Patent Application
20200399693
The present disclosure relates generally to nanopores and more specifically to systems and methods using nanopores assembled with non-membrane proteins for detecting analytes.
Biological nanopores are protein channels embedded in a substrate, typically lipid membranes. A wide variety of protein complexes in nature form elegant channel-like structures, some of which have been explored as nanopores, exemplified by α-hemolysin, MspA, aerolysin, FluA, Omp F/G, CsgG, ClyA, and PA63. For example, the biological protein nanopore Δ-hemolysin (ΔHL) from Staphylococcus aureus has been used for single molecule detection.
Recently, researchers have adopted biological, solid-state, DNA origami, and hybrid nanopores in single-molecule analyses. Biological nanopores have advantages compared to their synthetic counterparts, mostly because they can be reproducibly fabricated and modified with an atomic level precision that cannot yet be matched by artificial nanopores. The existing use of biological nanopores, however, also has drawbacks. For example, they are not versatile in providing different shapes, sizes, and hydrophilic/hydrophobic properties in order to detect different analytes with high sensitivity and specificity.
Accordingly, there remains a strong need for a robust nanopore system amendable for detecting analytes with distinct properties.
This disclosure addresses this need in the art by providing a nanopore assembly for detecting an analyte. The nanopore assembly comprises a channel formed of a plurality of subunits. Each of the subunits comprises a non-membrane protein capable of forming a protein channel In some embodiments, each of the subunits comprises a polypeptide having a polypeptide sequence at least 75% identical to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1-35. In some embodiments, the polypeptide comprises a polypeptide sequence at least 75% identical to SEQ ID NOs: 4-12.
In some embodiments, the polypeptide may include at least one residue substituted with cysteine. In some embodiments, the polypeptide is derived from phi29 portal or tail protein. In some embodiments, phi29 tail protein may include one or more of E595C, K321C, and K358C substitutions. In some embodiments, the phi29 tail protein may include one or more of K134I, D138L, D139L, D158L, E163V, E309V, D311V, K321V, K356A, K358A, D377A, D381V, N388L, R5241, R539A, and E595V substitutions.
In one aspect, the nanopore assembly further comprises a probe for detecting an analyte. The probe is operably linked to at least one of the subunits. The probe can be one of chemicals, carbohydrates, aptamers, nucleic acids, peptide, protein, antibodies, and receptors. In some embodiments, the probe comprises a sequence at least 75% identical to a sequence selected from the group consisting of SEQ ID NOs: 36-79. In some embodiments, the probe is an anti-PSA antibody. The probe may be operably linked via covalent bonding to at least one of the subunits. The covalent bonding includes a disulfide linkage, an ester linkage, or a sulfhydryl linkage. In some embodiments, the probe is operably linked to a location in proximity to an entrance of the channel or a location at an interior side of the channel
The analyte can be one of nucleic acids, amino acids, peptides, proteins, polymers, and chemical molecules. In some embodiments, the analyte is one of PSA, CEA, AFP, VCAM, MiR-155, MiR-22, MiR-7, MiR-92a, MiR-122, MiR-192, MiR-223, MiR-26a, MiR-27a, and MiR-802.
According to some embodiments of the nanopore assembly, the channel is embedded in a polymersome. In some embodiments, the channel is inserted in a membrane. The membrane may include a polymer membrane or a lipid membrane. The polymer membrane may include an alternating copolymer, a periodic copolymer, a block copolymer, a di-block copolymer, a tri-block copolymer, a terpolymer, or a combination thereof. In some embodiments, the polymer membrane comprises PMOXA-PDMS-PMOXA. In some embodiments, the nanopore assembly may further include cholesterol and/or porphyrin.
In another aspect, this disclosure also provides an apparatus for detecting an analyte. The apparatus includes the nanopore assembly described above and optionally a support for the nanopore assembly. The apparatus may further include an electrode, to which the nanopore assembly is tethered.
In another aspect, this disclosure further provides a kit that includes the nanopore assembly described above and optionally instructions for using the nanopore assembly.
In another aspect, the disclosure provides a method of detecting an analyte. The method includes: (1) contacting a sample containing an analyte with the nanopore assembly as described; (2) applying an electrical current across the channel of the nanopore assembly; (3) determining the electrical current passing through the channel at one or more time intervals; and (4) comparing the electrical current measured at one or more time intervals with a reference electrical current, wherein a change in electrical current relative to the reference electrical current indicates a presence of the analyte in the sample. The analyte may be any one of nucleic acids, amino acids, peptides, proteins, polymers, and chemical molecules. In some embodiments, the reference electrical current is measured with a sample that does not contain the analyte. In some embodiments, the nanopore assembly is placed on a support.
The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
This disclosure provides a robust nanopore system and method adaptable for detecting different analytes. It allows for detection of a single molecule and of a target molecule present with other contaminants. The system offers a label-free, amplification-free, real-time detection. It requires very low sample amount and can be used for high-throughput analysis. The system can be adapted to detect a variety of analytes (e.g., small molecules, polymers, polypeptides, and nucleotides) with different shapes, sizes, and hydrophilic/hydrophobic properties, with high sensitivity and specificity.
This disclosure addresses the need in the art by providing nanopores formed of non-membrane proteins, such as proteins derived from bacteriophages. To generate the disclosed nanopores, bacteriophage proteins were expressed, purified, self-assembled, and inserted in lipid or polymer membranes. Non-membrane protein channels, however, are more difficult to be inserted into lipid bilayer or polymer membrane directly. Unlike membrane protein channels, non-membrane protein channels generally lack hydrophobic layer in the middle and hydrophilic layers in both ends. This invention overcomes this limitation by employing a series of methods for inserting various protein channels either directly or through highly efficient fusion mechanism in polymer membranes. In addition, in some scenarios, protein engineering, such as site-directed mutagenesis, insertion, and deletion of amino acids, and introduction of functional modules are carried out to tune nanopore properties to meet different detection needs.
