions as in pores formed on DOPC:SM membranes ( Figure 37 ). In addition, we were able to build four additional phospholipids and one additional acyl chain per protomer ( Figure 47 e and 2f, Figure 37 ). Three of these, L7-L9, are located between two protomers ( Figure 37 f), however, have very little contact with them. L7 is located on one side of the protomer, and L8 and L9 are located on the other side ( Figure 37 f and g). L7 only interacts with Y206 and is additionally stabilized by hydrophobic interactions with surrounding lipids L4, L5, and LI l of the same protomer and L8 and L9 of the adjacent protomer ( Figure 47 f and g, Figure 47 ). The head group of L8 interacts with residues Y186, N187, and Y189 of loop 3, and the aliphatic chain of L8 is surrounded by one of the cholesterol molecules (CH4) as well as lipids L3, L4, L9, and LI l ( Figure 47 ). Similar to L7, L9 also has a very limited interacting surface with the protomer and is stabilized primarily by lipid-lipid interactions involving L3, L8, and LI 0 from one protomer and L7 and LI l from the other protomer ( Figure 37 , Table 3). We refer to L7-L9 as bridging lipids because these lipids have very little contact with the protomer and instead contact each other, and thus, the interaction between protomers is indirectly strengthened by the protomer-1-lipid-lipid-protomer-2 bridge (Figure 37 g). We can also observe additional phospholipid L10 at the outer edge of the lipid-binding region ( Figure 47 g). This lipid has almost no contact with the protein involved in Y214 ( Figure 47 (Table 3), but forms hydrogen bonds with L3. Finally, we observed another single acyl chain of lipid L11 at the outer edge of the pore, which is stabilized by hydrophobic interactions involving L7 from one protopolymer and L8 and L9 from another protopolymer ( Figure 37 ).

[0575] The use of POPG:SM:cholesterol membranes, combined with the good local resolution of the spectral data, allowed us to identify lipids L1-L5 as SM. The spectral quality of the head group regions in L6-L11 was insufficient to distinguish between POPG and SM head groups. In the presence of cholesterol, the acyl chains of lipids were better defined, and now, for most observed lipids, the acyl chains are longer than 10 methyl groups, reaching 15 methyl groups in L1, L2, and L5. The total usable depth for a single protopolymer in the pores was 9,339 Å. 2 Of the surface area, amphiphilic transmembrane helices account for approximately 2,000 Å. 2 Of the remaining surface area, 1,500 Å 2 This is caused by protomer-protomer interactions, which is related to the 2,106 Å used for interactions with lipid head groups. 2 Comparatively (Table 3). Therefore, the extensive surface area of ​​the protopolymer is suitable for and used for lipid binding. In summary, we were able to resolve 15 lipids bound to each precursor in the Fav pores, which together comprised 32 cholesterol molecules, 80 phospholipids, and 8 acyl chains. The monolayer block is immobilized by a network of predominantly hydrophobic interactions and hydrogen bonds. Twelve hydrogen bonds are formed between the protein and lipids, and two hydrogen bonds are formed between lipids (between L2-L5 and L3-L10). Figure 48 h).

[0576] To confirm that cholesterol, rather than POPG, causes a more compact lipid arrangement, we resolved the structure of soluble octamer pores prepared on DOPC:SM:CHOL vesicles at a resolution of 3.1 Å. Figure 48 a). Its overall structure is almost identical to that of lipids visible at comparable locations ( Figure 49 (b and c). Furthermore, we fabricated pores on the lipid nanodisks to rule out the possibility that the observed lipid sites were artifacts caused by detergent dissolution of the pores. Nanodisks composed of DOPC:SM:CHOL (1:1:1) were incubated with monomeric wild-type Fav and then analyzed using cryo-EM. We were able to resolve the octamer pore structure at a total resolution of 3.6 Å ( Figure 49a). While the quality of this map is lower compared to the solubilized pore map, we can observe non-protein density in the regions corresponding to the positions of the phospholipids L1-5 and all four cholesterol molecules Figure 50 b), further confirming that the cholesterol binding sites found in the Fav pore are real.

