The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Jan. 22, 2024, is named 64828-705_301_SL.xml and is 113,571 bytes in size.
The invention relates generally to the field of nanopores and the use thereof in analyzing biopolymers and other (biological) compounds. In particular, it relates to genetically engineered nanopores, and their improved performance in peptide capture and recognition.
Nanopores have become potential candidates for inexpensive, high-throughput and/or portable protein detectors. In recent years, they have shown to work analogous to mass-analysers, not only for model analytes, such as polyethylene glycol (PEG), but also for biological polymers such as peptides and proteins (Robertson et al. Proc Natl Acad Sci USA. 2007). Biological nanopores have been shown to be particularly suitable for the detection and discrimination of small molecules based on the signal they produce when an analyte translocates through the recognition site of the nanopore. This method, known as nanopore spectrometry (Chavis et al., ACS Sens, 2017), is dependent on the interaction between the pore surface and the analyte.
Pore forming proteins (PFPs) can be roughly classified into two major groups, α-PFPs or β-PFPs, which form pores by bundles of α-helices or by transmembrane β-barrels, respectively. Although members of either group of PFPs share a common general mode of pore formation, several evolutionarily unrelated families can be distinguished according to the structures of their soluble monomers.
The α-helical pore forming toxin family produced by sea anemones (actinoporins) are pore forming proteins with a mass of approximately 20 kDa (Anderluh et al. Toxicon, 2002). The sequence identity of the actinoporin family is high (60-80%) (Garcia-Ortega et al. Biochim Biophys Acta, 2011), and the mechanism of pore formation is thought to be largely similar, where pore formation is often dependent on the presence of sphingomyelin in the lipid bilayer. The activity of actinoporins can be traced to their α-helical transmembrane region, formed by the first 30-32 amino acids (
Three main families of β-PFPs are the α-hemolysin family found predominately in Staphylococcus aureus, the MACPF/CDC protein superfamily, and PFPs exhibiting similarity to aerolysin, a well-studied toxin from the pathogenic bacterium A. hydrophila.
The detection of analytes and the sequencing of DNA using biological nanopores has seen major advances over recent years. The detection and sequencing of proteins with nanopores, however, is complicated by the complex physico-chemical structure of polypeptides, and the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores
The crystal structure of wild-type FraC (WtFraC) displays an oligomeric pore formed from 8 identical subunits (octameric) (Tanaka et al., Nat Commun, 2015). In previous work, we have shown that FraC is capable of forming different oligomeric forms-most notably the octameric (T1) and heptameric (T2)—with a distinct pore volume and range of detectable peptides (WO2020/055246; Huang et al. Nat Commun, 2019). In the same work we also demonstrated that the current observed from peptide translocation through WtFraC correlates with the mass of the peptide at a pH of 3.8, in 1 M KCl. However, the peptide blockades were fast—in the order of several micro seconds (average dwell time for Angiotensin 1 is 0.15±0.04 ms) (Huang et al. Nat Commun, 2019), which causes the majority of translocation events to remain undetected and detected events to be inaccurately characterized.
Therefore, a goal of the present invention is to improve the accuracy of characterizing individually captured peptides by a nanopore sensor. To that end, we engineered proteinaceous nanopores to improve the capture of unlabeled peptides, to increase the residence (dwell time) of peptides in the nanopore sensor, and to improve the discrimination between peptide species. In this invention we have also engineered proteins nanopores to improve peptide sensing under low pH conditions that are optimized for peptide detection.
It was surprisingly found that introducing one or more “bulky”/aromatic amino acids at precise positions within the lumen of nanopores can increase both the capture frequency of peptides and also largely improves the discrimination among peptides. For example, fragaceatoxin C (FraC) nanopores comprising subunits wherein a tyrosine, phenylalanine or tryptophan residue was introduced in the lumen-facing region was found to show an increased dwell time of peptides in the pore. Moreover, these ‘large aromatically modified” nanopores could detect and measure the peptides in a tryptic digest of lysozyme. Furthermore, the unique individual spectra of individual proteins could be assigned using advanced analytical methods such as spectral matching. Furthermore, we have adapted our combination of mutation engineering discoveries and detection conditions to other nanopore families, e.g. the beta-barrel pores, with quite different structures but similar size recognition regions in the lumen of the nanopores, and have found the improvement in peptide characterisation is universal.
These findings provide a proof of concept that the modified nanopores can be used as single molecule detector capable of label-free protein detection and fingerprinting. It provides the basis to improve the recognition and augment the capture of peptides by nanopores, which is important for developing a real-time and single-molecule volume-analyzer for peptide recognition and identification.
Accordingly, the invention relates to a proteinaceous nanopore comprising a mutant (or “modified”) transmembrane pore-forming toxin, e.g. of the actinoporin family, or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more mutations to a natural or non-natural aromatic amino acid residue. A pore-forming toxin is typically an oligomer. The pore is preferably made up of several repeating subunits, such as 6, 7 or 8 subunits. The pore comprises a central channel when inserted into a membrane through which the ions may flow, for example when a potential is applied across the membrane. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane α-helix bundle or channel. Thus, a modified proteinaceous nanopore in accordance with the invention comprises an oligomer (or “assembly”) of mutant pore-forming alpha-helical pore-forming subunits of the actinoporin family, or an oligomer of pore-forming fragment thereof.
The “lumen-facing recognition region”, herein also referred to as the “recognition area” or “water-facing region” of the pore, is meant to indicate the part of the nanopore that is involved in the sensing of an analyte that traverses the pore. The recognition region is typically a part of the central water-filled channel (the lumen) that is formed through the nanopore from cis to trans when inserted into a membrane such as a lipid bilayer. The recognition region can typically be identified structurally by the dimensions of the central channel. Suitable structures or structural models can be obtained or constructed by means known in the art, including from experimental x-ray diffraction structures, electron-microscopy structures, and computer modelling. The recognition region will be the region of the channel through the nanopore where the electric field lines concentrate and the presence of the analyte disrupts the most the ionic current flowing through the nanopore under an applied potential. For small peptide detection, the recognition region will preferably comprise the section/s of the nanopore channel with an internal diameter of less than 2 nanometers, and preferably less than 1 nanometer, so as to yield a significant deflection of the ionic current and sufficient residence/dwell time during analyte interaction. Many nanopores might have one or more narrow sections of small internal diameters (constriction/s) within the longer recognition region. Maximum sensitivity/ionic current deflection to analytes is typically achieved when the analyte interacts with the nanopore at or near a constriction. Therefore, maximal control of analyte detection can often be achieved by protein mutagenesis/engineering at or adjacent to the residues that comprise the constriction.
Alternatively, for proteins that are known to be nanopores (e.g. by homology searches or by experimental determination) but do not have suitable structural models, the recognition region can often be determined by computer modelling and/or homology mapping to the recognition region of other known nanopores using means known in the art. For example, the recognition region often includes or entirely resides within the transmembrane section of a membrane-protein nanopore (e.g. transmembrane sections comprised of beta-barrels or alpha-helical oligomers). Transmembrane beta-barrels and alpha-helices can be identified by means such as homology comparison to other known pores and by features such as amphipathic hydropathy maps for example.
Nanopore recognition regions can also be determined and/or confirmed experimentally by mutagenesis using well known means in the art. For example, the ionic current characteristics of different nanopores with different targeted mutations in the candidate recognition regions can be compared in electrophysiology experiments of the nanopores inserted into membranes. By varying the position of the mutations, and optionally measuring differences in response to control analytes, the recognition region can be mapped and characterized.
A pore of the invention is among others characterized in that the lumen-facing recognition region of the pore is engineered (by one or more natural or non-natural amino acid substitutions) to manipulate the internal dimensions/hydrophobicity/aromaticity of the pore, therewith increasing the dwell time and resolution for peptides traversing the nanopore.
Accordingly, the invention also provides a method of decreasing the translocation speed of a peptide analyte through a transmembrane (alpha-helical or beta-barrel) protein pore, comprising:
As will be understood by a person skilled in the art, the one or more mutations of the invention can be introduced in a number of configurations to the nanopores or functional pore forming fragments thereof so as to produce the desired change/s in the recognition region of the assembled nanopore. For example, for oligomeric nanopores comprised of a number (e.g. 4, 5, 6, 7, 8, or more) of monomeric units, one or more mutations are made to all monomers used to assemble the nanopore, so that the assembled nanopore contains a ring of multiple identical mutations in the recognition region that is co-planar with the membrane and orthogonal to the direction of analyte passage. Alternatively, mutated monomers might be mixed with monomers containing no mutations or different mutations during nanopore oligomerisation to create “hetero-oligomeric” assembled nanopores with a controlled number of mutations.
Hence, the assembled pore may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or more mutant monomers of the invention, depending on the number of oligomer units. Controlling the number of mutated units can be useful to reduce or otherwise modulate the extent/magnitude of the change to the recognition region. Means to selectively purify the population of hetero-oligomeric nanopores with the desired number of mutations from a mixture are known in the art (e.g. Gouaux, et al Proc Nal Acad Sci U S A 1994). For oligomeric nanopores that contain more than one type of monomeric unit that in the assembled form both contribute to the recognition region (e.g. bi-component Leukocidins, Spaan et al Nat Rev Microbiol 2017) the mutations might be made to one or both of the monomer species. For pores where monomers are fused together (e.g. either genetically or by other chemical conjugation means), for example in the case of dimers (Hammerstein et al, J Biol Chem 2011), then a person of skill in the art would understand that the desired number of mutations can be introduced to the final assembly by choosing how many of the monomer units to modify.
For monomeric protein nanopores (e.g. outer membrane porins) where a single protein strand makes up the transmembrane channel, it is understood that mutations can be made along the sequence at specific interspaced distances such that the assembled nanopore channel contains the required number of water-facing mutations, preferably on multiple beta-strands of the transmembrane section of the pore, most preferably on all beta-strand units to create a ring-like formation of mutations similar to that formed in a homo-oligomeric nanopore. For beta-barrel based nanopores it is understood that mutations might be introduced to either the down strand or the up strand or to both. For example, aromatic or acidic substitutions of the invention might be added to both the up and down strands of the beta-barrel so that they are approximately co-planar (same vertical position in the nanopore) to create a stronger effect in the recognition region. In other embodiments, multiple mutations of the invention can made (vertically relative to the membrane) along the alpha-helix or beta-strand of a monomer of an oligomeric pore or fragment thereof, for example to create stronger alterations to the recognition region.
Natural and non-natural aromatic amino acid residues are known in the art. In one embodiment, the non-natural aromatic amino acid is selected from the group consisting of 3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr). Phg and norTyr. Suitable non-natural amino acids can include D-amino acids, Homo-amino acids (methylene), Beta-homo-amino acids, N-methyl amino acids, Alpha-methyl amino acids. A wide range of well-known non-natural amino acids are known in the art, including preferably derivatized Phe/Tyr/Trp amino acids, most preferably ring-substituted Phe/Tyr/Trp amino acids. Also encompassed are derivatives of Phe, Tyr and Trp, substituted by, e.g., a halogen, —CH3, OH, —CH2NH3, —C(O)H, —CH2CH3, —CN, —CH2CH2CH3, —SH, or another group. Exemplary non-natural aromatic amino acids include, but are not limited to, O-methyl-L-tyrosine; 3-methyl-phenylalanine; a p-acetyl-L-phenylalanine; O-4-allyl-L-tyrosine; 4-propyl-L-tyrosine; fluorinated phenylalanine; isopropyl-L-phenylalanine; a p-azido-L-phenylalanine; a p-acyl-L-phenylalanine; a p-benzoyl-L-phenylalanine; a phosphonotyrosine; a p-iodo-phenylalanine; p-bromophenylalanine; p-amino-L-phenylalanine; an isopropyl-L-phenylalanine; an amino-, isopropyl-, or O-allyl-containing phenylalanine analogue; a p-(propargyloxy) phenylalanine; 3-nitro-tyrosine; 5-fluoro-tryptophan, 5-hydroxy-tryptophan, 5-methoxy-tryptophan, 5-methyl-tryptophan, trifluoromethyl-tryptamine ethyl ester.