The disclosed nanopore system has a wide variety of applications, including but not limited to single molecule detection, DNA/RNA/peptide sequencing, sensing of chemicals, biological reagents, and polymers, and disease diagnosis. As will be further described below, the methods, kits, and detection devices employing the disclosed nanopore system are also within the scope of this disclosure.
One aspect of the present disclosure relates to nanopores formed of non-membrane proteins. The non-membrane proteins suitable for forming nanopores may be derived from cellular DNA translocases, helicases, terminase, ATPases, and fragments thereof. The non-membrane proteins suitable for forming nanopores may also include proteins involved in DNA repair, replication, recombination, chromosome segregation, DNA/RNA transportation, membrane sorting, cellular reorganization, cell division, bacterial binary fission, and other processes. The nanopore may include a plurality of subunits, each comprising a non-membrane protein. For example, the nanopore may include 10 to 15 subunits of phage portal proteins or 5 to 10 phage tail proteins. A non-membrane protein channel forming nanopores can be derived from bacteriophage portal proteins including, but not limited to T3, T4, T5, T7, SPP1, P22, P2, P3, Lambda, Mu, HK97 and C1. For example, a non-membrane protein channel forming nanopores can be derived from bacteriophage tail proteins including, but not limited to phi29, C1, Neisseria meningitidis serogroup B, T4, phiX174, lambda, SPP1, T5, Mu, F4-1, P2, Serratia phage KSP90, Enterobacteria phage T7M, Bacteriophage HK97.
Also within the scope of this disclosure are the variants and homologs with significant identity to bacteriophage proteins (e.g., bacteriophage portal or tail proteins) including, but not limited to T3, T4, T5, T7, SPP1, P22, P2, P3, Lambda, Mu, HK97, and C1. For example, such variants and homologs may have sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity over the sequences of the bacteriophage portal proteins described herein.
Biological pore mutagenesis can be used to optimize protein pores for assembly or use in a composition or method set forth herein. For example, protein engineering and mutagenesis techniques can be used to mutate biological pores and tailor their properties for specific applications. Accordingly, the nanopore assembly may include a channel formed of a plurality of subunits. Each of the subunits comprises a polypeptide having a polypeptide sequence having at least 75% identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1-35. In some embodiments, the polypeptide comprises a polypeptide sequence at least 75% identical to SEQ ID NOs: 4-12.
The subunits may include one or more substitutions, deletions or insertion to facilitate functionalization of nanopores or insertion in membranes. For example, the subunit may include at least one residue substituted with cysteine, such that a functional group can be linked to the subunit via a disulfide bond. For example, cysteine residues can be used for conjugating chemicals such as porphyrin to the nanopore. In some embodiments, the subunit may include phi29 gp9 protein having one or more of E595C, K321C, and K358C substitutions.
In addition, the subunit may include the phi29 gp9 protein having one or more of K134I, D138L, D139L, D158L, E163V, E309V, D311V, K321V, K356A, K358A, D377A, D381V, N388L, R524I, R539A, and E595V substitutions. Substitution of charged residues (e.g., K, R, D, E) with hydrophobic residues (e.g., A, V, L) increases the hydrophobicity of the protein locally and globally.
In some embodiments, the process of incorporating a protein channel in a membrane or a nanodisc can benefit from an affinity tag (e.g., polyhistidine affinity tags) on the protein and from the purification of the mixed nanopore population over an affinity column (e.g., a Ni column). Affinity tags and chemical conjugation techniques (e.g., modifications of cysteines set forth above) can be used to attach tethers to nanopores for use in a variety of methods, compositions or apparatus set forth herein. For example, the resulting tethers can be used to attract or attach a nanopore to a solid support or an electrode.
Neisseria
meningitidis
Escherichia phage
Escherichia phage
Escherichia phage
Escherichia phage
Lactococcus
Escherichia phage
Serratia phage
Enterobacteria
A polypeptide “variant,” as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the disclosure and evaluating one or more biological activities of the polypeptide as described herein and/or using any of some techniques well known in the art.
For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, accordingly, its underlying DNA coding sequence, whereby a protein with like properties is obtained. It is thus contemplated that various changes can be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.
Variant sequences include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of this disclosure Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. Such conservative modifications include amino acid substitutions, additions, and deletions. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
“Sequence identity” or “homology” refers to the percentage of residues in the polynucleotide or polypeptide sequence variant that are identical to the non-variant sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. In particular embodiments, polynucleotide and polypeptide variants have at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% polynucleotide or polypeptide homology with a polynucleotide or polypeptide described herein.
Polypeptide variant sequences may share 70% or more (i.e. 80%, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity with the sequences recited in this disclosure. Polypeptide variants may also include polypeptide fragments comprising various lengths of contiguous stretches of amino acid sequences disclosed herein. Polypeptide variant sequences include at least about 5, 10, 15, 20, 30, 40, 50, 75, 100, 150, or more contiguous peptides of one or more of the sequences disclosed herein as well as all intermediate lengths therebetween.
In one aspect, the nanopore assembly further comprises a probe for detecting an analyte. The probe is operably linked to at least one of the subunits. Such a probe is conjugated to the nanopore assembly to allow for selective binding of one or more analytes. A probe may be conjugated to the nanopore assembly at various stoichiometry (molar ratios between the probe and the nanopore assembly). In one example, the probe may be conjugated to each of the subunits. In another example, only one probe is conjugate the nanopore assembly. Also contemplated is a nanopore assembly including two or more different types of probes. Such functionalized nanopore assemblies can be used to detect analytes, such as nucleic acids, amino acids, peptides, proteins, polymers, and chemical molecules. In some embodiments, the analyte is one of PSA, CEA, AFP, VCAM, MiR-155, MiR-22, MiR-7, MiR-92a, MiR-122, MiR-192, MiR-223, MiR-26a, MiR-27a, MiR-802 or a fragment thereof.