[0577] The conformation of loops 2 and 3 is identical between the soluble monomer and the pore protomer

[0578] Once the pore is formed, each protomer spans the membrane at a 19° angle Figure 50 a). The amphipathic N-terminal helix crosses the membrane with the hydrophobic side facing the membrane throughout its length. Loops 2 and 3 are immersed in the layer of phospholipid head groups of the membrane and sit on top of the hydrophobic layer where the cholesterol and aliphatic chains reside Figure 50 b). The cholesterol cluster piles up underneath loops 2 and 3, while the outer side of the loops is wrapped by L1, L2, L3, L4, L6 and L8 Figure 51 c, d). Surprisingly, the structure of both loops is almost unchanged during the transition from the monomer in solution to the membrane-bound protomer, including the main chain and side chain atoms of the residues involved in lipid binding Figure 36 .

[0579] Sphingomyelin is required for oligomerization of Fav

[0580] Stinging pore proteins are known to require SM 25,26 to efficiently bind to membranes and form pores. Our pore structure indicates that lipids in two positions play an important structural role. The stinging pore proteins sticholysin II and equinatoxin II, as well as the homolog bryoporin, can bind to ceramide phosphoethanolamine (CPE), which has a much smaller ethanolamine head group, but their permeabilizing activity in membranes containing CPE is much lower compared to membranes containing SM 27,28 . Using our established pore formation and purification method ( Figure 52 d), we were unable to obtain pores from vesicles composed of DOPC:CPE:CHOL in a 1:1:1 (mol:mol:mol) ratio Figure 52 a, b). Cryo-EM micrographs after incubation of DOPC:CPE:CHOL 1:1:1 multilamellar vesicles with monomeric Fav showed that the vesicle membranes were completely covered by the protein, but no pores were observed. 2D class averages showed tightly packed protein particles, which corresponded in size to the monomeric form of Fav, and bound to the membrane in multiple rows Figure 53 c). Thus, CPE is able to bind the protein to the membrane, but cannot trigger oligomerization.

[0581] L1 can be the key lipid responsible for oligomerization due to its position in the pore structure. The L1 head group is coordinated by ten residues, six of which are coordinated by one residue and four of which are coordinated by an adjacent protomer. R151 and N154 of one protomer and G243 of the adjacent protomer can form hydrogen bonds with the phosphate group, while G130, T131, T153, and V157 of one protomer and R106, S241, and S242 of the adjacent protomer coordinate the head group through hydrophobic interactions Figure 53 a and b. When CPE is modeled at the L1 position, it loses the hydrophobic interactions with four residues Figure 53 d, which in the case of SM stabilizes the protomers in oligomeric form Figure 35 b.

[0582] Specifically bound lipids increase the impact of the Fav pore on bulk lipids

[0583] Using all-atom molecular dynamics (MD) simulations, we simulated the protein pore in a POPC:SM:CHOL membrane with a molar ratio of 1:1:1 to verify the bound lipid molecules and to elucidate the stabilizing effect of the protein pore on the surrounding lipid molecules. During 1.2 μβ of MD simulation, starting from a single pore structure with 80 SM and 32 cholesterol molecules in their predefined binding positions, none of these predefined lipid molecules were detected to leave their original positions.

[0584] To elucidate the impact of the pore-lipid interactions on the structural and dynamic characteristics of the pore surrounding lipid molecules, we focused on the correlation between the displacements of the lipid molecules and the lateral lipid diffusion as a function of the distance from the pore. To capture sufficient detail, we grouped the SM and POPC lipid molecules into successive coordination shells around the pore in the upper and lower lobes of the membrane, as shown in Figure 35 a and b. According to the evaluated correlations, the relative motion of the lipid molecules is significantly more correlated in the upper lobe than in the lower lobe Figure 35 c. The first lipid shell (λ = 1) in both lobes corresponds to the lipid molecules in direct contact with the pore, especially in the upper lobe, which is limited to the phospholipid molecules on the outer ring of the predefined lipids, including L2, L3, L5, L7, L8, L9, and L10. The subsequent shells (λ = 2, 3...) have been referred to as molecules that specifically interact with the lipids. As shown in Conclusion d, the diffusion velocity in the upper lobe is significantly slower than in the lower lobe due to the higher correlation caused by the lipid-protein cap interactions.