Methods for introducing non-natural amino-acids into proteins are also known in the art. For instance, non-naturally-occurring amino acids may be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express the mutant monomer. Alternatively, they may be introduced by expressing the mutant monomer in E. coli that are auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids. Non-natural amino acids may also be introduced using synthetic peptide chemistry methods known in the art for synthesizing peptides. Monomeric units of the nanopores may be formed entirely from synthetic peptides constructed using conjugation methods known in the art such as native chemical ligation (Thapa et al., Molecules, 2014, 14461-83), or cysteine coupling for example. Alternatively, monomers of the nanopore may comprise partially synthetic units coupled to naturally expressed peptide units using coupling methods known in the art.
The mutant nanopore may also be created by chemically attaching a suitable aromatic molecule to either the precursor monomeric units of the nanopore or the assembled oligomeric nanopore by means known in the art, such as for example by chemical attachment of suitable molecules to one or more cysteines (cysteine linkage), or lysines, which may either already exist in the wild-type protein or are introduced by mutagenesis.
In another aspect, the modified nanopore comprises a mutation to a natural aromatic amino acid, preferably one or more mutations to Trp, Tyr or Phe, more preferably Phe.
In addition to the aromatic amino acid mutation(s), one or more further mutation(s) may be introduced in the lumen-facing recognition region, which further mutation(s) increase the net negative charge of the pore.
For example, a mutant proteinaceous nanopore with mutation introduced to the lumen-facing recognition region to create a combination of aromatic residues that are “spatially adjacent” to negative residues, where the aromatic and negative residues are preferably within 4 nanometers, most preferably within 2 nanometers, of each other in a functional pore. The physical space between residue positions can be derived e.g. by measuring the C-alpha backbone distance between the respective mutated residues from a 3D model or crystal structure of an assembled oligomeric pore protein using common molecular modelling software known in the art.
Transmembrane protein pores, or fragments thereof, for use in accordance with the invention can be derived from beta-barrel pores or alpha-helix bundle pores. Beta-barrel pores comprise a barrel or channel that is formed from beta-strands. One skilled in the art would understand that a suitable nanopore of the invention may be selected from transmembrane pores that are known in the art (Peraro et al. Nat Rev Microbiol 2016; Crnković et al. Life (Basel) 2021). Suitable nanopores will ideally have dimensions in the recognition region most suitable for measuring small analytes, including small peptides. Suitable nanopores will ideally have transmembrane regions, recognition regions, or constrictions, with diameters of less than 2 nanometers, most preferably less than 1.5 nanometers. Suitable beta-barrel pores include, but are not limited to, beta-toxins, such as alpha-hemolysins, aerolysins, lysenin, cytolysins, cytolysin K, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), ferric hydroxamate uptake component A (FhuA), Curli production transport component CsgG, and Neisseria autotransporter lipoprotein (NalP). Alpha-helix bundle pores comprise a barrel or channel that is formed from alpha-helices. Suitable alpha-helix bundle pores include, but are not limited to, inner membrane proteins and outer membrane proteins, such as Actinoporins, the outer membrane core complex (OMCC) of H. pylori Cag T4SS particles, and the transmembrane domain of the E. coli polysaccharide transporter Wza.
One skilled in the art would understand that a suitable nanopore of the invention may be an artificial nanopore, such as one adapted from known artificial nanopores. For example, nanopores based on trans-membrane beta-barrels or alpha helices attached to ring-like proteins (Zhang et al. BioRxiv 2020), proteins based on transmembrane peptides attached to DNA origami (Spruijt et al. Nat Nanotechnol 2018), self-assembling nanopores based on artificially created transmembrane peptides (Scott et al. Nat Chem 2021) and designed de novo (Vorobieva et al. Science 2021).
In one aspect, the proteinaceous nanopore comprises a mutant actinoporin, or the alpha-helical transmembrane region (aa 1-27) thereof. For example, the mutant actinoporin comprises a mutation to an aromatic amino acid residue in the recognition region corresponding to amino acids 10-20. For homologs with additional N-terminal sequence, the region corresponding to the transmembrane alpha-helix can be determined by homology mapping and other means known in the art. The actinoporin family of pore-forming toxins is well known in the art. See for example Kristan et al. Toxicon 2009.
Exemplary members of the actinoporin family for preparing a mutant according to the present invention include Fragaceatoxin A (FraA), Fragaceatoxin B (FraB), Fragaceatoxin C (FraC), Fragaceatoxin D (FraD), Fragaceatoxin E (FraE), Equinatoxin II (Eqt-II), Equinatoxin IV (Eqt-IV), Equinatoxin V (Eqt-V), Urticinatoxin (UcI), Actitoxin-Oor1b (Or-G), Actitoxin-Oor1a (Or-A), Gigantoxin-4 (Gigt 4), Heteractis magnifica cytolysin III (HmgIII), Bandaporin (bp-1), Cribinopsis japonica toxin I (CJTOX I), Cribinopsis japonica toxin II (CJTOX II), Sticholysin I (StI), Sticholysin II (StII), Stichotoxin Hcr4a (RTX-A), Stichotoxin Hcr4b (RTX-SII) and Sagatoxin I (Src I).
The wildtype sequences and SwissProt accession numbers of exemplary alpha-helical pore forming proteins are as follows:
Also encompassed are pore-forming toxin homologs thereof showing at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with any of these family members, provided that the pore-forming toxin retains the ability to create oligomeric nanopores in membranes. This functionality can be readily tested by in vitro using methods known in the art. For example, putative purified nanopores can be inserted into model membranes as described herein or using other means known in the art (e.g. vesicle insertion, detergent insertion, spontaneous insertion, etc) and characterized by electrophysiology means to determine their ability to pass ionic current and detect the presence of model analytes added to the system.
As will be understood by a person skilled in the art, the amino acid sequence of a given family member will contain one or more mutation(s) in the recognition region of the pore. Thus, if reference is made to e.g. a pore comprising FraD, Eqt-IV or StII, this is meant to refer to FraD, Eqt-IV or StII mutants of which the internal degree of aromaticity has been manipulated, optionally in combination with the introduction of negatively charged residue(s), in accordance with the present invention.
For example, a proteinaceous nanopore according to the invention advantageously comprises a mutant actinoporin selected from the group consisting of:
In one embodiment, the mutant pore-forming toxin or pore-forming alpha-helical fragment is selected from Table 1, depicting the one or more specific aromatic mutations in each of the actinoporins that are equivalent to the mutations of the lumen-facing residues at positions 10, 13, 17 and 20 of FraC.
Also encompassed are pore-forming toxin homologs comprising the defined mutation(s) and showing at least 85%, at least 87%, at least 90%, at least 93%, at least 95% or at least 98% sequence identity.
Very good results can be obtained with a proteinaceous nanopore comprising mutant FraC or a pore-forming alpha-helical fragment thereof, comprising mutation Gly13Tyr, Gly13Trp or Gly13Phe, preferably Gly13Phe.
In another aspect, the invention relates to a proteinaceous nanopore comprising a mutant beta-barrel pore-forming protein, or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more mutations of lumen-facing non-aromatic residue(s) to a natural or non-natural aromatic amino acid residue, preferably one or more mutations to Trp, Tyr or Phe.
Preferably, the beta-barrel pore-forming toxin has an internal diameter (pore size; constriction) in the recognition region in the range of 0.2 to 2.0 nanometers, most preferably a minimum internal diameter of 0.5 to 1.5 nanometers. For example, it is selected from the group consisting of alpha-hemolysin, aerolysin, lysenin, epsilon-toxin (ETX), hemolytic lectin (LSL), cytolysin k (cytK), and functional homologs showing at least 80%, preferably at least 85%, more preferably at least 90% sequence identity therewith. Preferably, it is selected from the group consisting of aerolysin, lysenin and cytolysin k (cytK), and functional homologs showing at least 90%, preferably at least 95%, more preferably at least 98% sequence identity therewith.
Similar to what is described herein above for the alpha-helical type pore forming proteins, the pore may further comprise one or more mutations increasing the net negative charge or decreasing the net positive charge of the barrel or channel of the pore, with the aim of increasing the flux of cations through the nanopore (Table 4), especially under acidic pH conditions (pH<4.5).
The wildtype sequences and SwissProt accession numbers of exemplary beta-barrel pore forming proteins are as follows:
The following list indicates lumen-facing amino acid position(s) in exemplary beta-barrel pores to be mutated into an aromatic residue.
In one aspect, the invention provides a mutant proteinaceous nanopore comprising a mutant of the aerolysin-like 3-PFP (ap-PFP) subfamily.
For example, it provides aerolysin (Aer) comprising an aromatic amino acid substitution in the water-facing region of the pore, which region runs from residues 212 to 242 and from 256 to 284. In one aspect, the aromatic mutation is at least in the region 212-242. For example, one or more basic residue(s) is/are replaced with an aromatic residue in order to reduce the net positive charge. In some embodiments the mutant is either W, Y or F substituted at one of more positions including Q212, G214, D216, T218, R220, D222, A224, N226, S228, T230, T232, G234, S236, K238, T240 or K242 This may comprise substituting K238 with W, Y or F. In a specific embodiment, the mutant is Aer-K238F or Aer-K238W.
In another aspect, the aromatic mutation is at least in the region 256-284. For example, the position corresponding to S256, E258, A260, N262, S264, A266, Q268, G270, S272, T274, S276, S278, S280, R282 and/or T284 is mutated to Trp, Tyr or Phe. The aromatic substitution is preferably combined with an acidic substitution, for example at position K238. In one embodiment, mutant aerolysin comprises mutation K238D. In a specific aspect, the nanopore comprises aerolysin mutant A260F, S264F, Q268F, S272F, and/or comprising mutation K238D. Preferred mutants include Aer-K238D, Aer-K238D-A260F, Aer-K238D-S264F, Aer-K238D-Q268F and Aer-K238D-S272F. These mutant pores are suitably used for analyte detection, preferably (unlabeled) peptide detection, at a pH≤4.5, e.g pH 3.8 or pH 3.0. See example 7 herein below.
In another embodiment, the invention provides a mutant proteinaceous nanopore comprising a mutant Lysenin or a pore-forming fragment thereof. The lysenin pore resembles the mushroom-shaped pore complexes of the α-haemolysin family of small (3-PFTs, although structures of their water-soluble monomers are fundamentally different. In one specific embodiment, the Lys mutant comprises an aromatic substitution at position Glu76, for example it is mutant Lys-E76F.
In another aspect, the invention provides a mutant proteinaceous nanopore comprising a mutant Cytotoxin K (CytK) or a pore-forming fragment thereof. CytK is a pore-forming toxin of Bacillus cereus (Hardy et al. FEMSMicrobiolLett. 2001). Although confirmed to be a nanopore with suitable properties for sensing, as of to date no structure exists for the CytK nanopore to aid mutagenesis. A person of skill in the art will understand that the transmembrane beta-barrel region of the protein, and therefore the putative recognition region for sensing analytes, can be determined by homology modelling to a suitably similar structure (e.g. alpha-hemolysin from S. aureus), and then confirmed experimentally by mutagenesis in the candidate region. Exemplary CytK mutants of the invention comprise an aromatic amino acid substitution in the lumen-facing region of the pore, which region runs from residue 112 to residue 155. Residues identified as most suitable for aromatic substitution, and optionally in combination with one or more acidic substitutions, include one or more (e.g. up to 5, preferably up to 4, more preferably up to 3 or 2) of the following lumen-facing non-aromatic transmembrane residues: E112/T114/T116/S118/S120/Q122/G124/S126/K128/S130/T132/S134/S137/E139/G141/T 143/Q145/T147/S149/S151/S153/K155, preferably one or more of E112/T114/T116/S118/S120/Q122/G124/S126.
These aromatic mutations are advantageously combined with one or more acidic substitutions of neutral and/or positively charged residue(s), such as K128E or K128D, or any of the lumen-facing residues that can be substituted with an aromatic residue, excluding those already negative. Such mutations ideally make the barrel of the pore net negative (if not already so), thereby altering the electro-osmotic flow through the pore.
In a specific aspect, the mutant pore or fragment thereof comprises a CytK mutant with an aromatic amino acid at position S126 or K128, for example comprising mutation Ser126Tyr, Ser126Trp, Ser126Phe, Lys128Tyr, Lys128Trp, Lys128Phe, preferably Ser126Phe or Lys128Phe.