The terms “specific binding,” “selective binding,” “selectively binds,” and “specifically binds,” refer to a probe on the nanopore assembly binds to an analyte in the sample more strongly than other contaminants present in the sample. For example, the probe may bind to an analyte with an equilibrium dissociation constant (Kd) of approximately less than 10−6 M, such as approximately less than 10−7 M, 10−8 M, 10−9 M or 10−10 M or even lower when determined by a binding assay, e.g., ELISA, equilibrium dialysis or surface plasmon resonance (SPR) technology in a BIACORE® 2000 surface plasmon resonance instrument.
The probe can be one of chemicals, carbohydrates, aptamers, nucleic acids, peptide, protein, antibodies, and receptors. In some embodiments, the probe may include a sequence at least 75% identical to a sequence of SEQ ID NOs: 36-79. In some embodiments, the probe is an anti-PSA antibody. The probe is operably linked via covalent bonding to at least one of the subunits. The probe can be linked to a subunit via one or more functional sites on the subunit. Such functional sites can be introduced by mutagenesis, for example, substitution with cysteine or other non-natural amino acids. The probe can also be linked to the channel via well-established chemical methods, including but not limited to ester linkage, click-chemistry, and sulfhydryl linkage. In some embodiments, the probe is operably linked to a location in proximity to an entrance of the channel or to a location at an interior side of the channel
In another aspect, this disclosure provides a nanopore assembly, in which the channel is inserted in the membrane. The polymer membrane can be of various compositions depending on the applications either in stand-alone form or in a microfluidic device. The polymer-membrane embedded channels display robust electrophysiological properties, a pre-requisite feature for single-molecule nanopore-based analysis. The membrane may include a polymer membrane (e.g., planar polymer membrane) or a lipid membrane. The polymer membrane can be either symmetric or asymmetric in nature. For example, the polymer membrane is an alternating copolymer (e.g., A-B-A-B- . . . ) or aperiodic copolymer (e.g., AA-BB-AA-BB . . . ). In another example, the polymer membrane can be a block copolymer comprising of two or more homopolymer subunits linked by covalent bonds. Alternatively, the polymer membrane can be a diblock or triblock copolymer (e.g., PMOXA-PDMS-PMOXA). The polymer membrane can also be a terpolymer consisting of three distinct monomers.
In some embodiments, the polymer membrane comprises an alternating copolymer, a periodic copolymer, a block copolymer, a di-block copolymer, a tri-block copolymer, a terpolymer, or a combination thereof. In some embodiments, the polymer membrane comprises PMOXA-PDMS-PMOXA.
In some embodiment, the channel is embedded in a polymersome of various sizes. In some embodiments, the polymersomes are fused with a planar polymer membrane to insert the channel Polymersomes are composed of hydrophilic-hydrophobic block copolymer, arranged in a bilayer vesicular system having a central aqueous core. They have a hydrophilic inner core and lipophilic bilayer. They differ from nanoparticles in that they contain a hydrophilic core rather than a lipophilic core, as in the case of nanoparticles. Although they have a bilayer structure, they offer more stability than liposomes due to the presence of a thick and rigid bilayer. They contain a hydrophilic core that provides a protein-affable environment.
In some embodiments, the channels are inserted into polymer membranes in the presence of any detergents of various composition (e.g., DDM, DOC, Tween, SDS, and Brij based detergents). In some embodiments, the protein channels are reconstituted into polymersomes in the presence of varying amounts of glycerol, CsCl, and/or sucrose for high-efficiency fusion.
In some embodiments, the protein channel is reengineered by mutagenesis (e.g., containing one to several substitutions) to facilitate insertion in (planar) polymer membranes. In addition, the protein channel is reengineered to introduce functional sites for site-specific labeling with chemicals or biopolymers for the purpose of inserting in polymer membranes. Such functional sites include cysteine residues for conjugating chemicals, such as porphyrin, or biological molecules, such as cholesterol or any hydrophobic lipid modules or nucleic acids of various lengths. The functional sites/groups may also include non-natural amino acids and linkage created by well-established chemical methods, including but not limited to ester, click chemistry, and sulfhydryl.
In some embodiments, the location of the conjugation is in the membrane anchoring layer of the channel, such as residues 70-80, 110-140, 155-245, 300-340, 410-435 of T4 gp 20 portal protein; residues 10-45, 250-300 of P22 gp1 portal protein; and residues 130-170, 300-325, 350-390, 530-595 of phi29 gp9 tail protein. Accordingly, to increase or decrease the hydrophobicity of the belt region for the purpose of membrane insertion, reengineering may include mutagenesis (e.g., substitution, insertion, deletion) of any one of the residues 70-80, 110-140, 155-245, 300-340, 410-435 of T4 gp 20 portal protein; residues 450-500, 350-380 of P22 gp1 portal protein; and residues 130-170, 300-325, 350-390, 530-595 of phi29 gp9 tail protein.
In some embodiments, the location of the conjugation is in the cis- and trans-hydrophilic layers of the channel, such as residues 465-515, 285-305 of T4 gp 20 portal protein; residues 450-500, 350-380 of P22 gp1 portal protein; and residues 20-50, and 250-300 of phi29 gp9 tail protein. Accordingly, to increase or decrease the hydrophobicity of the belt region for the purpose of membrane insertion, reengineering may include mutagenesis (e.g., substitution, insertion, deletion) may include any of the residues 465-515, 285-305 of T4 gp 20 portal protein; residues 450-500, 350-380 of P22 gp1 portal protein; and residues 20-50, and 250-300 of phi29 gp9 tail protein.
In another aspect, this disclosure also provides a kit and a detection apparatus for detecting an analyte. The kit may include the nanopore assembly as described, optionally a buffer, and optionally instructions for using the nanopore assembly.
