Regulation of interactions between target molecular lipid bilayers

Abstract

A combination of lipid-binding molecules and / or lipid-binding proteins with lipid components (i.e., mispids) is provided for use in modifying the interaction between target molecules and lipid membranes. This includes, for example, the use of lipid-binding molecules and / or mispids to improve the sequencing efficiency and throughput of nanopore-based sequencing systems. To sequence target molecules such as nucleic acid sequences or nucleic acid substitute polymers derived therefrom, lipid-binding molecules and / or their mispids are combined with the target molecules. The mixture is then applied to a nanopore-based sequencing chip. The target molecules are then sequenced in the presence of lipid-binding molecules and / or nanodiscs, thereby improving the capture, arrival time, and effective concentration of the target molecules across the chip membrane. Such improved efficiency is particularly beneficial, for example, when the concentration of the target molecule is low.

Description

[Technical Field]

[0001] Description regarding sequence listings The sequence listing relating to this application is provided in XML format instead of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is P37738-WO_Sequence_Listing.xml. The XML file is 6500 bytes in size and was created on March 13, 2024.

[0002] The present invention generally relates to regulating the interaction between a target molecule and a lipid membrane, and includes modifying the interaction between the target molecule and the lipid membrane of a nanopore-based sequencing chip to improve sequencing efficiency and throughput. [Background technology]

[0003] Over the past two decades, biological membranes have emerged as a crucial tool in a variety of biomedical applications. This includes the use of lipid membranes in nanopore-based sequencing applications, where nanopores provide invariant and reproducible physical openings that can guide and sequence target molecules.

[0004] Generally, a nanopore is associated with a tip-based flow cell, which has an inlet into which a solution containing the target molecule is added. The flow cell also includes multiple wells, each well having a lipid bilayer membrane and a single nanopore embedded within the membrane. Each well is also associated with a detection electrode that can detect changes in current and / or voltage across the membrane of the well.

[0005] When a solution containing the target molecule is introduced into the flow cell sample port, the solution flows across the lipid bilayer membrane of the well, allowing nanopores within the membrane to capture the target molecule. The membrane, which exhibits high electrical resistance, allows ions to pass through the pores and generate an electric current when a suitable potential is applied across the membrane. The ionic current passing through the nanopore also drives the target molecule through the nanopore. As the target molecule passes through the nanopore, it causes a change in the ionic current, which can be detected, for example, via a detection electrode associated with the well of the flow cell. Then, for example, each base of a nucleic acid passing through the nanopore can be identified in real time through the characteristic discontinuity it causes in the current.

[0006] One challenge when using such biological nanopore-based sequencing platforms is the inefficiency associated with the uniformity of the distribution of target molecules across the biomembrane of the chip (when target molecules flow across the membrane). This is particularly true for target molecules that have an affinity for lipid bilayer membranes, because solutions containing such target molecules can deplete the target molecules early in their flow across the flow cell when the target molecules first encounter the wells of the flow cell. These inefficiencies become even more problematic when the initial amount of target molecules is low, for example, when the initial concentration of a DNA sample is low.

[0007] Therefore, it is necessary to improve the flow of target molecules on the lipid bilayer membrane of nanopore-based sequencing chips. In particular, there is a need for methods and systems to improve the rate at which target molecules reach the nanopores and the uniformity of target molecule capture by the nanopores. When the initial concentration of the target molecule is low, it is also necessary to improve nanopore-based sequencing. More broadly, there is a need to deliver molecules (such as metabolites) that can be detected by overall modifying the interaction between the target molecule and the lipid membrane, for example, by regulating the dynamics of the nanopores and / or the behavior of the pores. [Overview of the Initiative]

[0008] In certain exemplary embodiments, compositions are provided for modifying the interaction between a target molecule and a lipid membrane. These compositions, for example, comprise a heterogeneous mixture of lipids and lipid-binding proteins, where the lipids and lipid-binding proteins form stable complexes (i.e., "mispids") of varying sizes and stoichiometric concentrations. In certain exemplary embodiments, the lipid-binding proteins are membrane scaffolding proteins (MSPs), such as apolipoproteins (e.g., Apo-A1) or derivatives thereof. Furthermore, the lipid-binding proteins may contain a predetermined number of alpha-helices, for example, 3 to 10 alpha-helices. In addition, in certain exemplary embodiments, the ratio of lipid-binding proteins to lipid components is approximately 1:200 to 1:1500, for example, 1:200.