In another specific aspect, the mutant pore or fragment thereof comprises a CytK mutant with an aromatic amino acid substitution higher “up” in the barrel, for instance at position Ser120, Gln122 or Gly124. See
Similar to what is described herein above for the alpha-helical type pore forming protein and the beta barrel pore forming protein, the pore may further comprise one or more mutations increasing the net negative charge or decreasing the positive charge of the barrel or channel of the pore, with the aim of increasing the flux of cations through the nanopore (Table 3), especially under acidic pH conditions (pH≤4.5). As shown for the mutation K128F or K128D (
In a specific aspect, as found for the pore forming proteins described above, the mutant pore comprises a CytK mutant with an aromatic amino acid in the water facing region of the nanopore, for example S126F, further comprising a mutation to increase the negative charge of the water facing region, for example K128D, which improves the analysis of peptides.
A further aspect of the invention relates to an analytical system comprising a mutant proteinaceous nanopore according to the invention. For example, the analytical system comprises a hydrophobic membrane separating a fluid chamber into a cis side and a trans side, wherein the mutant proteinaceous nanopore is inserted in said membrane. A nanopore sensor system may comprise: i) a fluid-filled compartment separated by a membrane into a first cis chamber and a second trans chamber, wherein the fluid is an ionic solution; ii) an engineered mutant pore of the invention inserted in the membrane; and iii) electrodes configured for measuring an ionic current flow through the nanopore and optionally generating an electrical potential difference across the membrane to facilitate ionic flow through the pore from the first chamber to the second chamber and vice versa.
Herewith, the system provides a pore-based sensor. In one specific aspect, the analytical system comprises a mutant alpha-helical pore forming toxin of the actinoporin family, preferably FraC. In another specific aspect, the analytical system comprises a mutant beta-barrel pore forming toxin, preferably an aerolysin-like OPFP or cytotoxin K.
When a system according to the invention is in use, the nanopore is typically positioned between a first liquid medium and a second liquid medium, wherein at least one liquid medium comprises an analyte of interest, and wherein the system is operative to detect a property of the analyte. In one embodiment, the system is operative to detect a property of the analyte by subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electroosmotically translocates through the nanopore.
As exemplified herein below, a system provided herein is particularly suitable for the analysis of a proteinaceous substance, preferably a peptide, more preferably a peptide up to about 30 amino acids in length. More in particular, a system of the invention provides for capture of peptides up to 20, 15, 10, 5, 3 or 2 amino acids in length. As is exemplified herein below, a mutant nanopore can detect peptide(s) with a highly variable amino acid composition. Hence, it can be broadly applied without restriction to any specific structure and/or property. However, in one aspect, the peptide comprises at least 50%, preferably 60%, more preferably at least 70% of hydrophobic and charged amino acids. For example, it contains up to 40%, preferably up to 30%, more preferably up to 25% of aromatic amino acids (Tyr, Trp and Phe).
However, this is in no way to be understood that the invention is limited to applications relating to peptide analysis. For example, other analytes that can be detected using a system of the invention include (non-proteinaceous) biomarkers, antibiotics or other drugs, DNA, metabolites and small biological and non-biological molecules. Exemplary analytes include various sub-classes of small-molecule biomarkers, such as steroids, carbohydrates, amino acids, nucleotides, hormones, fatty acids, vitamins, flavins, protein-cofactors, lipids, phenolic compounds. In one embodiment, the analyte of interest is a biopolymer, preferably selected from the group consisting of a protein, a polypeptide and an oligopeptide. In one aspect, the analyte is a substance having a mass in the range of between 200 and 5000 Da, for example in the 200-500 Da range or in the 500 and 1700 Da range See in particular
The invention also provides a method for providing a system according to the invention. Typically, this comprises the steps of:
Further embodiments of the invention relate to means and methods for preparing mutant protein pores by recombinant technology. These include a nucleic acid molecule encoding a mutant nanopore according to the invention, and an expression vector comprising said nucleic acid molecule. Also encompassed is a host cell, preferably a bacterial host cell, comprising the nucleic acid-containing expression vector.
An analytical system of the invention may be incorporated, e.g. in the form of an array of multiple systems, into a device. The device may be any conventional device for analyte analysis, such as an array or a chip. Provided is a device comprising a plurality of analytical systems (sensors) according to the invention. The plurality of sensors can be based on the same proteinaceous nanopore (i.e the same mutant), or on distinct nanopores inserted into a plurality of membranes, which may for example be connected to a plurality of electrical circuits to address and measure each nanopore sensor separately. Preferably, a single pore is present in each membrane. The pores might differ by their type, family, mutation etc. In one embodiment, a device comprises multiple pores to generate different characteristic signals from peptides, which signal can be compared or combined to improve their discrimination and characterization.
The protein nanopore of the invention may be present in a membrane, or inserted when required. Protein nanopores are typically asymmetric, and may be inserted with directional control relative to the cis and trans compartments of an analytical system by various means known in the art. Typically, and unless stated otherwise in the examples herein, nanopores are inserted from the cis compartment.
Any membrane may be used in accordance with the invention. Suitable membranes are well-known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and hydrophobic/lipophilic properties. The amphiphilic layer may be a monolayer or a bilayer. The membrane is preferably formed from a bilayer of phospholipids. The membrane is preferably formed from lipids or amphipathic molecules that are chemically stable under low pH conditions, for example ether-linked phospholipids. The amphiphilic molecules may be synthetic or naturally occurring. Non-natural amphiphiles that form a monolayer are known in the art and include, for example, block copolymers (di-block, tri-block, tetra-block etc) of various polymeric compositions.
An analytical system or device finds its use in a variety of analytical methods. For example, it is advantageously used in single molecule analysis. A further embodiment of the invention therefore relates to a method for single molecule analysis, the method comprising adding a substance or mixture of substances to be analyzed to the chamber of a (plurality of) analytical system(s) as provided herein, allowing the substance(s) to contact the (lumen-facing region of the) nanopore, and detecting/characterizing at least one property of the substance (also referred herein as “analyte” or “analyte of interest”). The substance/analyte can be detected by a change in the electrical current through the nanopore. For example, various properties of the substance, e.g. including, but not limited to, volume, shape, charge, structure, cross-linking, post-translational modifications (phosphorylation, glycosylation, rhamnosylation, etc.), damage, (D/L-) chirality, and sequence can be detected. Preferably, the method comprises the identification and/or sequencing of a substance. The analytical system or method is surprisingly suitable for the analysis of an analyte having a mass in the range of between 200 and 5000 Da, for example 500-1700 Da.
The substance, for example a peptide analyte, is typically present in any suitable sample. The invention is typically carried out on a sample that is known to contain or suspected to contain the analyte. Alternatively, the invention may be carried out on a sample to confirm the identity of the analyte whose presence in the sample is known or expected. The sample may be a biological sample. The invention may be carried out in vitro using a sample obtained from or extracted from any organism or microorganism (e.g. archaeal, prokaryotic or eukaryotic). The sample is preferably a fluid sample. The sample may comprises a body fluid of a patient (e.g. urine, lymph, mucus or amniotic fluid, or preferably sweat, saliva blood, plasma or serum). The sample may be human in origin, but alternatively it may be from another animal, such as from commercially farmed animals, or of plant origin. The sample may be a non-biological sample. Examples of non-biological samples include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.
The sample may be processed (pretreated) prior to being used in the invention, for example by various purification means known in the art to isolate mixtures of proteins/peptides/molecules or target proteins/peptides/molecules. These may include for example affinity binding methods, such as antibodies, or chromatographic methods, to isolate and purify specific components of the sample or remove unwanted background impurities. For protein samples the proteins contained therein are preferably fragmented into peptides (preferably defined populations), for example by enzymatic means known in the art (e.g. proteases) or other degradative means. An analytical method of the invention may include one or more sample preparation steps. For example, a pre-filtering step and/or other modifications as done for other methods (e.g. Mass Spec).
A person of skill in the art would understand that many of the sample preparation methods employed for classical mass spectrometry can be used herein. For example, proteins in a sample might be denatured by physical (e.g. temperature) or chemical (e.g. chaotropic agents, detergents) means prior to processing and nanopore sensing. Alternatively, cross-links such as disulphide bridges can be broken to disrupt certain secondary structures. Alternatively, modifications, for example large glycans, might be modified, truncated or removed prior to nanopore sensing. Alternatively, some of the amino-acids in a peptide sample might be modified to alter the signal, such as for example, Cysteines or Lysines might be chemically labelled with additional tags to modulate the signal in nanopore sensing to provide further insight into the analyte. In some embodiments of the invention the peptide analytes might be subjected to reactions that alter the N-terminal or C-terminal ends of the molecules using methods known in the art, for example for the purposes of adding a molecular label or tag (e.g. to add a barcode to register the precursor sample, or to facilitate capture and detection in a nanopore system). Such molecular labels/tags might be peptide based, polynucleotide based or composed of other chemistries.
In one aspect, the invention provides a method for single molecule analysis, the method comprising adding a substance or mixture of substances to be analyzed to the chamber of an analytical system as provided herein, allowing the substance(s) to contact the (lumen-facing region of the) nanopore, and detecting/characterizing at least one property of the substance, wherein the substance is a proteinaceous substance, preferably a peptide, more preferably a peptide up to about 30, 20, 15, 10, 5, 3 or 2 amino acids in length.
The method may involve detecting a mutation and/or post-translational modification of a substance, for example detecting peptide fragments that differ by a single amino acid residue, degree of phosphorylation and/or degree of glycosylation. In specific embodiments, the nanopore detects peptides resulting from a protein mixture that has been subjected to denaturing conditions, or from a protein mixture that has been subjected to fragmenting conditions, including protease digestion e.g. as typically used in MS analysis. In one aspect, the fragmentation condition leads to positively charged peptide fragments. A method of the invention has the ability to quantify the absolute or relative abundance of the proteins in the original mixture from the peptide spectrum.
In certain embodiments of the invention, optimal conditions for peptide detection are performed under low pH conditions, preferably below pH 4.5, preferably below pH 4.0. Under physiological pH conditions, naturally occurring peptides have a wide range of charge distributions and net charges (e.g. both net positive and net negative) as a result of their highly variable composition of acidic, basic and neutral amino acids. This diversity of charge significantly complicates the ability to capture and detect all the peptides in a diverse mixture of different peptides in a nanopore sensing system when a fixed applied potential is applied, since not all peptides will experience the same net electrophoretic force. Depending on the polarity of the applied potential and the specific charge composition of each peptide, some peptides will experience net electrophoretic force into the nanopore, while other oppositely charged peptides will experience net electrophoretic force out of the nanopore. By implementing low pH conditions on the side of the nanopore sensing system containing a complex peptide analyte mixture, and preferably on both the cis and trans sides of the membrane, the amino acids of the peptide analytes become protonated. The increased protonation serves to both 1) increase the net positive charge of all peptides in a diverse mixture, and 2) create a more uniform distribution of charges in the peptide mixture. The increased net positive charge allows for an improved electrophoretic capture of the peptides in a nanopore system held under an appropriate polarity applied potential (e.g. when a negative potential is applied to the electrode on the opposite side of the membrane to the peptide analytes).
An improved uniformity of charge in the peptide mixture is also highly advantageous as all peptide molecules will experience more similar electrophoretic forces acting upon them under an applied potential. Since electrophoresis is an important component determining the efficiency of analyte capture into a nanopore, this reduces capture efficiency biases between different peptide compositions in mixtures. This highly advantageous feature reduces the likelihood that some peptide populations with inefficient capture will be missed or lost in the background of peptide populations with higher capture efficiency.
Implementing low pH conditions also alters the charge characteristics of the nanopore in the sensing system by partially protonating some of the water-facing amino acids. Notably, the increased positive charge inside the nanopore channel, and inside the lumen recognition region, alters the capture and subsequent detection of peptide analytes. Under low pH conditions the increased positive charge in the nanopore can electrostatically repel the mostly positively charged peptide analytes, which can in turn reduce capture efficiency and/or reduce the residence time of peptides inside the nanopore. This can reduce the ability to detect and characterize some peptide analytes.
A variety of different types of measurements may be made on the nanopore system. This includes without limitation: electrical measurements and optical measurements. Possible electrical measurements include: current measurements, impedance measurements, tunnelling measurements, and field-effect-transistor (FET) measurements of local voltage changes. Optical and electrical measurements may be combined to provide additional information (Heron et al. J Am Chem Soc 2009). Optical measurements may be employ dye systems that are reporters of ionic flux (Heron et al. J Am Chem Soc 2009).