The apparatus may include the nanopore assembly as described and a support for the nanopore assembly. The detection apparatus can further include electrodes embedded in the solid support. The electrodes can be used to monitor assembly of protein nanopores into membranes. The electrodes can also be used for data collection during analyte detection steps. Electrodes used for monitoring and detection need not be embedded in the support and can be provided, for example, in a separate application-specific integrated circuit (ASIC) chip.
As used herein, the term “support” refers to a rigid substrate that is insoluble in an aqueous liquid and incapable of passing a liquid absent an aperture. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers. Particularly useful supports comprise modified silicon such as SiN membranes on a Si substrate. For some embodiments, supports are located within a flow cell apparatus or other vessels.
In some embodiments, nanopores are fabricated on substrates such as chips, disks, blocks, plates and the like. Such substrates can be made from a variety of materials including but not limited to silicon, glass, ceramic, germanium, polymers (e.g., polystyrene), and/or gallium arsenide. The substrates may or may not be etched, e.g., chips can be semiconductor chips.
In particular embodiments, a detection apparatus can include a reservoir in contact with the array of nanopores. The reservoir can contain electrodes located to apply a current through the apertures formed by the protein nanopores.
In some embodiments, the detection apparatus of the present disclosure may include (a) an electrode; (b) a nanopore tethered to the electrode; and (c) a membrane surrounding the nanopore. Multiplex embodiments are also provided. For example, a detection apparatus can include (a) a plurality of electrodes; (b) a plurality of nanopores, each of the nanopores tethered to an electrode in the plurality of electrodes; and (c) a membrane surrounding each of the nanopores.
A nanopore can be tethered to an electrode (e.g., via a dielectric pad) using covalent moieties or non-covalent binding moieties. An example of a covalent attachment is when a nucleic acid tether is covalently attached to the nanopore and covalently attached to the dielectric pad. Other tethers can be similarly used for covalent attachment, including, for example, non-nucleic acid tethers such as polyethylene glycol or other synthetic polymers. An example, of a non-covalent attachment, is when a nanopore has an attached affinity moiety, such as a polyhistidine tag, Strep-tag or other amino acid encoded affinity moiety. Affinity moieties can bind non-covalently to ligands on a dielectric pad such as nickel or other divalent cations that bind polyhistidine, or biotin (or analogs thereof) that bind to Strep-tag. In some embodiments, such amino acid affinity moieties need not be used.
As described herein, the nanopores (whether hybrid nanopores or tethered nanopores) can be coupled with a detection circuit, including, for example, a patch clamp circuit, a tunneling electrode circuit, or a transverse conductance measurement circuit (such as a graphene nanoribbon, or a graphene nanogap), to record the electrical signals in methods of the present disclosure. In addition, the pore can also be coupled with an optical sensor that detects labels, for example, a fluorescent moiety or a Raman signal generating moiety, on the polynucleotides.
A detection apparatus of the present disclosure can be used to detect any of a variety of analytes including, but not limited to, ions, nucleic acids, nucleotides, polypeptides, biologically active small molecules, lipids, sugars or the like. Accordingly, one or more of these analytes can be present in or passed through the aperture of a protein nanopore in an apparatus set forth herein.
Other detection techniques that can be applied to an apparatus set forth herein include, but are not limited to, detecting events, such as the motion of a molecule or a portion of that molecule, particularly where the molecule is DNA or an enzyme that binds DNA, such as a polymerase. For example, Olsen et al, JACS 135: 7855-7860 (2013), which is incorporated herein by reference, discloses bioconjugating single molecules of the Klenow fragment (KF) of DNA polymerase I into electronic nanocircuits so as to allow electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Or, for example, Hurt et al., JACS 131: 3772-3778 (2009), which is incorporated herein by reference, discloses measuring the dwell time for complexes of DNA with the KF atop a nanopore in an applied electric field. Or, for example, Kim et al., Sens. Actuators B Chem. 177: 1075-1082 (2012), which is incorporated herein by reference, discloses using a current-measuring sensor in experiments involving DNA captured in an α-hemolysin nanopore. Or, for example, Garalde et al., J. Biol. Chem. 286: 14480-14492 (2011), which is incorporated herein by reference, discloses distinguishing KF-DNA complexes based on the basis of their properties when captured in an electric field atop an α-hemolysin pore. Other references that disclose measurements involving α-hemolysin include the following, all to Howorka et al., which are incorporated herein by reference: PNAS 98: 12996-13301 (2001); Biophysical Journal 83: 3202-3210 (2002); and Nature Biotechnology 19: 636-639 (2001).
In some embodiments, the invention relates to channel proteins assembled into a lipid bilayer membrane. The presence of an analyte is monitored by the ionic current that passes through the pore at a fixed applied potential with an interruption of current indicating interactions of the analyte with the channel protein. In some embodiments, a stabilized sensor chip contains a single protein nanopore protein. The protein nanopore sensor chip can be applied to measurements at the single-molecule level, i.e., stochastic sensing. By monitoring the ionic current that passes through the pore at a fixed applied potential, various analytes can be distinguished on the basis of the amplitude and duration of individual current-blocking events.
In another aspect, the disclosure provides a method of detecting/sensing an analyte. The method includes: (1) contacting a sample containing an analyte with the nanopore assembly as described; (2) applying an electrical current across the channel of the nanopore assembly; (3) determining the electrical current passing through the channel at one or more time intervals; and (4) comparing the electrical current measured at one or more time intervals with a reference electrical current, wherein a change in electrical current relative to the reference electrical current indicates a presence of the analyte in the sample. In some embodiments, the reference electrical current is measured with a sample that does not contain the analyte. In some embodiments, the nanopore assembly is placed on a support.
In some embodiments, the method may include measuring the ion current and/or a change in current signature, induced by translocation of each unit of the analyte or polymer through the channel In some embodiments, the method may include measuring the ion current and/or a change in current signature, induced by transient or permanent binding of each unit of the analyte or polymer to probes coupled on the channel which cause a change in current signature.