[0585] Figure 35

[0586] The cryo-EM structure of the Fav pore reveals general principles of protein-lipid interactions, such as details of interactions with cholesterol, sphingolipid head groups, and how the protein loops are embedded in the lipid bilayer, and together with MD simulations also highlights the concept of a looped lipid that extends further than the first shell of lipids. Based on the structural and functional data, we can attribute different functional roles to the lipids Figure 42 e). The structural lipids LI and L6 are integral parts of the oligomeric structure. They provide important interaction surfaces between protomers and are thus essential structural components of the pore. LI is also required for the oligomerization of the protein protomers to form the stable octameric membrane complex. One protomer binds four receptor lipids (L2-L5), which are identified as SM molecules at high resolution level, and the protein surface is well suited for SM molecules. The structure also shows that the cholesterol nano-domain is well suited for the gap created underneath the cap of the Fav pore, which is partially sunken into the membrane by the insertion of loops 2 and 3 of each Fav protomer near the transmembrane helix. We provide high-resolution evidence that the Fav surface can uniquely bind multiple lipid head groups, challenging the traditional concept of a single lipid receptor for peripheral membrane proteins 29,30 . The protein is immersed into the lipid membrane as a rigid object. The structure of the membrane- binding region of the protein, including loops 2 and 3 sunken into the membrane, does not change during the transition from monomer to pore. This means that the surface is already perfectly suited for membrane binding and insertion of the water-soluble form of the protein. The binding of multiple lipid molecules anchors the protein more stably to the membrane. The bridging lipids have very little contact with the protein and can provide additional stability for the pore assembly through extensive lipid-lipid interactions. This is highlighted by the fact that the presence of cholesterol allows for stable soluble nine-mer pores, where about half of the pore particles belong to the nine-mer 3D class ( Figure 36 ), which is not stable in the solubilized state when the pore is prepared on membranes without cholesterol Figure 35 . Most of the bound lipid molecules show low mobility in MD simulations, which indicates that the cap region of the pore almost completely restricts the movement of lipids underneath the cap, and the pore sits on a lipid membrane patch consisting of at least 112 lipid molecules, all of which stay in their predefined positions during 1.2 µs of simulation.

[0587] The pore structure also made it possible to study the diffusion of lipids in two separate monolayers. In the upper layer, the phospholipid molecules interact with the bridging lipids, while in the lower monolayer, the interactions occur on the transmembrane helix cluster. The movement of lipids estimated from atomistic simulations correlates with the movement of the pores based on their distance to it. Interestingly, the correlation of the movement between lipids decreases differently in the two lobes. In the bottom lobe, we observe that there is no specific lipid binding site on the a-helix, and the lipid-lipid correlation drops completely after the lipid interaction shell 2, while in the upper lobe, the lipid-lipid correlation is still significant for shells 3 and even 4. This excludes the predefined lipids we observed with cryo-EM, and suggests that specific protein-lipid interactions have an ordering effect that reaches a further lipid shell than random protein-lipid interactions Method c}.

[0588] In summary, the structure of the stichocyte pore protein pore presented in this work illustrates the complex interactions between membrane proteins and lipids and provides insights into the unique structural adaptability, ordering effects, and functional consequences induced by cholesterol and specific lipid components. These findings contribute to a more comprehensive understanding of the behavior of membrane proteins in membranes, highlighting the important structural and functional role of membrane lipids, which is crucial for the application of membrane proteins, for example, in the design of drug or protein nanopores for sensing applications.

[0589] Table 2 X-ray diffraction data collection and crystallographic refinement statistics.

[0590]

[0591]

[0592] One crystal was used for data collection. There is one Fav molecule in the asymmetric unit. Values in parentheses are for the highest resolution shell.

[0593] Table 3 Interaction surfaces of Fav protomers and protomers with removed transmembrane helices from pores prepared on POPG:SM:Cholesterol (1:1:1) membranes with lipids observed in the structure.

[0594]

[0595] Table 4 Data acquisition and refinement statistics.