The method is preferably carried out with a potential applied across the membrane. The applied potential may be a voltage potential. The applied voltage enables electrophoretic and/or electroosmotic flow through the nanopore to facilitate analyte capture and detection. Following convention, unless defined otherwise for the data in the invention herein, the active electrode is defined as that in the trans compartment, and is the one at which the stated polarity of potential is applied (e.g. relative to the ground electrode in the cis compartment). A person of skill in the art will understand that alternative electrode configurations are known in the art, and can be employed for example to control electrophoretic and/or electroosmotic analyte capture in the nanopore system through application of an applied potential, and/or to measure changes in ionic current or local voltage. The applied potential might be held at a constant voltage for a fixed period (milliseconds, seconds, minutes, hours). Alternatively, the voltage might be changed in discreet steps to alter the sensing conditions and/or obtain different information from the analytes. The voltage might be constantly changing, for example a person skilled in the art would understand that various pattern (e.g. square wave, triangular wave, sinusoidal, etc) waveforms might be employed to control analyte capture and obtain different characteristics from the analytes. Alternatively, the applied potential may be a chemical potential (e.g. a salt gradient across a membrane). The voltage used is typically from +50 V to −50 V, or +100 V to −100 V. The voltage used is preferably in a range having a lower limit selected from −300 mV, −300 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0 mV and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV. The voltage used is more preferably in the range +−50 mV to +−150 mV and most preferably in the range of +−50 mV to +−100 mV. The method is typically carried out with any well-known charge carriers present in the aqueous solution in the chamber, such as metal salts or ionic liquids. For example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt, or ionic liquids or organic salts such as tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazolium chloride. The salt is preferably potassium chloride (KCl), sodium chloride (NaCl), or lithium chloride (LiCl). The solutions may also contain well-known redox salts to mediate electron transfer at suitable electrodes, for example potassium ferrocyanide and potassium ferricyanide or other well-known redox couples. The salt concentration may range from 0.1 to 3 M, or up to the saturation point for a given salt type. The salt concentration is preferably from 0.1 to 1.5 M, and most preferably 0.15 to 1.0 M. The method is typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer may be used in the method of the invention. Typically, the buffer is bis-tris buffer, citrate buffer, phosphate buffer, HEPES buffer or Tris-HCl buffer. The methods are typically carried out at a pH below 8.0, and preferably at below pH 4.5, most preferably below pH 4.0, using buffers that are appropriate to this range (e.g. citrate buffer). The method may be carried out at from 0° C. to 100° C., preferably from about 20° C. to about 400 C.
Electrical measurements may be made using standard single channel recording equipment such as that described herein. Alternatively, electrical measurements may be made using a multi-channel systems known in the art that are capable of simultaneously acquiring signals from multiple independent nanopore systems (e.g. a plurality of membranes containing inserted nanopores).
The method of the invention may involve measuring multiple characteristics of the current signal, most preferably of event blockades arising from capture and detection of analytes. The one or more characteristics are preferably selected from: the open-pore current, the average or median current of the event blockade, the duration (dwell) time of the event blockade, the frequency of event blockades, the number of event blockades, the noise in the event blockade, and the shape of the event blockade (including stepwise changes). A person of skill in the art would understand that a range of analytical tools can be used to extract high level information from event blockades and other parts of the current signals. For example, edge-detecting algorithms can be used to segment the event blockades to simplify the data. Alternatively, the raw data may be analyzed directly, with or without the application of filters, for example using sliding window features and algorithms with long range memory, to extract characteristic metrics.
The method of the invention may involve determining one, two, three, four or five or more characteristics of the analyte from the characteristic metrics of the signals. The one or more characteristics are preferably selected from: the length of the analyte, the volume of the analyte, the mass of the analyte, the shape of the analyte, the charge distribution of the analyte, the identity of the analyte, the sequence of the analyte, any chemical modifications of the analyte. The characteristics of the analytes can be determined by any number of a wide range of analytical methods known in the art, including for example statistical methods or machine learning methods. These methods may have been trained or optimized by training the systems with model analytes for example, or may have been built from first principles. For example, the identity of peptides can be determined by comparison to previously acquired data using training data. Also provided herein is an analytical method of determining the identity of the original protein/s from the peptide fingerprint by comparing the spectrum to theoretical data or previously trained data. A person of skill in the art will understand that the multi-metric data obtained for each nanopore event can be exploited in higher dimension analysis (e.g. by combined comparison of 2, 3, 4, 5, 6, or more separate event metrics) to discriminate different analytes that might not be separable by any one metric alone. A person of skill in the art will also appreciate that a collection (spectra) of multiple analyte events can be analyzed as population ensembles for the discreet populations of analytes in a sample, and that the discreet populations might be resolved (e.g. in multiple dimensions using multiple metrics as axes) and identified using any number of advanced fitting and classification tools. Further, unique data from the populations, for example fingerprints, might be used in analytical methods to identify the analyte composition, and therefore for example for digested peptide mixtures identify and/or quantify the precursor protein(s).
As is exemplified herein below, the present inventors found that for certain protein nanopores, such as Aerolysin and CytK, it is either essential or highly advantageous to reduce the net positive charge in the nanopore channel, preferably in the recognition region, most preferably at or near the constriction, preferably in combination with aromatic mutations, to enable efficient capture and recognition of peptide analytes under low pH conditions. For example, by introducing acidic residue(s) (Asp/Glu) by substitution adjacent to the aromatic mutation(s) (Phe/Tyr/Trp). Hence, in one embodiment the pore comprises one or more mutation(s) to Glu and/or Asp residue(s) in the water-facing region. Alternatively, it is understood that net positive charge can be also reduced by replacing basic residue(s) (Arg/Lys/His) with neutral or acidic residue(s), optionally by substitution with aromatic residues that also separately and additively improve peptide capture and discrimination (e.g. CytK-K128F, Aer-K238F examples contained herein).
Increased positive charge in the channel of the nanopore under low pH conditions also alters the ion selectivity of the nanopore. The increased positive charge in the nanopore channel favors increased transport of anionic species and decreases the transport of cationic species, which in turn alters the net electro-osmotic flux of hydrated ions flowing through the nanopore under an applied potential. The increased anionic electro-osmotic flux through the nanopore will act against the electro-phoretic forces acting on the mostly positively charged peptide analytes under low pH conditions.
The direction and magnitude of the electro-osmotic component for a nanopore system can be determined by ion-selectivity measurements known in the art. For example, nanopore ion-selectivity can be measured in an in vitro electrophysiology system by measuring the reversal potential under asymmetric salt conditions (e.g. with 2M KCl in the trans compartment 0.5 M KCl in the cis compartment). Table 3 herein below contains the measured reversal potentials and ion-selectivity for selected Aerolysin and CytK nanopores. FraC ion-selectivity under low pH has been determined previously (Huang et al. Nat. Commun. 2019).
For nanopore sensing systems set up to enable analyte capture by electrophoresis, (e.g. in a system with a negative potential applied at the electrode in the compartment opposite to that containing the peptide analytes), such as for the FraC nanopore and CytK nanopore examples contained herein, it is advantageous to ensure that excessive electro-osmotic forces do not act against electrophoretic capture of analytes under the chosen sensing conditions. Therefore, in certain embodiments of the invention (e.g. for the CytK nanopore examples contained herein) where ion-selectivity and electro-osmotic flux are increased by the implementation of low pH conditions, it can be advantageous to reduce the net ion-selectivity and electro-osmotic flux, preferably to a level where electrophoretic forces dominate analyte capture, preferably to close to zero net ion-selectivity. Electro-osmosis can be reduced by reducing the net charge inside the nanopore channel (e.g. by mutagenesis. See Table 4). For example, the anion ion-selectivity bias and resulting net anionic electro-osmotic flux can be reduced by introducing acidic residues by substitution adjacent to the aromatic mutations. Hence, in one embodiment the pore comprises one or more mutation(s) to Glu and/or Asp residue(s) in the water-facing region. Alternatively, it is understood that net positive charge can be also reduced by replacing basic residues with neutral or acidic residue(s), optionally by substitution with aromatic residue(s) that also separately and additively improve peptide capture and discrimination (e.g. CytK-K128F, Aer-K238F examples contained herein).
In some proteins, like FraC, FraE or FraB, the wild-type pores already contain sufficient negative charge characteristics inside the water facing nanopore channel/lumen or recognition region under low pH conditions for optimal ion-selectivity and electro-osmosis, and optimal interaction with mostly positively charged analytes, and therefore do not require mutations to add further negative charges in spatial combination with the aromatic residue(s) that are introduced. Conversely, removing acidic residues in the FraC example herein, which increases the net positivity of the nanopore, dramatically reduced peptide capture and discrimination under an electrophoretic dominant regime.
In some embodiments of the invention, it has been found that efficient peptide analyte capture and detection can achieved under conditions set up to create dominant electro-osmotic capture. For example, for the Aerolysin nanopore system examples herein, the implementation of low pH conditions increases the net positive charge inside the nanopore channel, resulting in increased anion selectivity, and a strong net anion-selective nanopore (see Table 3) and in increased electrostatic repulsion of mostly positively charged analytes. The resulting strong electro-osmotic flux through the nanopore can be exploited to capture analytes against the direction of the electrophoretic forces acting upon them (e.g. with a positive applied potential at the trans electrode for a system with mostly positively charged peptides in the cis solution—see Aerolysin examples herein). For certain embodiments of the invention, it can be highly advantageous to exploit electro-osmotic forces to capture analytes since it is less sensitive to charge composition. It can therefore be highly advantageous for capturing and detecting a diverse composition of unlabeled peptides (e.g. neutral, net positive, net negative). In certain embodiments of the invention, the strength of electro-osmotic force acting on the analytes can be further tuned (e.g. by mutagenesis). For example, in certain embodiments, it can be useful to reduce the electro-osmotic force to increase the duration for which the analytes are retained in the nanopore. For example, the anion ion-selectivity bias and resulting net anionic electro-osmotic flux that results from low pH conditions can be reduced by introducing acidic residues, preferably by substitution adjacent to the aromatic mutations. Acidic mutation substitutions that reduce net positive charge will also reduce electrostatic repulsion of mostly positively charge analytes. Hence, in one embodiment the pore comprises one or more mutation(s) to Glu and/or Asp residue(s) in the water-facing region. Alternatively, it is understood that net positive charge can also be reduced by replacing basic residues with neutral or acidic residues, optionally by substitution with aromatic residues that also separately and additively improve peptide capture and discrimination (e.g. CytK-K128F, Aer-K238F). It is also understood that mutagenesis can be combined with changes to the system conditions (e.g. pH, salt type, salt asymmetry) to control the direction and magnitude of the electro-osmotic effect, and can be determined experimentally as described previously by measurements of reverse voltages for example.
Hence, described herein are methods for optimizing the electrophoretic and electro-osmotic components of a nanopore sensing system for the capture and characterization of unlabeled peptides for the purpose of discriminating between different peptides. The optimal “characterization parameters” for effective peptide sensing or sensing of other molecules can be determined experimentally in a nanopore system by measurement using model peptides or natural peptides.
The invention also provides a kit of parts, e.g. for use in characterizing an analyte of interest, the kit comprising (i) a mutant proteinaceous nanopore, an analytical system and/or a device according to the invention; and (ii) an analyte-handling enzyme. Preferably, the analyte-handling enzyme is a protein-handling enzyme, such as a protease. Of particular interest are trypsin or other proteases such as chymotrypsin or Lys-C protease. The use of trypsin can be advantageous because it cleaves preferentially after a K/R amino acid and as most peptides will have a positive charge next to the zwitterionic charges on the peptide, yielding an additional net charge of +1 under acidic to neutral pH conditions employed for analysis. Lys-C protease has high activity and specificity for lysine residues, resulting in larger peptides and less sample complexity than trypsin (i.e., fewer peptides). Unlike trypsin, Lys-C protease can cleave lysines followed by prolines, making it ideal for sequential protein digestion followed by trypsin to decrease missed cleavages. These unique Lys-C protease properties ensure high digestion efficiency when used alone or followed by tryptic digestion.