The analyte that can be detected by the disclosed methods of the present disclosure may be any one of nucleic acids, amino acids, peptides, proteins, polymers, and chemical molecules. A nucleic acid detected in the methods can be single stranded, double stranded, or contain both single stranded and double stranded sequence. The nucleic acid molecules can originate in a double-stranded form (e.g., dsDNA) and can optionally be converted to a single-stranded form. The nucleic acid molecules can also originate in a single stranded form (e.g., ssDNA, ssRNA), and the ssDNA can optionally be converted into a double-stranded form.
To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted here that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. The terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.
As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.
The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
As used herein, the terms “nanopore” and “channel” are used to refer to structures having a nanoscale passageway through which ionic current can flow. The inner diameter of the nanopore may vary considerably depending on the intended use of the device. Typically, the channel or nanopore will have an inner diameter of at least about 0.5 nm, usually at least about 1 nm and more usually at least about 1.5 nm, where the diameter may be as great as 50 nm or longer, but in many embodiments will not exceed about 10 nm, and usually will not exceed about 2 nm.
As used herein, the term “analyte” refers to a substance or chemical constituent that is undergoing analysis or sought to be detected. It is not intended that the present invention be limited to a particular analyte. Representative analytes include ions, saccharides, proteins, nucleic acids, and nucleic acid sequences.
As used herein the term “membrane” refers to a sheet or other barrier that prevents passage of electrical current or fluids. The membrane is typically flexible or compressible in contrast to solid supports set forth herein. The membrane can be made from lipid material, for example, to form a lipid bilayer, or the membrane can be made from non-lipid material. The membrane can be in the form of a copolymer membrane, for example, formed by diblock polymers or triblock polymers, or in the form of a monolayer, for example, formed by a bolalipid. See, for example, Rakhmatullina et al., Langmuir: the ACS Journal of Surfaces and Colloids 24: 6254-6261 (2008), which is incorporated herein by reference.
As used herein, the term “lipid membrane” means a film made primarily of compounds comprising saturated or unsaturated, branched or unbranched, aromatic or non-aromatic, hydrocarbon groups. The film may be composed of multiple lipids. Examples of lipids include, but are not limited to, fatty acids, mono-, di-, and triglycerides, glycerophospholipids, sphingolipids, steroids, lipoproteins, and glycolipids.
“Nucleic acid” or “nucleic acid sequence” or “nucleic acid molecule” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term nucleic acid is used interchangeably with gene, complementary DNA (cDNA), messenger RNA (mRNA), oligonucleotide, and polynucleotide. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). The terms encompass molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions, in some aspects, are achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081, 1991; Ohtsuka et al., J. Biol. Chem. 260: 2605-8, 1985; Rossolini et al., Mol. Cell. Probes 8: 91-8, 1994). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
“Polypeptide” is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, polypeptides, and proteins are included within the definition of polypeptide, and such terms can be used interchangeably herein unless specifically indicated otherwise. This term also includes post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. A polypeptide can be an entire protein or a subsequence thereof.
The terms “identical” or percent “identity” as known in the art refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). “Substantial identity” refers to sequences with at least about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity over a specified sequence. In some aspects, the identity exists over a region that is at least about 50-100 amino acids or nucleotides in length. In other aspects, the identity exists over a region that is at least about 100-200 amino acids or nucleotides in length. In other aspects, the identity exists over a region that is at least about 200-500 amino acids or nucleotides in length. In certain aspects, percent sequence identity is determined using a computer program selected from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm.
The term “similarity” is a related concept but, in contrast to “identity,” refers to a measure of similarity which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If, in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% ( 15/20). Therefore, in cases where there are conservative substitutions, the degree of percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.
It also is specifically understood that any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as about 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. The values listed above are only examples of what is specifically intended.
Ranges, in various aspects, are expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When values are expressed as approximations, by use of the antecedent “about,” it will be understood that some amount of variation is included in the range.
The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
As used herein, the term “purified” or “substantially purified,” as used herein, refers to the desired protein is enriched by at least 20%, more preferably by at least 50%, even more preferably by at least 75%, and most preferably by at least 90%, or even 95%.
Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure.
Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Recitation of ranges of values herein are merely intended to serve as a shorthand method for referring individually to each separate value falling within the range and each endpoint unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.
All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise.
In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.
The section headings as used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.
This example describes the materials and methods to be used in the subsequent examples.
For a non-membrane protein channel, it is difficult to insert into lipid bilayer or polymer membrane directly because unlike membrane protein channels, they usually lack hydrophobic layer in the middle and hydrophilic layers in both ends and hence require extensive engineering. In order to be used for biosensing, or sequencing, non-membrane protein channel also need to be engineered and functionalized with different probes. Although non-membrane protein channel may originate from various source with different sequences, shapes, structures, or properties, the strategies and methods employed to reengineer and enable them to be used as nanopore share some common feature. As shown in
Although non-membrane protein channel may originate from various source with different sequences, shapes, structures, or properties, the strategies and methods employed to express and purify them share some common features.
First, the reengineered non-membrane protein channel gene was cloned into an expression vector with or without a tag on the terminal. Then the vector was transformed into suitable protein expression host, e.g., E. coli system. After the protein channel was expressed in the host, the host was lysed and series of steps were taken to remove the host debris Finally, non-membrane proteins could be purified by one or combination of following methods, such as affinity chromatography, exchange chromatography, size exclusion chromatography, or other commonly used purification methods.