[0009] In certain exemplary embodiments, a method is provided for modifying the interaction between a target molecule and a lipid bilayer. This method includes, for example, preparing a lipid membrane and contacting the lipid membrane with a plurality of mispids, for example, a solution of the mispids and a plurality of target molecules, for example, a solution of the target molecules. The target molecules may have an affinity for the lipid membrane, for example, but when the lipid membrane is contacted with the mispids, the affinity of the target molecules to the lipid membrane decreases. Therefore, the interaction between the target molecule and the lipid membrane is modified by the use of mispids.

[0010] In a further exemplary embodiment, a method for sequencing target molecules is provided. This method includes, for example, preparing a chip, the chip comprising a plurality of wells, each well comprising a detection electrode, a lipid membrane positioned adjacent to or near the detection electrode, and nanopores positioned within the lipid membrane. The chip comes into contact with a plurality of mispids, e.g., a solution of mispids. The chip also comes into contact with a plurality of target molecules, e.g., target molecules, each having affinity for the lipid membrane. A voltage is applied across the membrane of the chip, and changes in current or voltage associated with the nanopores are measured via one or more detection electrodes. The sequence of target molecules is then determined based on one or more of the measured changes in current or voltage associated with the nanopores, with the help of, for example, a computer processor.

[0011] In yet another exemplary embodiment, a method is provided for sequencing target molecules, optionally including mispids. Such a method includes, for example, providing a chip comprising a plurality of detection electrodes and a lipid membrane positioned adjacent to or near the detection electrodes. For example, a plurality of nanopore assemblies are also placed within the lipid membrane. The chip is brought into contact with a plurality of lipid-binding proteins and optionally a plurality of mispids. For example, the chip can be brought into contact with lipid-binding proteins only. However, the chip also comes into contact with a plurality of target molecules, such as a solution containing the target molecules. In a particular exemplary embodiment, each target molecule has an affinity for the lipid membrane. A voltage is applied to the membrane of the chip, and one or more changes in current or voltage related to the nanopore assemblies are measured, for example, via one or more detection electrodes associated with the chip. The sequence of target molecules is then determined, for example, with the help of a computer processor, based on one or more of the measured changes in current or voltage related to the nanopore assemblies. In a particular exemplary embodiment, at least a portion of the lipid-binding proteins form complexes with lipid components to form a plurality of mispids, and the chip comes into contact with the mispids.

[0012] In certain exemplary embodiments, the target molecule is a nucleic acid, a modified nucleic acid, or a nucleic acid substitute. An example of a nucleic acid substitute is Xpandomer.

[0013] In certain exemplary embodiments, the lipid-binding protein is a membrane scaffolding protein (MSP). For example, the MSP is apolipoprotein A1 or a derivative thereof. In certain exemplary embodiments, the lipid-binding protein comprises a predetermined number of alpha helices, such as 3, 4, 5, 6, 7, 8, 9, or 10.

[0014] In certain exemplary embodiments, the ratio of lipid-binding protein to lipid component is approximately 1:200. In certain exemplary embodiments, the ratio of lipid-binding protein to lipid component is approximately 1:200 to 1:1500. For example, the ratio of lipid-binding protein to lipid component is approximately 1:1200. In certain exemplary embodiments, the ratio of lipid-binding protein to lipid component is approximately 1:200.

[0015] In certain exemplary embodiments, the lipid component includes phosphatidylcholine lipids, phosphoethanolamine lipids, derivatives thereof, or combinations thereof. For example, the lipid component may include 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DphPE), derivatives thereof, or combinations thereof.