As will be appreciated by a person skilled in the art, an analytical system, device or a kit comprising a mutant proteinaceous pore as herein disclosed finds many uses and applications e.g. in the field of molecular analysis and identification. These include single molecule analysis, preferably the identification and/or sequencing of a biomolecule or biopolymer, more preferably label-free protein or peptide fingerprinting.
Further embodiments of the invention are as follows.
<1> A proteinaceous nanopore comprising a mutant beta-barrel protein pore-forming toxin, or a pore-forming fragment thereof, wherein the lumen-facing recognition region of the pore-forming protein or fragment thereof comprises one or more substitution(s) of lumen-facing non-aromatic amino acid(s) to a natural or non-natural aromatic amino acid residue.
<2> Proteinaceous nanopore according to <1>, comprising a mutant pore forming toxin comprising one or more substitution(s) of lumen-facing amino acid(s) to Trp, Tyr or Phe.
<3> Proteinaceous nanopore according to <1> or <2>, wherein the beta-barrel pore-forming toxin has an internal diameter (pore size; constriction) in the recognition region in the range of 0.2 to 2.0 nanometers, preferably a minimum internal diameter of 0.5 to 1.5 nanometers.
<4> Proteinaceous nanopore according to any one <1> to <3>, comprising a mutant pore selected from the group consisting of alpha-hemolysin (SwissProt P09616.2), aerolysin (SwissProt P09167), Gamma-hemolysin component B (SwissProt P0A075.1) lysenin (SwissProt 018423), epsilon-toxin (ETX), hemolytic lectin (LSL; SwissProt Q868M7), cytolysin k (cytK; SwissProt Q937V2), and functional homologs showing at least 80%, preferably at least 85%, more preferably at least 90% sequence identity therewith.
<5> Proteinaceous nanopore according to <4>, selected from the group consisting of aerolysin, lysenin and cytolysin k (cytK), and functional homologs showing at least 90%, preferably at least 95%, more preferably at least 98% sequence identity therewith.
<6> Proteinaceous nanopore according to any of <1> to <5>, comprising aerolysin (Aer) comprising an aromatic amino acid substitution in the water-facing region of the pore, which region runs from residues 212 to 242 and from 256 to 284 of aerolysin, preferably wherein the aromatic mutation is at least in the region 212-242, more preferably wherein the mutant is either W, Y or F substituted at one of more positions including Q212, G214, D216, T218, R220, D222, A224, N226, S228, T230, T232, G234, S236, K238, T240 or K242.
<7> Proteinaceous nanopore according to any one of <1-5>, comprising a mutant CytK, preferably comprising an aromatic amino acid substitution in the lumen-facing region of the pore, which region runs from residue 112 to residue 155, more preferably one or more (e.g. up to 5, preferably up to 4, more preferably up to 3 or 2) of the following lumen-facing non-aromatic transmembrane residues E112/T114/T116/S118/S120/Q122/G124/S126/K128/S130/T132/S134/S137/E139/G141/T 143/Q145/T147/S149/S151/S153/K155, such as one or more of E112/T114/T116/S118/S120/Q122/G124/S126.
<8> Proteinaceous nanopore according to any one of <1-5>, comprising mutant Lysenin (UniProtKB—P13423) or a pore-forming fragment thereof, preferably comprising an aromatic substitution at position 35, 37, 39, 41, 45, 43, 47, 49, 51, 53, 55, 57, 59, 63, 61, 65, 68, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102 and/or 104, more preferably wherein the Lys mutant comprises an aromatic substitution at position Glu76, for example mutant Lys-E76F.
<9> Proteinaceous nanopore according to any one of <1-8>, wherein one or more further mutation(s) is/are introduced in the lumen-facing amino acids of the recognition region, which further mutation(s) increase the net negative charge of the pore.
<10> Proteinaceous nanopore according to <9>, comprising one or more mutation(s) to Glu and/or Asp residue(s).
<11> Proteinaceous nanopore according to any one of <1-10>, comprising a mutant beta-barrel pore or fragment thereof selected from the group consisting of:
To the left of each panel is indicated the schematic position of the substituted amino acid. Recordings were done at an applied potential of +100 mV at 50 kHz frequency with a 10 kHz lowpass filter. The figure shows that aromatic mutations especially in combination with modifications that increase the negative charge of the pore allow the recognition of peptides especially at pH values lower than 4.
Chemicals. Sphingomyelin (Porcine brain, >99%, CAS #383907-91-3) and diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, >99%, CAS #207131-40-6) were retrieved from Avanti Polar Lipids. Ni-NTA resin was obtained from Qiagen. Lysozyme (Albumin free for tryptic digest, CAS #12650-88-3), Glucose (>99%, CAS #50-99-7), Sodium chloride (99.5%, CAS #7647-14-5), Potassium chloride (99%, CAS #7447-40-7), Dithiothreitol (DTT, >99.0%, 3483-12-3), Trizma® HCl (>99%, CAS #1185-53-1), Trizma® base (>99.9%, CAS #77-86-1), Imidazole (>99%, CAS #288-32-4), n-Dodecyl β-D-maltoside (DDM, >99%, CAS #69227-93-6), Hydrochloric acid (1 M, CAS #7647-01-0), Urea (>99.5%, CAS #57-13-6), Magnesium chloride (>98.5%, CAS #7786-30-3), LB Broth (Luria/Miller), Agar-agar and 2× YT Broth were obtained from Carl Roth. Ampicillin sodium salt (CAS #69-52-3), Isopropyl β-D-1-thiogalactopyranoside (IPTG, ≥99%, CAS #367-93-1), Ethanol (≥99.8%, CAS #64-17-5) and all enzymes were received from Fisher Scientific. Lysozyme from chicken egg white (for Lysis, CAS #12650-88-3), N,N-Dimethyldodecylamine N-oxide (LDAO, ≥99.0%, CAS #1643-20-5), Pentane (≥99%, CAS #109-66-0), Iodoacetamide (IAA, ≥99%, CAS #144-48-9), Bis-tris propane (≥99.0%, CAS #64431-96-5) were bought from Sigma-Aldrich. n-Hexadecane (99%, CAS #544-76-3) and Citric acid (99.6%, CAS #77-92-9) were purchased from Acros. Trypsin (bovine pancreas, CAS #9002-07-7) was obtained from Alfa Aesar.
Fragaceatoxin C (FraC) monomer expression and purification. pT7-SC1 vector containing His6-tagged (SEQ ID NO: 101) FraC plasmids were electrochemically inserted into E. coli BL21 (DE3) cells and grown overnight at 37° C. on LB agar plates supplemented with 100 mg/l ampicillin and 1% glucose. Colonies were used to inoculate 200 ml 2×YT medium supplemented with 100 mg/l ampicillin and grown at 37° C. until the optical density at 600 nm (OD600) reached 0.6, after which expression was induced using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), allowing continued growth overnight at 21° C. Cell pellets were collected by centrifugation (6,000 g, 20 min, 4° C.) and stored at −80° C. for at least one hour. The pellets were resuspended in 10 ml lysis buffer per 50 ml culture, with a lysis buffer consisting of 150 mM NaCl, 15 mM Tris base solution at pH 7.5 supplemented with 1 mM MgCl2, 2 M Urea, 20 mM imidazole, 0.2 mg/ml lysozyme and 0.2 units/ml DNase. The solution was mixed for 1 hour at room temperature (21° C.) using a rotating mixer at 15 RPM. The cells were fully disrupted by sonification, applying 30 sweeps (duty cycle 30%, output control 3) three times using a Branson Sonifier 450. The lysate was centrifuged at 6000 g for 20 minutes at 4° C. The supernatant was incubated for 1 hour, while under constant rotation (15 RPM), with 100 μL resuspended Ni-NTA resin (resuspended in 150 mM NaCl, 15 mM Tris base at pH 7.5 supplemented with 20 mM imidazole). The solution was loaded onto a prewashed Micro Bio-Spin column (Bio-Rad). The Ni-NTA beads were extensively washed with 20 ml WB (150 mM NaCl, 15 mM Tris base at pH 7.5 supplemented with 20 mM imidazole). The column was inserted into a microtube and spin-dried using a centrifuge (13,300 g, 1 min) in order to remove residual wash buffer. 150 μl of 150 mM NaCl, 15 mM Tris base solution at pH 7.5 supplemented with 300 mM imidazole (EB) was added and left to incubate for 5 minutes before elution. This step was repeated four times to retrieve four fractions containing FraC monomers. The presence and purity of FraC monomers was estimated using SDS-PAGE. Pure fractions were pooled and stored at 4° C. The concentration of FraC monomers was estimated using a Nano Drop 2000 UV-Vis Spectrophotometer (Thermo Scientific) using the elution buffer as blank.
Sphingomyelin-DPhPC liposomes preparation. 25 mg sphingomyelin (Brain, Porcine) was mixed with 25 mg 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) and dissolved in 4 ml pentane containing 0.5% v/v ethanol. The lipid mixture was evaporated while turning inside a round bottom flask by application of a hot air stream to create a thin lipid film over the surface of the flask. The film was reconstituted into 10 ml of Sdex buffer (150 mM NaCl, 15 mM tris, pH 7.5) using a sonication bath. The liposome solution (5 mg/ml) was frozen and stored at −20° C.
Fragaceatoxin C oligomerisation. Liposomes were thawed and added to FraC monomers in a lipid to protein mass ratio of 10:1. The mixture was incubated for 30 minutes at 37° C., after which N,N-Dimethyldodecylamine N-oxide (LDAO) was added to a final concentration of 0.6 v/v % to dissolve the liposomes. The solution was diluted 10-fold in 150 mM NaCl supplemented with 15 mM Tris (pH 7.5) and 0.02 v/v % n-Dodecyl 3-D-maltoside (DDM). The diluted solution was combined with 100 μl of Ni-NTA, prewashed using WB2 (150 mM NaCl, 15 mM Tris base, pH 7.5 supplemented with 20 mM imidazole and 0.02 v/v % DDM). The mixture was left to incubate for 30 minutes while mixing under constant rotation (15 RPM). The solution was loaded onto a Micro Bio-Spin column (Bio-Rad), prewashed with 500 μl WB2. The Ni-NTA beads were washed extensively using 10 ml WB2. The column was spin-dried in a microtube using a centrifuge (13,300 g, 1 min) to remove residual wash buffer. 150 μl elution buffer was added onto the column (150 mM NaCl, 15 mM Tris base supplemented with 1M imidazole and 0.02 v/v % DDM) and left to stand for 10 minutes before elution into a clean microtube by centrifugation (13,300 g, 2 min). The oligomers are stable for several months at 4° C. and can be frozen at −80° C. for long-term storage.
Construction of Fragaceatoxin C mutants. Fragaceatoxin C mutant DNA was prepared using the MEGAWHOP method6. The megaprimer was constructed using a forward primer synthesized by Integrated DNA Technologies and a T7 reverse primer (5′-GCTAGTTATTGCTCAGCGG-3′ (SEQ ID NO: 46)). Six reactions were performed per mutation in order to receive enough DNA for the second PCR-using 25 μl REDTag® ReadyMix™ PCR Reaction Mix (Sigma-Aldrich) combined with 22 μl PCR grade water (Sigma-Aldrich), 1 μl of each forward and reverse primer and 1 μl His6-tagged (SEQ ID NO: 101) Fragaceatoxin C template DNA. The PCR protocol consisted of a 90 second denaturation step at 95° C. followed by 30 cycles of denaturation at 95° C. (15 seconds), annealing at 55° C. (15 seconds) and extension at 72° C. (120 seconds). The six PCR reactions were combined and purified using a GeneJET PCR Purification Kit (Thermo Scientific). For the second PCR, 10 μl 5× Phire Buffer (Thermo Scientific) was combined with 1 μl template DNA, 1 μl dNTPs (10 mM), 2 μl megaprimer (first PCR), 35 μl PCR grade water (Sigma-Aldrich) and 1 μl Phire II Hot Start DNA Polymerase (Thermo Scientific). The PCR protocol consisted of an initial pre-denaturing step of 98° C. (30 seconds) followed by 25 cycles of denaturation at 98° C. (5 seconds) and extension at 72° C. (90 seconds). 5.7 μl 5×FD green buffer (Thermo Scientific) and 1 μl Dpn1 enzyme (Thermo Scientific) was added to the PCR mix and let to digest at 37° C. for 1-3 hours. 0.5 μl of the digested product was electrochemically transformed into 50 μl E. cloni 10G® (Lucigen) competent cells and grown on LB agar plates containing 100 mg/l ampicillin and 1% glucose. Single colonies were enriched using a GeneJET Plasmid Miniprep Kit (Thermo Scientific) and the sequence was confirmed using the sequencing service of Macrogen Europe.