As an example, the non-membrane protein channel gene and variants gene was cloned into a PET23a vector with His-tag at the N-terminus. After transforming into BL21 (DE3) cells, one BL21(DE3) colony was inoculated in 5 mL fresh LB medium in the presence of antibiotics and cultured at 37° C. in a shaker for several hours until the OD reached 0.8. The flask was then kept at 4° C. for cooling down. Then 0.5 mM IPTG was added into the flask for induction. The bacteria were cultured at 16° C. in a shaker overnight. The bacteria were harvested in 8000 rpm for 10 mins, supernatant discarded, and then the pellet suspended with lysis buffer. The bacteria solution was sonicated until the solution became transparent and not sticky. The solution was centrifuged after sonicating with 12000 rpm for 30 mins, the supernatant discarded and the pellet collected. 10 ml urea (8 M) was added to the precipitation and oscillated at low speed on the oscillator until the precipitation was completely dissolved in urea. The solution was centrifuged at 12000 rpm for 10 mins The supernatant was added to 100 ml protein renaturation buffer and stirred overnight. The solution was centrifuged after refolding at 12000 rpm for 30 mins, the pellet discarded and the supernatant collected. The supernatant was passed through 0.45 um syringe filter to discard the denatured protein. The supernatant was added to a dialysis bag, and the dialysis fluid replaced three times. The fluid in the dialysis bag is collected and centrifuges for 12000 rpm for 10 minutes, the pellet discarded and the supernatant collected. Nickel beads were equilibrated with lysis buffer, and the supernatant added to the beads. The beads were then washed with washing buffer. The protein was eluted using elution buffer for 7˜10 column volumes. The eluent was collected and concentrated to 5 mL. The eluent was centrifuged at 12000 rpm for 10 mins, and then the supernatant was absorbed and injected with a syringe into AKTA FPLC. Before injection, the sample loop was washed with 10 mL lysis buffer. The protein was collected after passing through a size exclusion column. An SDS-PAGE gel was run to check the protein sample after SEC, and stored at −80° C.
To functionalized with different probes or enhance the hydrophobicity or hydrophilicity of non-membrane protein channels, various conjugation methods could be employed. Although non-membrane protein channel may originate from various source with different sequences, shapes, structures, or properties, the strategies and methods employed for conjugation share some common feature. To conjugate a hydrophobic moiety to non-membrane protein channel, the reactions could be done on cysteine group on the protein channel The protein channel contains one or multiple cysteines per subunit, which are located in the middle layer of the channel and accessible to the environment. The protein solution was prepared in a buffer containing 0.5 M NaCl, 50 mM Tris, 15% glycerol with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation at room temperature, 4 μL of 4 mM cholesterol-PEG-maleimide was added drop-wise to the protein solution, and the reaction mixture was incubated in the dark at room temperature for 2 hours. Excess cholesterol-PEG-maleimide was removed by a NanoSep 100K spin column. The labeling of the protein was checked with 12% SDS-PAGE.
To conjugate probes to non-membrane protein channel, click chemistry, such as Tetrazine-Alkene ligation, or Azide-Alkyne click chemistry is employed. For Tetrazine-Alkene ligation, the protein solution was prepared in a buffer containing 0.5 M NaCl, 50 mM Tris, 15% glycerol with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation, 1.6 μL of 10 mM Methyltetrazine-PEG4-maleimide was added drop-wise to the protein solution, and the mixture was incubated in the dark at room temperature for 1 hour. Excess Methyltetrazine-PEG4-maleimide was removed by desalting using a desalt spin column. A 50 μL of probe solution was prepared in a buffer containing 0.5 M NaCl, 50 mM Tris with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation, 6 μL of 25 mM TCO-PEG3-maleimide was added drop-wise to the oligo solution, and the mixture was incubated at room temperature for 2 hours. Excess TCO-PEG3-maleimide was removed by desalting column. The labeling of the probe was verified by 20% urea-PAGE gel.
For Azide-Alkyne ligation, TCO-modified probe was mixed with Methyltetrazine-labeled protein at different molar ratios to achieve optimal protein labeling efficiency. The mixture was incubated at room temperature for 1 hour. The conjugation was verified with 12% SDS-PAGE. To A 40 μL of 20 μM protein solution was prepared in a buffer containing 0.5 M NaCl, 50mM Tris, 15% glycerol with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation, 1.6 μL of 10 mM Sulfo DBCO-PEG4-maleimide was added drop-wise to the protein solution, and the mixture was incubated in the dark at room temperature for 1 hour. Excess maleimide reagent was removed by desalting using a desalt spin column. The DBCO-modified protein was mixed with Azide-modified probe at equal molar concentration, and the reaction mixture was incubated at room temperature for 2 hours. The conjugation was verified with 12% SDS-PAGE gel.
Although non-membrane protein channel may originate from various source with different sequences, shapes, structures, or properties, all non-membrane protein channels or variants could be applied on the similar nanopore setup or device. Typically, the setup comprises a sensor chip or array which have one or multiple apertures. The sensor chip is capable of supporting lipid or polymer membrane formation which could separate the compartment into cis-(top) and trans-(bottom) compartments. Both compartments were filled with conducting buffer. Input electrode was embedded into one of the compartment, and a ground electrode was embedded into another compartment. Furthermore, the setup maybe also combined with a fluidic system to enable sample flow from one container to the sensor device. To insert non-membrane protein channel into lipid or polymer membrane, the protein channel was suspended in their respective storage buffer is diluted 50-100 fold in the conducting buffer (typically 1 M KCl or 1 M NaCl, 5 mM HEPES or Tris, pH 7.6) and added to the top compartments. Under an applied potential (constant holding voltage or ramping voltage) with or without detergent, direct insertion of the protein channels in planar membranes can be observed. When no analyte presents, the current is stable and clean. When the analyte presents, the interaction with probe results in a current change which was recorded.