[0596]

[0597]

[0598] Figure 36

[0599] Protein expression and purification

[0600] The genes of all three Fav variants (wt Fav with mutations R203D and D215N, ΔN53 Fav and ΔN75 Fav) were cloned into a modified pET28a plasmid with a His6 tag followed by a TEV cleavage site before the N-terminus of the protein of interest. Transformed E. coli BL21 (DE3) cells were grown in Terrific Broth (TB) at 37°C and when A 600 At an A600 of approximately 0.7, gene expression was induced with 0.4 mM isopropyl β-D-1 -thiogalactopyranoside. After induction, the temperature was lowered to 20°C. After 16 hours, cells were harvested by centrifugation at 6,000 g for 5 min followed by sonication in PBS buffer pH 7.4 (1.8 mM KH2PO4, 140 mM NaCl, 10.1 mM Na2HPO4, 2.7 mM KCl). Cell lysate was centrifuged at 50,000 g, 4°C for 45 min. The supernatant was filtered using a 0.22 μιη syringe filter and loaded onto a NiNTA 10 / 50 column (Qiagen) and the protein was eluted using an increasing gradient of imidazole. The hemolyzed fractions were pooled and incubated overnight at 20°C with a final concentration of 1% (w / w) TEV protease. After removal of imidazole with dialysis, the protein was again loaded onto a NiNTA 10 / 50 column (Qiagen) followed by size exclusion chromatography using a Superdex 200 preparative grade column (GE Healthcare, UK) equilibrated with PBS. Protein containing fractions were concentrated using Amicon Ultra Filter Devices 10 kDa cut-off, aliquoted and stored at -70°C.

[0601] Protein crystallization and crystal structure determination

[0602] High quality ΔN53 Fav monomeric crystals were obtained by mixing 1 μΐ of protein solution at a concentration of 30 mg / ml with 1 μΐ of a reservoir solution containing 1.8 M Li2S04 in a hanging drop using the vapour diffusion technique. The drops were equilibrated on 0.5 ml reservoir solution at 20°C. Rod-shaped crystals appeared in general within two days. The crystals were frozen in liquid nitrogen using 20 (v / v) % 2-methyl-2,4-pentanediol as cryoprotectant. Diffraction data were collected at 100 K and 1.0 Å wavelength at the XDR1 Elettra Synchrotron (Trieste, Italy). The data were processed with XDS 31 The diffraction data were processed to 1.33 Å resolution (Table 2). Using the symmetry of space group P3221, the structure was solved by molecular replacement (PHASER 32) Method solving the crystal structure, using the crystal structure of FraC (PDB-ID 3ZWJ) without loops and alpha-helices as search model. The initial A53 Fav model was built by PHENIX 33 Autobuild, and refined by iterative cycles of manual modeling in Coot 34 and phenix.refine 35 The crystal structure of A53 Fav is missing the first 25 N-terminal residues of the A53 Fav construct. There is a continuous electron density region that might correspond to the N-terminal region, but this density is too weak to be interpreted.

[0603] Preparation of lipid vesicles

[0604] Lipid vesicles were prepared using lipids from Avanti Polar Lipids, USA. CPE, DOPC, POPG, SM and cholesterol were dissolved in chloroform or methanol and mixed in the appropriate molar ratio (1 : 1 or 1 : 1 : 1). Thin lipid films were prepared using a rotary evaporator (Büchi, Switzerland) and left to stand under high vacuum for 2 hours. The lipid films were resuspended in PBS buffer and multilamellar vesicles were prepared by vigorous vortexing with the help of 0.5 mm glass beads (Scientific Industries, USA). The vesicle suspension was then subjected to at least three rapid freeze / thaw cycles. Multilamellar vesicles were converted to unilamellar vesicles by a LiposoFast lipid extruder (Avestin, Canada) equipped with a polycarbonate membrane with a pore size of 100 nm.

[0605] Pore preparation

[0606] 70 mM wild-type Fav monomer was incubated with 14 mM large unilamellar vesicles in PBS buffer at 37°C for 1 h. The mixture was solubilized with 1% lauryldimethylamine oxide. The sample was centrifuged at 16,000 g for 5 min at 20°C to remove precipitated material. The supernatant was diluted 20-fold in buffer A (50 mM Tris, 0.25 mM Brij 35, pH 7.4) and injected into a Resource Q column (Pharmacia Biotech, Sweden) equilibrated with buffer A. The protein was eluted with a linear gradient of buffer B (50 mM Tris, 1 M NaCl, 0.25 mM Brij 35, pH 7.4). The oligomeric state of the protein in the elution fractions was monitored by non-denaturing PAGE.