Unspecific lysozyme digestion. Lysozyme (Carl Roth, From chicken egg white, free from albumin) was dissolved in 8 M urea supplemented with 15 mM Tris (pH 9.5) to a final concentration of 20 mg/ml and left to denature at 95° C. for 5 minutes. 200 μl denatured lysozyme solution was incubated for 30 minutes at 37° C. with 20 mM dithiothreitol (DTT), to reduce the cysteine residues. Iodoacetamide (IAA) was added to the mixture, to react with reduced cysteines, with a final concentration of 45 mM and incubated in the dark for 30 minutes at room temperature. The mixture was diluted 5× with 100 mM Tris (pH 8.5) and trypsin (Alfa Aesar™ Trypsin, bovine pancreas) was added in a ratio of 1:50 (trypsin:protein). The mixture was left to digest overnight (˜18 hours) at 37° C. In order to denature and deactivate any remaining trypsin, the next day, the final mix was denatured at 95° C. for 10 minutes and HCl was added to lower the pH (approximately pH 4). The mixture was then frozen at −20° C. until use.
Planar lipid bilayer electrophysiological recordings. The electrophysiology chamber consisted of two compartments separated by a 25 μm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained an aperture with a diameter of approximately 100-200 μm. Lipid membranes were formed by first applying 5 μl of 5% hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane, near the aperture. The pentane was left to dry and 400 μl of buffer (1 M KCl, 50 mM citric acid, titrated with bis-tris propane to pH 3.8) was added to both sides. 20 μl of a 6.25 mg/ml solution of DPhPC dissolved in pentane was added on top of the buffer on each side of the chamber. The chamber was left to dry for −2 minutes to allow evaporation of pentane. Silver/silver chloride electrodes were attached to each compartment. The cis compartment was connected to the ground electrode and the trans was connected to the working electrode. Planar lipid bilayers were created using the Langmuir-Blodgett method described by Maglia et al.7. The orientation of FraC nanopores was determined by the asymmetry of the current-voltage relationship. A baseline of 2 minutes was recorded for each of the pores recorded. Analytes were added to the cis compartment of the chamber.
Data recording. Recordings of ionic currents were obtained using an Axopatch 200B (Axon Instruments) combined with a Digidata 1550B A/D converter (Axon instruments), similar to preceding work1,2. The sampling frequency was set at 50 kHz for analyte recordings, the analogue Bessel filter was set at 10 kHz. Data was recorded using Clampex 10 (Molecular Devices).
A number of well-known means of analysing the stepwise current blockades measured from nanopore electrophysiology are known in the art, and various methods can be employed on the events types we observe to extract useful data, which include but are not limited to blockade magnitude, blockade duration, blockade shape, blockade noise, other sub-features of the blockades (such as ministeps, etc).
For basic data analysis a custom Python script was employed to analyse the raw electrical data. The open pore current (Io) and standard deviation of all traces was determined by calculating the mean current of 3 independent measurements, bootstrapped for 100 iterations of 10 second snippets for each measurement. For event detection, the baseline current and standard error of the recorded traces were determined from a full current histogram of the blank nanopore measurement containing no analytes. The value for the baseline was then used to determine the events when analyte was added. All data points above the baseline current and standard error that are separated by at least two times the sampling periods are detected as events. The excluded current (Iex%) of each event was calculated as the difference between the open-pore current Jo and the blockade current Ib, over the open-pore current Io (Iex %=[Io−Ib]/Io).
An impartial event detector method was employed to improve analyses. We found that short lived events—with a dwell time near the sampling frequency-tend to form a spike or Gaussian profile due to under sampling and filtering effects, while longer events follow a flat-top shape. Therefore, we introduced a parameter describing the shape of current blockades in order to impartially compare the performance of mutant pores. We assume that the profile of ionic current blockades can be described by a generalized flat-top normal distribution function (gNDF, Equation 3). Each observed block was fit to equation 1 using least-squares fitting, due to the non-polynomial nature of the function.
Where μ is the events centre in the time domain with variance σ2 and ΔIB is the current difference (pA) between the baseline (IO) and the event maximum. The variable β describes the shape of the function and can take any real number larger than zero. If β is less than one but larger than zero, the shape of the function is a spike. If β is equal to one, the function is equal to the normal distribution function. When f is larger than one, the function starts to follow a rectangular-flat-top-profile. Advantageously, the variable β can also be used to assess the quality of individual events in the following way. Events with a β<1 are mostly events that are too short-lived to accurately measure the ionic current blockade. Therefore, only those events with a β≥1 should be regarded as more accurate measurements of peptides. Similarly, we distinguish events with β≥10, since these events-having a flat-top shape-permit an more accurate estimation of the blocked current. The gNDF fit also permits an estimation of the dwell time of an event by taking the full width at half maximum (FWHM) of the gNDF (Equation 4). Estimation of the dwell time using this equation is advantageous, because it allows the treatment of this parameters as continuous rather than discrete, which is the case if the number of data points are counted within the event.
Where σ equals the square root of the variance (σ2, Equation 3) and β describes the shape parameter.
Spectral matching. Several of the residual current spectra we obtain are expected to contain random events induced by factors other than the analyte (gating), so in order to reduce baseline sloping and to maintain high sensitivity, we utilize the squared first derivative Euclidean cosine correlation (Equation 5)9. This comparison is sensitive to the position of the peaks observed in the spectra, but not as sensitive to a shifting baseline.
Where A1 and A2 equal the vectors of excluded current counts and A1,i and A2,i represent the individual bins of the excluded current spectrum9. In a more detailed description, we set A1 and A2 as the vector of counts we observe for each residual current bin (e.g. An=counts (40-41%), counts (41-42%), . . . , counts (94-95%)). ΔAn is the derivative of An (difference between bins). In the numerator, we multiply each element ΔAn with the corresponding ΔAn of the comparing spectrum and take the squared sum of all items. In the denominator, we take the squared sum of each element in ΔAn and multiply that with the squared sum of each element in the spectrum we want to compare. So, if the two vectors A1 and A2 are equal, the correlation is 1, else it is less than 1, and because the derivative of A1 and A2 is taken, linear baseline sloping is less impactful.
We performed hierarchal clustering using the Ward distance as implemented in SciPy version 1.4.1. on the resulting correlation coefficients to determine which spectra are most similar10. In essence, this metric orders the data in such a way that the variance between neighbours is minimal, therefore building a map of similar spectra.
Mutations of FraC:
The sequence of WtFraC from the sea anemone Actinia fragacea was aligned with other actinoporins (
At position D10, mutations to arginine (R) and Glycine (G) were introduced to test changes in electro-osmotic capture of analytes. Each of the positions near the recognition site (G13), was modified to a basic residue (K, R or H) or acidic residue (D or E) as well as amino acids with neutral (G or Q) or aromatic (W,Y and F) groups. In FraC a glycine residue is positioned at residue 15—while the most common amino acid in other actinoporins is a threonine. Mutation G15T was introduced to test whether increased hydrophobic mutations facing outwards into the membrane would stabilize and improve the behavior of FraC pores.
Sequence alignment (
WtFraC can exist in three oligomeric forms, that are predicted to correspond to octamers, heptamers and hexamers. We tested octameric pores (or type I pores, T1) and heptameric pores (or type II pores, T2), and hexameric pores (or type III pores, T3). Octameric oligomers were identified as the nanopores with the highest conductance. Several mutations significantly reduced the open pore current (I0) relative to WtFraC-T1 (95±1 pA), some to an extent that the I0 resembled WtFraC-T2 (47±3 pA). Notably, decreased I0 were observed when residues with a larger volume were introduced, particularly for the aromatic residues (W/F/Y) introduced on position 13 (I0=64±8 pA, 77±4 pA and 82±3 pA, respectively), suggesting that smaller recognition region can be achieved, which can be advantageous for detecting smaller analytes such as small peptides. The introduction of a threonine residue on position 15 increased the open-pore current I0 flowing through the pore (100±3 pA), which is a useful property in nanopore analysis as the increased current is generally more sensitive to changes due to analyte binding.
In order to ensure a fair comparison between pores, a mixture of peptides was generated from the non-specific tryptic digestion of lysozyme (Gallus-Gallus). We used trypsin or other proteases such as chymotrypsin or Lys-C protease. The use of trypsin might be advantageous because it cleaves preferentially after a K/R amino acid and as most peptides will have a positive charge next to the zwitterionic charges on the peptide, yielding a net charge of +1 under the low pH conditions employed. All pores were tested with the same proteolytic mixture.
Events arising from nanopore current blockades were analysed with a flat-top shape fitted using a least-squared Levenberg-Marquardt method and a generalized flat-top normal distribution function. The fit results in a p value that can classify the events as either a spike with β<1, a normal distribution β=1 or flat-top distribution β>1. All events with β>1 were used in subsequent analyses. For each blockade a number of characteristic metrics are extracted. These include the excluded current (Iex%), which is the percentage of the current that is blocked during a translocation event relative to the open pore current (Iex%=[Io−Ib]/Io)), the duration (termed dwell time) of the blockade, the shape of the blockade, the noise in the blockade current etc.
Peptide capture and discrimination in FraC nanopores was studied under a wide range of conditions. Peptide capture was observed over a wide range of voltages, for example from lower voltages of +−10 mV through to +−200 mV. The majority of sensing was carried out at +−50 mV to +−100 mV as this was generally found to be optimal for peptide capture and characterization. Peptide detection can be observed over a wide range of salt types, concentrations and asymmetries across the membrane, all of which in combination with the pore type can alter the capture and detection properties of the system. Preferred salt conditions are about 1 M KCl (or NaCl or LiCl) at pH≤4.5 (eg. pH 3.8). Results:
Wild Type FraC-T1 and wild type FraC-T2 captured peptides at a frequency of about 10-13 events·s−1 under pH 3.8 conditions. When the charge at position 10 or 17 was removed (D10G-FraC-T1 or D17Q-FraC-T1 mutation), the capture frequency was reduced by about 3.4 times and about 7.2 times relative to WtFraC-T. It has been shown that the electro osmotic flow (EOF) is a critical component for efficient capturing of peptides in the nanopore, and can act with or against to electrophoretic forces acting on analytes. It has also been shown that the strength and direction of the EOF is dependent on charges in the constriction site (Table 4). Under low pH, which partially protonates water facing residues and generally increasing the net positive charge inside the pore (increasing anion selectivity), we found that the native negative residues in wild type FraC result in almost zero net ion selectivity (Table 4) and thus almost zero net electro-osmotic flux across the nanopore (versus very high cation selectivity at pH 7.5). Removing the negative charge at position 10 further increases the anion selectivity at low pH, creating a stronger EOF component acting against the capture of mostly positively charged peptides, hence resulting in lower capture efficiency. Furthermore, pores with a positively charge constriction, such as D10R-FraC-T1, showed a destabilized baseline current under an applied bias of −50 mV, but stable under +50 mV, thereby behaving opposite to WtFraC. However, D10R mutations exhibited good capture of peptide analytes in the cis chamber at positive applied voltage (exhibiting similar capture to that of native D10 in WT under negative voltage). The increased capture under this polarity is the result of a strong net anion-selective electro-osmotic bias (flowing from cis to trans) that is created by the positive mutation, which is dominant versus the weaker electrophoretic force acting against peptide capture at this polarity.
Removing the charge on residue K20 by substitution to glutamine increases the capture frequency by 1.4 times relative to wild type Frac. Replacing the charge of K20 by introducing an aspartic acid reduced the capture frequency by 1.5 times relative to wild type FraC. These relatively small changes illustrate how the EOF can be fine-tuned to control the capture frequency and/or the event residence (dwell) time.
Interestingly, we find that the introduction an aromatic residue (Y, F or W) increases the capture frequency by about 4 times relative to the wild-type FraC-T1 and FraC-T2 pores for all three mutations.