Although non-membrane protein channel may originate from various source with different sequences, shapes, structures, or properties, the strategies and methods employed for data analysis share common feature. Typically, ˜40,000+ current blockage events (either translocation or single molecule binding events) are analyzed to ensure the result is within statistical significance. MATLAB or PYTHON-based custom algorithm was developed for quantitative fast processing of events. Generally, two parameters are used: (1) Current blockage fraction, represented as [(Currentunblocked−Currentafter analyte block)/Currentunblocked]; and (2) Dwell time: τoff (duration of an event) and τon (time between consecutive events). From the τon and τoff , the κon (association rate constant) and κoff (dissociation rate constant) can be obtained and finally Kd (equilibrium dissociation constant). A calibration curve was constructed showing the capture rate as a function of analyte concentration. Upon introducing an unknown concentration of the analyte to the nanopore, the mean capture rate can be calculated, and the analyte concentration can be determined from the calibration curve. Analysis of clinical samples requires further tuning of the analysis algorithm to clearly discriminate between ‘contaminative’ or non-specific signals and true analyte induced events. To standardize the analyte detection system, an ‘endogenous’ normalizer with a ‘spiked-in’ control was generally employed. The platform data was then cross-validated using standard assays commonly used in the diagnostic field, such as immunoassay and qRT-PCR. Finally, statistical sample size/power analyses were based on two-sample t-tests for two-group comparisons and two-way analysis of variance (ANOVA) for a combination of two factors.
The purified phi29 gp-9 tail protein and its variants in SDS PAGE are shown in
Many bacteriophages contain a long contractile or non-contractile, or short non-contractile tail. The tail plays a critical role in the process of host cell recognition, membrane penetration, and viral genome ejection. The tail proteins are derived from (including but not limited to) phi29, T4, T3, T5, T7, SPP1, P22, P2, P3, Lambda, Mu, HK97 and C1.
These protein channels of the invention are ideal for biosensing and sequencing of a biological molecule, such as disease-related biomarker, polynucleotide and polypeptide sequences. With improved membrane capability, the modified protein channels could insert into lipid or polymer membrane efficiently and serve as nanopore for biosensing and sequencing. By conjugating with various probes, the modified protein channels have the capacity to detect specific disease-related biomarker with high sensitivity and specificity. The pore of the invention may be present in a homologous or heterologous pore.
The crystal structure of phi29 bacteriophage tail (gp9) shows that six gp9 subunits form a hexameric or cylindrical-like tube structure. Inside the structure, the distal end is blocked by six flexible hydrophobic loops before DNA ejection is triggered. In order to deliver the genomic dsDNA into the host cell cytoplasm, the phi29 tail needs to penetrate the cell membrane. The length of the tube is about 12.5 nm. The tube has an inner diameter of approximately 4 nm and an outer diameter of approximately 9 nm and. The wall of the tube is comprised of largely β-sheets with about 2.5 nm thickness.
A clone of full-length gp9 and series of mutants were constructed (
Portal proteins exist not only in bacteriophages phi29, T3, T4, T5, T7, SPP1, and P22, but also in other viral systems such as Adeno and Herpes viruses. The portal channel, termed the connector, is a pore-like protein structure with a central channel that acts as a pathway for genomic DNA to enter the viral capsid during packaging and exit during infections. Although structural studies indicate significant differences in sequence homology and sizes among different viral connector proteins, they all are topologically similar with a truncated cone shape. The stoichiometry of the connectors derived from overexpressed proteins often varies depending on the expression conditions. These protein channels of the invention can be ideal for biosensing and sequencing applications if they can be directly inserted into robust polymer membranes. Since the structures of the connector from different viral portal proteins show similar characteristics, the principle outlined above also applies to all of them by extension.
P22 is a tailed bacteriophage which assembles empty precursor capsids that are subsequently packaged with viral DNA by a powerful packaging motor. P22 portal protein forms a channel-like structure for bidirectional passage of viral DNA. Podoviridae family of short-tailed dsDNA bacteriophages includes members of the P22-like subgroup, such as Sf6, CUS-3, epsilon34, and APSE-1. The portal barrel is highly dynamic and susceptible to proteolysis in solution. The P22 portal protein is composed of 12 identical subunits, arranged symmetrically around the central channel. The overall height is ˜30 nm with a funnel-shaped core of diameter ˜17 mm, which is connected to an ˜20 nm long α-helical tube. The average internal diameter of the channel varies from 3.5 nm to 7.5 nm throughout the structure.
Series of mutants were constructed (
The T4 portal mostly exists as a 12-mer (some 11-mer or 13-mer depending on protein expression conditions) ring that is 14 nm long with 7 nm wide, and an interior channel of 3 nm in diameter. Since the channel assembles from 12 subunits, altering residue(s) in one monomer will trigger the effect in the entire channel with the mutation present in the same plane of the molecule. The invention includes (
Planar bilayer lipid membranes (BLMs) or polymer membrane were generated in (a) BCH-1A horizontal BLM cell (Eastern Scientific) or (b) home-made custom chamber. A Teflon partition with a 100 or 200 μm aperture was placed in the apparatus to separate the BLM cell into cis-(top) and trans-(bottom) compartments. The home-made chamber has pre-drilled 100 or 200 um apertures separating the cis- and trans-compartments.
Lipid membrane: A planar lipid bilayer of varying composition was formed by pre-painting the aperture with lipids in hexane (concentration: 0.5 mg/ml) followed by painting with lipids in n-decane (concentration: 20-30 mg/ml). Examples of lipid composition include: (i) zwitterionic lipids such as 100% DPhPC or DOPC or POPC; (ii) 0-50% anionic lipids such as DPhPG/DOPG/POPG or DPhPS/DOPS/POPS mixed proportionately (final ratio adds up to 100%) with composition (i); (iii) 0-25% cholesterol mixed with composition (i) and (ii) proportionately. The exact lipid composition depends on the properties of the protein. Typical lipid membrane composition used include: 100% DPhPC; 100% DPhPC; 30% DPhPS; 70% DPhPC: 28% DPhPG: 2% cholesterol.