[0016] In certain exemplary embodiments, the mispipes and target molecules are mixed together before the tip or membrane is brought into contact with the mispipes and target molecules. In yet other exemplary embodiments, the membrane or tip is brought into contact with the mispipes and target molecules in that order, or with the target molecules and mispipes in that order.

[0017] In certain exemplary embodiments, contacting a chip or membrane with a mispit and a target molecule improves the throughput associated with sequencing reactions involving the target molecule. In yet another exemplary embodiment, contacting a chip or membrane with a mispit and a target molecule improves the flow cell capture of the target molecule in sequencing reactions involving the target molecule.

[0018] In certain exemplary embodiments, each of the methods described herein may use a nanodisc, or in certain exemplary embodiments, a mixture of a nanodisc and a mispid, or in certain exemplary embodiments, a mixture of a lipid-binding protein, a mispid, and / or a nanodisc. For example, a membrane or chip, such as a membrane associated with a sequencing chip, can be brought into contact with the lipid-binding protein described herein, the mispid described herein, the nanodisc described herein, or a combination thereof. Such contact of the membrane and / or chip modifies, for example, the interaction between the target molecule and the membrane and / or chip. For example, if the target molecule has affinity for the membrane, contact of the membrane lipid-binding protein, a mispid, a nanodisc, or a combination thereof can reduce the interaction between the target molecule and the membrane.

[0019] These and other aspects, purposes, features and advantages of the exemplary embodiments described will become apparent to those skilled in the art when considering the following detailed description of the exemplary embodiments. [Brief explanation of the drawing]