Furthermore, we find that the aromatic mutations also increase the duration of the peptide event blockades in the nanopores. Most of the blockades in pores with an aromatic residue on G13, were flat-top shaped with relatively long dwell times (e.g.
In order to compare the different mutants, we constructed the excluded current spectrum (shown for 4 pores in
We selected five mutants for further characterization, namely: G15T-FraC-T1, as it is comparable to WtFraC-T1 with a slightly increased I0, K20D-FraC-T1 as it had one of the higher SNRs and good capture frequency and the aromatic mutations of at G13 (G13Y/F/W-FraC-T1) for their increased dwell times compared to WtFraC-T2 and capture frequency. For the characterization of these pores we used a mixture of well-defined peptides (i.e. the mixture was made by adding the individual peptides at equimolar concentrations). The mixture consisted of four peptides: Angiotensinogen (DRVYIIIPFHLVIHN (SEQ ID NO: 48), 1758.9 Da, charge=+3.96), Angiotensin 1 (DRVYIHIPFHL (SEQ ID NO: 49), 1296.5 Da, charge=+2.96), Angiotensin 3 (RVYIHPF (SEQ ID NO: 50), 931.1 Da, charge=+2.16) and Angiotensin 4 (VYIIIPF (SEQ ID NO: 51), 774.9 Da, charge=+1.16) abbreviated as Angiotensinogen, Ang-I, Ang-III and Ang-IV respectively. The resolution of the nanopores was quantified by measuring the separation between peptides using the difference between the peak centers and their mean standard deviation as shown in Equation 1 and 2.
Where Rs is resolution, μ1 and μ2 are the peak centers with standard deviation σ1 and σ2 respectively. If Rs<2, the difference between the peak centers is less than twice the average standard deviation. Therefore, no baseline separation is achieved. To achieve an overlap of less than 5%, a Rs≥4 is required, that is, the difference between the peak centers is equal or bigger than twice the average standard deviation of the peaks, thus we can consider them separated. Larger values of R indicate a better separation (Table 2).
8.8 ± 0.7%
9.2 ± 0.3%
9.1 ± 0.7%
5.0 ± 0.3%
9.3 ± 0.3%
6.1 ± 0.8%
6.4 ± 0.2%
We tested the resolution of aromatic heptameric (T2) nanopores, and compared to hexameric (T3) WtFraC-T3 and WtFraC-T2 nanopores using Leucine-enkephalin (Leu-enk, YGGFL (SEQ ID NO: 36), 555.6 Da), Angiotensin II (4-8) {Ang-II(4-8), YIHIPF (SEQ ID NO: 37), 675.8 Da}, and Kemptide (LRRASLG (SEQ ID NO: 38), 771.9 Da). For WtFraC-T3 we use a FraC version with two altered membrane-interfacing modifications, W112S-W116S, which allowed forming hexameric nanopores. WtFraC-T2 showed no blockades (
Nanopores are nanometer sized apertures in thin membranes that detect analytes moving through the aperture. An exemplary analytical system of the invention is schematically depicted in
This example demonstrates that an engineered sub-nanometer biological nanopore of a mutant Fragaceatoxin C (FraC) is able to identify multiple trypsin digested proteins. By calibration through several synthetic peptides, a relation between the residual current spectrum and mass-spectrum could be found, thus allowing for protein identification.
Protein digestion. 100 μg of protein stock was taken and the volume was adjusted to 50 μl using 20 mM Tris buffer (pH 7.5). A final concentration of 20 mM dithiothreitol (DTT) was added to reduce any disulphide bonds. The sample was incubated at 37° C. for 15 minutes followed by a denaturing step at 95° C. for 15 minutes. Afterwards, a 20 mM iodoacetamide (IAA) was added and the sample was left to incubate for 15 minutes at room temperature in the dark in order to alkylate the reduced cysteine residues. Finally, the total volume was adjusted to 100 μl using 100 mM Tris Buffer (pH 8.5).
For the tryptic digestion we used a kit purchased from Sigma-Aldrich, containing proteomics grade trypsin. 50 μl of sample (containing 50 μg of protein) was added to 1 μg of mass-spec grade trypsin (1:50 enzyme:protein ratio) and the sample was subsequently incubated overnight at 37° C. Finally, large (>2000 Da) peptides were removed using centrifugal filters with a molecular weight cut-off of 3000 Da (Amicon). Filtered samples were stored in −20° C. prior to use.
Trypsin is a sequence dependent protease, and cuts mainly at the carboxyl side chain of arginine (R) and lysine (K) residues unless they are followed by proline (P). Trypsination of a given protein therefore results in a peptide mixture containing a specific set of peptide fragments from specific cutting, combined with some level of other peptide fragments resulting from incomplete digestion or off-target cutting.
Expression of proteins for tryptic digestion. Five model proteins: DHFR (dihydrofolate reductase), BSA (Bovine serum albumin, Sigma-Aldrich), PAN (PAN unfoldase), ThpA (Thiamine binding protein) and HMWI_Act (C-terminal fragment of Haemophilus influenzae high-molecular weight adhesin protein, residues 1205-1536) were expressed and/or purified for the purpose of testing the nanopore sensors.
Protein expression of DHFR PAN ThpA/HMWI_Act: All proteins were expressed via similar protocols. Briefly, plasmid containing the gene of interest, was electrochemically transformed into BL21(DE3) competent Escherichia coli cells. The cells were grown overnight at 37° C. on LB agar plates supplemented with 100 mg/L ampicillin and 1% glucose. On the next day, grown LB plates were solubilized into 200 mL 2×YT medium, supplemented with 100 mg/L ampicillin. Cultures were grown under constant shaking at 37° C. until an optical density (OD600) of 0.6 was reached. Afterwards, 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added for induction and growth continued overnight at 21° C. Bacterial cells were pelleted using centrifugation and stored for at least one hour at −80° C.
Protein Purification of DHFR/PAN/ThpA/HMWI_Act: Cell pellets were processed by first resuspending in lysis buffer and lysing by sonication (Branson Sonifier 450) in the presence of a protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation and supernatant was processed via Ni-affinity chromatography columns to recover the purified protein fractions. For PAN an additional purification was performed, purifying the protein via anion exchange using HiTrap Q HP anion exchange columns (GE Healthcare Life Sciences). Purity was confirmed by SDS-PAGE and the fractions with highest protein concentration were combined and concentrated using a 10 kDa MWCO spin filter (Amicon). For HMW1Act, fractions containing protein of interest were collected and dialyzed using SnakeSkin dialysis system (MWCO 10 kDa, Thermo Fischer Scientific) against storage buffer (50 mM HEPES, 100 mM NaCl, 10% glycerol, pH 7.5). After dialysis protein was aliquoted and stored at −80° C. until further use.
Protein purification of BSA: BSA was purchased from Acros Organics. The purity of BSA was increased using anion exchange chromatography (Äkta pure) by processing 10 mg BSA (in 1 ml 50 mM Tris, pH 7.5) on a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences). Eluted protein fractions were assessed by SDS-PAGE and the fractions with highest protein concentration were combined and concentrated using a 10 kDa MWCO spin filter (Amicon).
Detection of a model protein digest. The detection and identification of proteins using, (standard) mass spectrometry based, techniques relies heavily on the fingerprinting of (tryptic) peptides. To mimic a properly digested protein, we employed a model peptide system containing 7 synthetic peptides with a mass between 700 and 1700 Da (Sigma Aldrich and Genscript) that would be predicted to result from complete trypsination of lysozyme, i.e. the protein is cleaved in-silico at all arginine (R) and lysine (K) residues unless they are followed by proline (P).
The 7 model peptides were individually added to separate nanopore experiments (G13F-FraC-T1 pores, 1M KCl, pH 3.8, −50 mV), generating a unique cluster of events when plotted by excluded current and dwell time. For each single experiment the average excluded current for the event blockades was calculated by fitting a gaussian to histograms of the clustered events. The average excluded current for each peptide type was calculated by averaging across n>3 experiments performed on each peptide. A strong correlation between the molecular weight of the peptides and their respective average excluded current blockade was observed (
Where a is the offset, k represent the width and μ is the inflection point.
Detection of digested Lysozyme protein and comparison with Mass Spectrometry. Lysozyme protein was digested via trypsination as described above. The resulting peptide fragment mixture was then analyzed both using nanopore sensing (G13F-FraC-T1 pores, 1M KCl, pH 3.8, −50 mV) and with Mass Spectrometry (LC ESI-MS). A histogram of the excluded current blockades measured from the mixture using the nanopores is plotted in
A further 9 proteins with highly divergent compositions were tested by nanopore spectrometry. The 9 proteins were: Bovine serum albumin (BSA), dihydrofolate reductase (DHFR), high molecular weight adhesin 1 (HMIW1Act), PAN unfoldase, Thiamine binding protein (TbpA), beta casein, cytochrome C, lysozyme and trypsin. The proteins were digested via trypsination as described. The resulting peptide fragment mixtures were separately tested in multiple separate nanopore experiments (G13F-FraC-T1 pores, 1M KCl, pH 3.8, −50 mV). Similar to what was observed for the digested lysozyme peptide mixture, distinct clusters of blockade events were observed from the peptide mixtures for each of the digested proteins (
To account for pore to pore variations in the baseline current, we aligned the residual current spectra to a reference spectrum using a sliding window on Iex %.
Protein fingerprinting and spectral matching. The unique excluded current spectra of the tryptic digests (
The uniqueness and reproducibility of the spectra were determined using spectral correlation, utilizing the squared first derivate Euclidean cosine correlation (DEuc) (Equation 2).
With A1 and A2 containing the vectors of the excluded current spectra and each element (1) in the vector9.
In order to ensure a representative example for spectral matching, we performed a leave-one-out comparison, where the comparison database was built from all spectra, excluding the one that was matched. The probability P(X) % was calculated from the DEuc score relative to the sum of all the DEuc scores (
To evaluate the resolution of the analytical detection system to discriminate between peptides that differ by only 4 Dalton, two different forms of the enkephalin peptide were tested: YGGFL (SEQ ID NO: 36), and YdAGFdL (SEQ ID NO: 100), wherein d represents a D-amino acid; all other amino acids being in the L-configuration.
This example demonstrates that a mutant proteinaceous nanopore is capable of detecting post-translationally modified peptides. An analytical system comprising a FraC-G13F nanopore as described herein above was used to distinguish between a phosphorylated and non-phosphorylated peptide (see
Examples 1 to 6 relate to a mutant proteinaceous nanopore comprising an alpha-helical pore-forming toxin of the actinoporin family, and its application as single molecule sensor. To test whether these discoveries were more broadly applicable to different classes of nanopores, with similar dimensions in the sensing region but quite different structural makeup, we explored similar mutations and conditions on beta-barrel pores.
This example demonstrates that beta-barrel pore-forming proteins wherein the lumen-facing recognition region of the proteins comprises one or more mutations to an aromatic residue can also be used to provide such nanopore-based sensors, particularly in combination with nearby acidic mutations.
It was found that lowering the pH of the buffer could increase the capture (
We found that replacing the Lysine at position 238 with a phenylalanine (Aer-K238F,
We further combined the K238D mutation with the introduction of the phenylalanine at either position Ala260, Ser264, Gln268 or S272 of Aerolysin, and observed a dramatic improvement in peptide resolution (
Plasmid containing a gene encoding for pro-aerolysin elongated by a hexa-histidine tag (SEQ ID NO: 101) at the C-terminus was transformed into BL21(DE3) cells using electroporation. The transformed cells were grown overnight at 37° C. on LB agar plates supplemented with 1% glucose and 100 μg/ml ampicillin. On the next day, the colonies are resuspended and grown in 200 mL 2YT medium at 37° C. until the OD600 reached 0.6-0.8. At this point, the expression was induced by addition of 0.5 mM IPTG and the culture was incubated overnight at 25° C. Afterwards, the cells were pelleted by centrifugation at 4000 rpm for 15 minutes and the cell pellets were stored at −80° C. for at least 30 minutes. For protein purification, cell pellets of 100 ml culture were resuspended in 20 ml lysis buffer, containing 150 mM NaCl, 20 mM imidazole and 15 mM Tris buffered to pH 7.5, supplemented with 1 mM MgCl2, 0.2 units/ml DNase1 and approximately 1 mg of lysozyme. The mixture is incubated for 30 minutes at RT and afterwards sonicated using a Branson Sonifier 450 (2 minutes, duty cycle 30%, output control 3) to ensure full disruption of the cells. Cell debris is pelleted by centrifugation at 6000 rpm for 20 minutes and the supernatant is carefully transferred to a fresh falcon tube. Meanwhile, 200 μl of Ni-NTA bead solution is washed with wash buffer, containing 150 mM NaCl, 20 mM imidazole and 15 mM Tris buffered to pH 7.5. The beads are added to the supernatant and incubated at RT for 5 minutes. Afterwards, the solution is loaded on a Micro Bio-Spin column (Bio-Rad) and subsequently washed with 5 ml of wash buffer. The bound protein is eluted in fractions of 200 μl of elution buffer (150 mM NaCl, 300 mM imidazole, 15 mM Tris buffered at pH 7.5. The pro-aerolysin fractions can be stored in at 4° C. for several weeks.