Polymer membrane: A planar polymer membrane of varying composition was formed by manual painting using membranes suspended in organic solvents such as Decane or Silicone oil (Polyphenyl-methylsiloxane based or Polydimethylsiloxane based with a viscosity of 20 mPa·s). The membrane composition is Polyoxazoline based triblock copolymers (
(i) PEOXA-PEO-PEOXA [Poly(2-ethyl oxazoline)-b-poly(ethylene oxide)-b-poly(2-ethyl oxazoline)];
(ii) PMOXA-PDMS-PMOXA [Poly(2-methyl oxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyl oxazoline)]; (with ethyl-benzyl or propyl or propyl-ethoxy link between blocks)
(iii) PMOXA-PB-PMOXA [Poly(2-methyl oxazoline)-b-poly(1,4-butadiene)-b-poly(2-methyl oxazoline)];
(iv) PMOXA-PE-PMOXA [Poly(2-methyl oxazoline)-b-poly(ethylene)-b-poly(2-methyl oxazoline)];
(v) PMOXA-PEO-PMOXA [Poly(2-methyl oxazoline)-b-poly(ethylene oxide)-b-poly(2-methyl oxazoline)]
Typical membranes used include PMOXA6-PDMS35-PMOXA6; PMOXA6-PDMS65-PMOXA6; PMOXA11-PDMS65-PMOXA11; PMOXA5-PDMS13-PMOXA5; PMOXA3-PDMS38-PMOXA3 (
Typically, the protein channels (tail proteins or portal channels) suspended in their respective storage buffer is diluted 50-100 fold in the conducting buffer (typically 1 M KCl or 1 M NaCl, 5 mM HEPES or Tris, pH 7.6) and added to the top compartments of the BLM cell. Under an applied potential (constant holding voltage or ramping voltage), direct insertion of the protein channels in planar membranes can be observed (
If necessary, the membrane compositions as described can also be used to generate vesicular polymersome structures with varying polydispersity to reconstitute the protein channels. For high insertion efficiency, varying amounts of glycerol, CsCl, and/or sucrose can be encapsulated within the polymersomes. The resulting proteo-polymersomes can then fuse with a planar bilayer of the same composition in a salt and voltage-dependent manner
Here T4 gp20 portal protein is used as an example to demonstrate how to conjugate hydrophobic moiety to the non-membrane channel via reactions with cysteine residues. The protein channel contains three cysteines per subunit, which are located in the middle layer of the channel and accessible to the environment (
Planar polymer membranes were generated. Under an applied voltage (constant holding voltage or ramping voltage), direct insertion of non-membrane protein channel carrying hydrophobic moiety was observed after adding protein channel to a cis chamber (
phi29 gp10 portal protein was used as a representative example.
Strategy 1: via Tetrazine-Alkene ligation: A 40 μL of 20 μM protein solution was prepared in a buffer containing 0.5 M NaCl, 50 mM Tris, 15% glycerol with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation, 1.6 μL of 10 mM Methyltetrazine-PEG4-maleimide was added drop-wise to the protein solution, and the mixture was incubated in the dark at room temperature for 1 hour. Excess Methyltetrazine-PEG4-maleimide was removed by desalting using a desalt spin column. A 50 μL of 30 μM thiol modified miRNA probe solution was prepared in a buffer containing 0.5 M NaCl, 50 mM Tris with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation, 6 μL of 25 mM TCO-PEG3-maleimide was added drop-wise to the oligo solution, and the mixture was incubated at room temperature for 2 hours. Excess TCO-PEG3-maleimide was removed by desalting column. The labeling of the miRNA probe was verified by 20% urea-PAGE gel. To conjugate the miRNA probe to the protein, the TCO-modified miRNA probe was mixed with Methyltetrazine-labeled protein at different molar ratios to achieve optimal protein labeling efficiency. The mixture was incubated at room temperature for 1 hour. The conjugation was verified with 12% SDS-PAGE.
Strategy 2: via Azide-Alkyne click chemistry: A 40 μL of 20 μM protein solution was prepared in a buffer containing 0.5 M NaCl, 50 mM Tris, 15% glycerol with pH 6.8. The solution was degassed, and 100-fold excess TCEP was added to the solution. After 20 min incubation, 1.6 μL of 10 mM Sulfo DBCO-PEG4-maleimide was added drop-wise to the protein solution, and the mixture was incubated in the dark at room temperature for 1 hour. Excess maleimide reagent was removed by desalting using a desalt spin column. The DBCO-modified protein was mixed with Azide-modified miRNA probe at equal molar concentration, and the reaction mixture was incubated at room temperature for 2 hours. The conjugation was verified with 12% SDS-PAGE.
The protein-miRNA probe conjugate was incubated with the target miRNA oligo at an equal molar concentration at room temperature for 30 min. The binding of the protein-miRNA probe with the target miRNA was verified with 12% SDS-PAGE (
To conjugate a protein probe to the nanopore channel, various methods have been tried and tested, including reaction of cysteine residue with Methyltetrazine-PEG4-maleimide and subsequent reaction with TCO-labeled probes, and SpyCatcher/Spytag protein conjugation system. How a single chain antibody against PSA conjugate to phi29 gp10 portal protein channel via SpyCatcher/Spytag was demonstrated. Phi29 gp10 protein channel with a C-terminal SpyTag peptide was constructed and purified. Single chain antibody against PSA with a C-terminal SpyCatcher was constructed and purified. The assembled protein channel-PSA single chain antibody was verified by gel (
To conjugate a nucleic probe to the nanopore channel, a variety of different methods have been tried and tested as outlined in Example 6. It was demonstrated that miR-21 probes can be conjugated to T4 gp20 portal protein channel The purified conjugated complex was inserted into a polymer membrane to test its capacity of binding corresponding microRNA. The procedure to setup electrophysiological experiments was described previously. After insertion, a series of different concentration of microRNA was added to the chamber, and a unique binding event was observed (
Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the invention, are given by way of illustration only. Additionally, it is contemplated that changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/629,604, filed Feb. 12, 2018, the disclosures of which are incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/17432 | 2/11/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62629604 | Feb 2018 | US |