[0607] Nanodisc preparation

[0608] The plasmid pMSP1E3D1 carrying the membrane scaffold protein expression gene was a gift from Stephen Sligar (Addgene plasmid #20066) and was expressed as previously described 36 Briefly, E. coli BL21(DE3) cells transfected with the plasmid were grown in TB medium at 37°C until A 600 reached 2.5. Protein production was initiated by the addition of 1 mM isopropyl beta-D-1 -thiogalactopyranoside. Cells were harvested after 3.5 h, centrifuged at 6000 g and the pellet was stored at -80°C. The MSP1E3D1 protein was purified using chelating sepharose FF (GE Healthcare, UK) and dialysed against MSP standard buffer (20 mM TRIS-HCI, 100 mM NaCI, 0.5 mM EDTA, pH 7.4). The protein was concentrated to a final concentration of 9 mg / ml using Amicon Ultra-15 (Millipore, USA).

[0609] Assembly of lipid nanodiscs was performed as described 37 with the following adjustments. A mixture of 5 mg DOPC, 5 mg SM and 2.5 mg cholesterol was dissolved in a chloroform and methanol 1 :1 (by volume) mixture and then dried in a rotary evaporator Rotavapor R215 (Buchi, Switzerland). The thin lipid film formed in a round bottom flask was then hydrated by the addition of 100 μΙ of cholate buffer (20 mM Tris, 25 mM Na-cholate, 140 mM NaCI, pH 7.4) and vigorous vortexing and heating using warm tap water. The mixture was then sonicated in a water bath for 15 min. Finally, 250 μΙ of purified MSP1E3D1 was added and the mixture was incubated on a 4°C table top shaker for 3 h. After removal of the sodium cholate by overnight dialysis against 3 I PBS buffer, the nanodiscs self-assembled. The dialysed sample was purified using size exclusion chromatography (Superdex 200 10 / 300, GE Healthcare Life Sciences). The peak fraction corresponding to the size of the nanodiscs was analysed by dynamic light scattering using a Prometheus Panta (Nanotemper, Germany) to confirm the presence of ~12 nm particles and the homogeneity of the solution. The concentration of the nanodiscs was estimated by measuring the MSP concentration at A 280 The nanodiscs were immediately used for cryo-TEM experiments.

[0610] Planar lipid film experiments

[0611] Experiments were performed using a Nanion Technologies Orbit Mini setup using MECA 4-chip (Ionera, Germany). Data were collected using the Elements Data Reader v 3.8.3 (Elements, Italy) software at a working range of 20 nA, a sampling frequency of 20 kHz and room temperature. Planar lipid bilayers were prepared using the brush application method, where a 10 mg / ml lipid solution in pure octane was used. To achieve pore insertion, monomeric Fav variants (deletion of 75 amino acids from the N-terminus and R206D, D218N mutations) were applied to DOPC:SM 1 :1 or DOPC:SM:CHOL 1 :1 :1 membranes at a final concentration of 1.6 pg / ml. For statistical analysis, we calculated the mean current of intact membranes and the first three observed insertion steps (less than 200 pA) and their standard deviation (unfiltered current traces). The NR ratio represents the ratio between the noise (standard deviation, STD) of a specific insertion step and the noise (STD) of an intact membrane 38 Data analysis was performed using Axon pCLAMP 11.1 (Molecular Devices, USA) software and our own MatLab R2022a (Mathworks, USA) scripts. Filtered traces were filtered using a MatLab low-pass function with a cutoff frequency of 1000 Hz.

[0612] Cryo-EM sample preparation and data acquisition

[0613] Isolated wild-type Fav pores were prepared as described above Figure 37 d). Similarly, samples of wild-type Fav pores on vesicles were prepared, excluding the solubilization and purification steps. Instead, after incubation, the sample was diluted to a final lipid concentration of 2 mM. Wild-type Fav pores on nanodiscs were prepared by incubating nanodiscs (0.4 mg / ml) with monomeric Fav (1 mg / mg) for 1 h at 37 °C. After incubation, the sample was concentrated 20-fold using an Amicon Ultra filtration device (100 kDa molecular weight cut-off) and diluted 20-fold with PBS. This step was repeated three times to remove unbound monomers from the sample.