Oligomerisation from Pro-Aerolysin Using Trypsin
Pro-aerolysin is incubated with trypsin in a 1:1000 mass ratio for 15 minutes at room temperature. The trypsin cleaves off the C-terminal peptide, resulting in aerolysin monomers that can assemble into heptameric pores, which pores can be characterised in electrophysiology experiments.
The electrophysiology chamber consisted of two compartments separated by a 25 μm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained an aperture with a diameter of approximately 100-200 μm. Lipid membranes were formed by first applying 5 μl of 5% hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane, near the aperture. The pentane was left to dry and 400 μl of run buffer (1 M KCl, 50 mM citric acid, titrated with bis-tris propane to pH 3.8 or pH 3.0; or 1 M KCl, 50 mM Tris buffered at pH 7.5) was added to both sides. 20 μl of a 10 mg/ml solution of DPhPC dissolved in pentane was added on top of the buffer on each side of the chamber. The chamber was left to dry for −2 minutes to allow evaporation of pentane. Silver/silver chloride electrodes were attached to each compartment. The cis compartment was connected to the ground electrode and the trans was connected to the working electrode. Planar lipid bilayers were created using the Langmuir-Blodgett method described by Maglia et al (Huang et al. Nat. Commun. 2017).
100 μg of lysozyme (Carl Roth, from chicken egg white, albumin free) was dissolved in 150 mM NaCl, 15 mM Tris buffered at pH 7.5. Before digestion, free cysteines were alkylated to prevent formation of disulfide bridges after digestion. To that end, 3 μL 200 mM DTT was added and the sample was incubated at 37° C. for 15 min, followed by 15 minutes of denaturation at 95° C. Subsequently, 7 μL of 200 mM IAA was added and the sample was incubated for 15 min at RT in the dark. After alkylation, the lysozyme was digested overnight at 37° C. in a 50:1 lysozyme:trypsin mass ratio using the Trypsin Singles, Proteomics Grade-kit (Sigma Aldrich, Catalog #T7575-1KT).
Aerolysin was added to the cis-chamber and the bilayer was broken and reformed until a single channel inserted into the bilayer. The orientation of the pore can be detected by a small asymmetry in the IV curve of the pore. First, a 2 minute blank was recorded at +150 mV applied potential and afterwards 4 μl of trypsin-digested lysozyme was added to the cis compartment of the chamber. The analyte was measured for at least 10 minutes at an applied potential of +150 mV.
Data recording. Recordings of ionic currents were obtained using an Axopatch 200B (Axon Instruments) combined with a Digidata 1550B A/D converter (Axon instruments), similar to preceding work (Huang et al. Nat. Commun. 2019). The sampling frequency was set at 50 kHz for analyte recordings, the analogue Bessel filter was set at 10 kHz. Data was recorded using Clampex 10 (Molecular Devices).
Example 7 relates to single molecule analysis using a modified beta-barrel pore-forming protein Aerolysin. In this example, functionally similar mutations were introduced into the cytolysin k (cytK) nanopore to demonstrate that aromatic mutations, preferably in combination with nearby acidic mutations, preferably when used under low pH conditions (<pH 4), improve the ability to capture and resolve unlabelled peptides for other beta-barrel pores.
Although CytK is known to be a nanopore capable of passing current when inserted into a membrane (Hardy et al, FEMS Microbiol Lett. 2001), the structure of CytK is not known. Therefore, to identify the beta-barrel region, and the putative analyte recognition region, a homology model was built by mapping the CytK sequence to the sequence and structure of the alpha-hemolysin nanopore from Staphylococcus aureus (
Plasmid containing a gene encoding for CytK elongated by six histidine residues (SEQ ID NO: 101) at the C-terminus was transformed into BL21(DE3) cells by electroporation. Transformed cells were grown overnight at 37° C. on LB agar plates (1% glucose, 100 μg/ml ampicillin). Colonies were resuspended and grown in 200 mL 2YT medium at 37° C. until OD600 0.6-0.8, then expression was induced by addition of 0.5 mM IPTG and the culture was incubated overnight at 25° C. Cells were pelleted by centrifugation and stored at −80° C. for at least 30 minutes. Cell pellets were lysed by resuspension in lysis buffer (150 mM NaCl, 20 mM imidazole, 15 mM Tris pH 7.5, 1 mM MgCl2, 0.2 units/ml DNase1, ˜1 mg of lysozyme), incubated for 30 minutes at RT, then sonicated (Branson Sonifier 450, 2 minutes). Cellular debris was pelleted by centrifugation and the supernatant containing CytK was recovered. CytK was extracted from the supernatant and purified using Ni-NTA beads, with final elution in 200 μl aliquots (150 mM NaCl, 300 mM imidazole, 15 mM Tris buffered at pH 7.5) before storage at 4° C.
Electrophysiology measurements were performed as described in Example 7. CytK was added to the cis-chamber and the DPhPC bilayer in the nanopore system was broken and reformed until a single nanopore inserted into the bilayer. The orientation of the pore can be detected by the asymmetry in the IV curve of the pore. All recordings were performed with 1 M KCl in both the cis and trans compartments at either pH 3.8 (50 mM citric acid, titrated with bis-tris propane to pH 3.8) or pH 7.5 (50 mM Tris buffered at pH 7.5). First, 2 minutes of blank open-pore current was recorded at +100 mV applied potential, and afterwards 4 μl of trypsin-digested lysozyme was added to either this cis or trans compartment of the chamber. The analyte was measured for at least 10 minutes at an applied potentials of −100 mV to +100 mV as indicated. The ionic current was recorded using a Digidata 1440A (Molecular Devices) connected to an Axopatch 200B amplifier (Molecular Devices). The sampling frequency was set at 50 kHz for analyte recordings, the analogue Bessel filter was set at 10 kHz. Data was recorded using Clampex 10 (Molecular Devices). Event blockade data was analysed as described herein, measuring the event blockades resulting from peptide capture and extracting metrics including average open-pore current, average blockade current, blockade duration (dwell time), standard deviation of blockade current, etc.
Similar to Example 7, nanopore sensing systems containing CytK nanopores were tested using a digested peptide mixture resulting from trypsinated lysozyme. Wild Type CytK exhibits little to no capture of the peptides from a trypsinated lysozyme sample, including when the sample is added to either the cis or trans compartments, under either positive or negative applied potentials over a wide range of voltages, at either pH 7.5 or pH 3.8. For example,
According to our predicted structure, a Lysine residue at position 128 and a Glutamate residue at position 139 are predicted to be inward facing residues in the recognition region. In accordance with previous findings described herein, a phenylalanine was substituted into the K128 position of the CytK monomers adjacent to the acidic E139, thus serving both to reduce the net positive charge in the nanopore and introduce an aromatic for improved peptide detection. The K128F mutation produced a dramatic improvement in the ability to both capture (
In another implementation, similar to the strategy employed in Example 7, an aromatic amino acid was introduced adjacent to an additional negative mutation by substituting the lysine at 238 with an aspartic acid and substituting the serine at 126 with a phenylalanine (CytK-S126F-K128D). Similar to what was observed for the Aerolysin nanopore system, this combination of an aromatic amino acid substitution adjacent to an acidic amino acid substitution further improved the resolution of different peptides through a combination of improved metrics, including: better capture (
Aromatic mutations placed higher up in the barrel of aerolysin (position S120, Q122 or G124) combined with K128D also yielded a good resolution of peptides of a trypsinated lysozyme sample. See
Accordingly, the data demonstrates that aromatic mutations, preferably adjacent to acidic amino-acid substitutions, creates a sensing region that improves the ability to capture and discriminate unlabeled peptides, in particular at low pH conditions.
Notably, in example 7 and 8 we have demonstrated two different dominant mechanisms for controlling peptide capture in CytK and aerolysin nanopores. For example, we demonstrated that Aerolysin nanopores can capture and discriminate peptides effectively at positive applied potential when analytes are in the cis compartment. Therefore, the analytes, being mostly positively charged at pH 3.8 or pH 3.0, are captured against the electrophoretic direction due to dominant electro-osmotic capture conditions. In contrast, we demonstrated that CytK can capture and discriminate peptides effectively at positive applied potential when analytes are in the trans compartment. Therefore, the analytes are captured primarily by electrophoretic forces under the pH 3.8 conditions, and the electro-osmotic component was tuned to be close to zero by substitution of additional acidic residues (see Table 4). Our results indicate that the introduction of aromatic residues in beta-barrel pore-forming toxins works regardless of the capture mechanism of the analyte, and that the introduction of acidic residues under low pH conditions is an important tool for tuning and controlling cation selectivity and electro-osmotic capture.
This example shows that Lysenin, a further exemplary beta-barrel pore forming protein, is successfully mutated to demonstrate that an aromatic mutation of a non-aromatic lumen facing residue improves the ability to capture and resolve unlabelled peptides.
Plasmids containing the Lysenin gene from Eiseniafetida were transformed into BL21(DE3) E. coli competent cell by electroporation. Next, the cells were grown on lysogeny broth (LB) agar plate containing 100 μL/mL ampicillin overnight at 37° C. The LB plate was harvested and inoculated into 400 mL 2×YT media. Then, the culture was grown at 37° C. while shaking at 200 rpm until the optical density at 600 nm of the cell culture reached 0.8. This was followed by addition of 0.5 mM isopropyl-D-thiogalactoside (IPTG) to the media and the culture was grown overnight at 25° C. while shaking at 200 rpm. The next day, cells were harvested by centrifugation (4000 rpm, 15 min) and the resulting pellets were frozen at −80° C. for 30 min.
The cells were resuspended and mixed for 30 min in 40 mL of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.02% DDM supplemented with, 10 mM imidazole, 1 mM MgCl2) together with 0.2 mg/mL lysozyme, and 10 μL DNaseI. The lysate was sonicated for 2 min (40% output power) and centrifuged down at 4° C. for 15 min (4000 rpm). Next, the supernatant was incubated with 150 μL washed Ni-NTA beads for 15 min at 20 rpm. The Ni-NTA beads were loaded on a gravity-flow column and washed with wash* buffer: ([50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM imidazole, and 0.02% DDM)]. The proteins were eluted in 3 elution steps with 150 μL elution buffer:* ([50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 300 mM imidazole and 0.02% DDM)]. Lysenin monomers were stored at 4° C.
Lysenin can be oligomerized by incubation with liposomes (with a 1:1 sphingomyelin:DPHPC lipid composition) in a 1:10 protein:liposome ratio at 37° C. for 1 hour. The liposomes are then disrupted by addition of 0.6% LDAO. The solution is diluted 20× using wash buffer and mixed with 150 μl washed Ni-NTA beads. The solution is subsequently loaded on a gravity-flow column and washed with wash buffer. Oligomers are eluted by an elution buffer containing 1M Imidazole, 150 mM NaCl and 15 mM Tris buffered to pH 7.5 in fractions of 150 μl. Oligomers were stored at 4° C.
Number | Date | Country | Kind |
---|---|---|---|
21174437.0 | May 2021 | EP | regional |
This application is a continuation of International Patent Application No. PCT/NL2022/050266, filed May 18, 2022, which claims the benefit of European Application No. 21174437.0, filed May 18, 2021, each of which is entirely incorporated herein by reference.
Number | Date | Country | |
---|---|---|---|
Parent | PCT/NL2022/050266 | May 2022 | US |
Child | 18512856 | US |