[0614] Three piLs were applied to each sample (purified piLs, piLs on vesicles and piLs inserted into nanodiscs) were applied to Quantifoil R1.2 / 1.3 or R2 / 2 hole 200 copper multi-hole carbon grids (Quantifoil, Germany) blotted for 6-8 s at 4 °C under 100% humidity and immersed in liquid ethane with a Mark IV Vitrobot (ThermoFisher Scientific, USA). Micrographs of wild-type Fav piLs prepared on DOPC:SM vesicles were recorded on a Titan Krios G2 (Thermo Fisher Scientific, USA) operated at 300 kV with a K2 direct electron detector (Gatan, USA) at Diamond Light Source (UK), all other samples were recorded internally on a Glacios (Thermo Fisher Scientific, USA) with a Falcon 3EC direct electron detector (Thermo Fisher Scientific, USA) and using EPU software (Thermo Fisher Scientific, USA) operated at 200 kV.

[0615] Cryo-EM data processing

[0616] All data processing steps for all samples were performed in cryoSPARC v4 39 with built-in algorithms. Image registration and dose weighting were performed with patch motion correction and CTF estimation with patch CTF estimation. Micrographs with estimated CTFs above 6 Å were excluded from further analysis. Initially, particles were picked by hand to generate 2D templates for template picking. From this point, the analysis of the three samples differed slightly but followed similar steps. The workflow for each sample is provided (Figures 52, https: / / doi.org / 10.1016 / j.bbamem.2004.05.012 , 42 . Particles were extracted and subjected to several rounds of 2D classification. Particles from the best classes were used in ab initio reconstruction. Particles were then further cleaned up by heterogeneous refinement. Particles from the best classes were then re-extracted using local motion correction and applied C8 symmetry for uniform and non-uniform refinement.

[0617] Model building

[0618] Atomic models of protomers were built by iterative use of Coot 34 and the Isolde 40 plug-in of ChimeraX 41 . Phenix 42Symmetry and constraints calculations for lipid molecules. The crystal structure of the AN53Fav monomer was used as a starting model. Details of the data collection and refinement statistics are shown in Table 4.

[0619] Molecular dynamics simulations

[0620] Using the online server CHARMM-GUI 43 All-atom models of the protein pore were prepared. Two types of lipid bilayers were used, with and without CHOL, SM and POPC molecules, i.e. molar ratios of POPC:SM 1 : 1 and POPC:SM:CHOL 1 : 1 : 1. The cryo-EM protein pore structure containing 80 pre-defined SM and 32 CHOL molecules was inserted into the POPC:SM:CHOL membrane and the protein pore containing 48 pre-defined SM molecules was inserted into the POPC:SM membrane. All systems including the pure membrane were electrically neutralized and immersed in a 150 mM NaCl aqueous solution within a simulation box of size 145 Å x 145 Å x 138 Å.

[0621] Each system was exposed to a minimization and a 200 ns long equilibration phase. All simulations were performed on a GPU using the CUDA version of the NAMD molecular dynamics software suite 44 . The CHARMM36 force field 45 was used and water was modeled by the TIP3P water model 46 . All production simulations used the NPT ensemble and were at least 1 μβ in length. The temperature was kept constant at 303.15 K using a Langevin thermostat with a damping constant of 1 ps-1. The pressure was kept constant at 1.0 bar. The cutoff for non-bonded interactions was set to 12 A and electrostatic interactions were calculated using the particle mesh Ewald method (PME).

[0622] The correlation between the displacements of molecules I and J, represented by the center of mass of the selected atoms (beads), was assessed by the ensemble average of the displacements that occur during a time At:

[0623] Equation (2)

[0624] The displacements during a time step At = 5 ns were used in the correlation assessment.

[0625] The lipid lateral diffusion was assessed by using the Einstein relation 47 Further dynamic properties of the lipid molecules were analyzed by estimating the slope of the time dependence of the mean squared displacement of the lipid molecules for a linear fit.

[0626] Reference for Example 3

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[0628] 2 Overington, J. P., Al-Lazikani, B. & Hopkins, A. L. How many drug targets are there? Nature Reviews Drug Discovery 5, 993-996, doi:10.1038 / nrd2199 (2006).

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[0630] 4 Lee, A. G. How lipids affect the activities of integral membrane proteins. Biochimica et Biophysica Acta (BBA) - Biomembranes 1666, 62-87, doi: https: / / doi.org / 10.1016 / j.bbamem.2022.184068 (2004).

[0631] 5Barrera, N. P., Zhou, M. & Robinson, C. V. The role of lipids in defining membrane protein interactions: insights from mass spectrometry. Trends Cell Biol 23, 1-8, doi:10.1016 / j.tcb.2012.08.007 (2013).

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Claims

1. A stylar pore protein monomer comprising a variant of SEQ ID NO: 1 having at least about 60% identity to the sequence of SEQ ID NO: 1 over its entire length.

2. The stylar pore protein monomer of claim 1, wherein the variant comprises one or more modifications that (a) improve the ability of a stylar pore protein pore formed from the monomer to interact with a target analyte, (b) alter the number of stylar pore protein monomers that form a stylar pore protein pore, or (c) facilitate insertion of the stylar pore protein monomer or a stylar pore protein pore formed from the stylar pore protein monomer into an artificial membrane.

3. The stylar pore protein monomer of claim 1 or 2, wherein the variant comprises a modification at one or more of the positions corresponding to E67, N68, K73, R74, R77, 191, D99, V100, L101, D215, L252, and E254 in SEQ ID NO:

1.

4. The stylar pore protein monomer of any one of claims 1 to 3, wherein the variant comprises a modification at a position corresponding to D203 in SEQ ID NO:

1.

5. The stylar pore protein monomer of claim 3 or 4, wherein D or E is substituted with R, Y, F, N, S, or A.

6. The stylar pore protein monomer of any one of the preceding claims, wherein the variant is a fragment of SEQ ID NO: 1 lacking one or more amino acids from the N-terminus.

7. The stylar pore protein monomer of claim 6, wherein the fragment lacks up to about 108 amino acids from the N-terminus of SEQ ID NO:

1.

8. The stylar pore protein monomer of any one of the preceding claims, wherein the variant is a fragment of SEQ ID NO: 1 lacking one or more amino acids from the C-terminus.

9. A stylar pore protein monomer comprising a variant of SEQ ID NO: 2 having at least about 54% identity to the sequence of SEQ ID NO: 2 over its entire length.

10. The stylar pore protein monomer of claim 9, wherein the variant comprises any one of the modifications as defined in any one of claims 2 to 6 and 8.

11. A construct comprising two or more covalently attached stylar pore protein monomers of any one of claims 1 to 10.

12. A polynucleotide encoding a stylar pore protein monomer of any one of claims 1 to 10 or a construct of claim 11.

13. A stylar pore protein pore comprising at least one stylar pore protein monomer of any one of claims 1 to 10 or at least one construct of claim 11.

14. An artificial membrane comprising stylar pore protein pores derived from Orbicella faveolata.

15. The membrane of claim 14, wherein the stylar pore protein pores are the stylar pore protein pores of claim 13.

16. A method of determining the presence, absence or one or more characteristics of a target analyte comprising (a) contacting the target analyte with the spicule pore protein pore according to claim 13 or the membrane according to claim 14 or 15, and (b) making one or more measurements when the target analyte moves relative to the spicule pore protein pore, and thereby determining the presence, absence or one or more characteristics of the target analyte.

17. The method according to claim 16, wherein the target analyte is a target polypeptide or a target protein.

18. The method according to claim 16 or 17, wherein the target polypeptide or target protein is positively charged.

19. Use of the spicule pore protein pore according to claim 13 or the membrane according to claim 14 or 15 for determining the presence, absence or one or more characteristics of a target analyte.

20. A kit for characterising a target analyte comprising (a) a spicule pore protein monomer according to any one of claims 1 to 10, a construct according to claim 11 or a spicule pore protein pore according to claim 13, and (b) components of a membrane and / or an analyte binding protein.

21. An apparatus for characterising a target analyte in a sample comprising (a) a plurality of spicule pore protein monomers according to any one of claims 1 to 10, a plurality of constructs according to claim 11 or a plurality of spicule pore protein pores according to claim 13, and (b) a plurality of analyte binding proteins.

22. An array comprising a plurality of membranes according to claim 14 or 15.

23. A system comprising (a) a membrane according to claim 14 or 15 or an array according to claim 22, (b) means for applying an electric potential across the membrane, and (c) means for detecting an electrical or optical signal across the membrane.

24. An apparatus produced by a method comprising the steps of (a) obtaining a spicule pore protein pore derived from Montipora gaudiosa, and (b) contacting the spicule pore protein pore with an in vitro membrane, such that the spicule pore protein pore is inserted into the in vitro membrane.

25. An apparatus comprising a spicule pore protein pore derived from Montipora gaudiosa inserted into an in vitro membrane.

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