[0020] [Figure 1A] Figure 1A shows the flow of target molecules across the lipid membrane of a flow cell well in the absence of mispipes, according to a particular exemplary embodiment. As shown, as target molecules flow across the flow cell well, they bind to and accumulate in the lipid bilayer membrane of the well. As a result, target molecules are depleted from the sample, and fewer target molecules flow across the flow cell. Pore capture is limited by how quickly the pore can attract target molecules from the bilayer over its lifetime. [Figure 1B] Figure 1B illustrates the flow of target molecules across the lipid membrane of a flow cell well in the presence of mispids, according to a particular exemplary embodiment. As shown, the mispids compete for space on the lipid bilayer of the flow cell well membrane. This reduces the binding of target molecules to the lipid membrane, resulting in more target molecules being released and flowing across the entire flow cell (to further wells in this example). This improves the confluence of target molecule capture across the flow cell. [Figure 2] Figure 2 is a block flow diagram illustrating a method for sequencing a target molecule in the presence of mispids, according to a particular exemplary embodiment. [Figure 3A] Figure 3A is a graph showing the size exclusion chromatography elution profile of a 1:200 MSP2N2:DPhPE mispit preparation according to a specific exemplary embodiment. As shown, the main peak of the mispit preparation roughly corresponds to elution at vacancy volume, indicating a very large and heterogeneous distribution of particles. [Figure 3B]Figure 3B is an image showing the gel electrophoresis of fractions eluted from size exclusion chromatography purification of a 1:200 MSP2N2:DPhPE mispid preparation (from Figure 3A) according to a particular exemplary embodiment. All lanes contain the MSP2N2 protein at approximately 40 kDa. [Figure 4A] Figures 4A - 4C are a series of histograms showing the sequencing metrics of Xpandomer (「XP」) molecules in the presence of three concentrations of mispid compared to Xpandomer sequencing alone according to a particular exemplary embodiment. More specifically, Figure 4A shows the throughput for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. Figure 4B shows the reach for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. Figure 4C shows the accuracy of three concentrations of mispid / Xpandomer conditions and the Xpandomer control. [Figure 4B] Figures 4A - 4C are a series of histograms showing the sequencing metrics of Xpandomer (「XP」) molecules in the presence of three concentrations of mispid compared to Xpandomer sequencing alone according to a particular exemplary embodiment. More specifically, Figure 4A shows the throughput for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. Figure 4B shows the reach for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. Figure 4C shows the accuracy of three concentrations of mispid / Xpandomer conditions and the Xpandomer control. [Figure 4C]Figures 4A-4C are a series of histograms showing sequencing metrics of Xpandomer ("XP") molecules in the presence of three concentrations of mispids, compared to single Xpandomer sequencing, according to certain exemplary embodiments. More specifically, Figure 4A shows throughput for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. Figure 4B shows the reach for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. Figure 4C shows the accuracy for three concentrations of mispid / Xpandomer conditions and the Xpandomer control. [Figure 5A] Figures 5A-5C are a series of heatmaps showing the spatial distribution of different metrics representing Xpandomer capture along the entirety of a flow cell, according to certain exemplary embodiments. [Figure 5B] Figures 5A-5C are a series of heatmaps showing the spatial distribution of different metrics representing Xpandomer capture along the entirety of a flow cell, according to certain exemplary embodiments. [Figure 5C] Figures 5A-5C are a series of heatmaps showing the spatial distribution of different metrics representing Xpandomer capture along the entirety of a flow cell, according to certain exemplary embodiments. [Figure 6A] Figures 6A-6C include a heatmap (Figure 6A) showing the spatial distribution of Xpandomer molecule capture frequency along a flow cell, and histograms (Figures 6B-6C) of Xpandomer capture metrics in the presence of increasing concentrations of saposin (lipid-binding protein only), according to certain exemplary embodiments. [Figure 6B] Figures 6A-6C include a heatmap (Figure 6A) showing the spatial distribution of Xpandomer molecule capture frequency along a flow cell, and histograms (Figures 6B-6C) of Xpandomer capture metrics in the presence of increasing concentrations of saposin (lipid-binding protein only), according to certain exemplary embodiments. [Figure 6C]Figures 6A–6C include a heatmap (Figure 6A) showing the spatial distribution of Xpandomer molecule capture frequencies along a flow cell according to a specific exemplary embodiment, and histograms (Figures 6B–6C) of Xpandomer capture metrics in the presence of increasing concentrations of saposins (lipid-binding proteins only). [Figure 7A] Figures 7A and 7B include a heatmap (Figure 7A) showing the spatial capture distribution of the alternative molecule fauXmer in the presence of increasing mispit concentrations according to a particular exemplary embodiment, and a histogram (Figure 7B) showing the total number of fauXmers (mt_count). [Figure 7B] Figures 7A and 7B include a heatmap (Figure 7A) showing the spatial capture distribution of the alternative molecule fauXmer in the presence of increasing mispit concentrations according to a particular exemplary embodiment, and a histogram (Figure 7B) showing the total number of fauXmers (mt_count). [Figure 8A] Figures 8A and 8B include a histogram showing the total number of high-quality Xpandomer captures (Figure 8A) and a heatmap showing Xpandomer captures across the entire flow cell (Figure 8B), according to a particular exemplary embodiment. [Figure 8B] Figures 8A and 8B include a histogram showing the total number of high-quality Xpandomer captures (Figure 8A) and a heatmap showing Xpandomer captures across the entire flow cell (Figure 8B), according to a particular exemplary embodiment. [Modes for carrying out the invention]

[0021] overview As described herein, lipid-binding molecules and / or mispids containing lipid-binding molecules are used to modify the interaction between a target molecule and a lipid membrane. The target molecule may be, for example, a nucleic acid sequence, in which the use of lipid-binding molecules and / or mispids improves the sequencing efficiency and throughput of a nanopore-based sequencing system. To sequence a target molecule such as a nucleic acid sequence, the lipid-binding molecule and / or its mispid is combined with the target molecule. The mixture is then applied to a nanopore-based sequencing chip. The target molecule is then sequenced in the presence of the lipid-binding molecule and / or mispid, thereby improving the capture, arrival time, and effective concentration of the target molecule across the chip membrane. Such improved efficiency is particularly beneficial, for example, when the concentration of the target molecule is low.

[0022] More specifically, the various embodiments described herein can be used with any biological system in which target molecules, such as target molecules having affinity for lipid membranes, are delivered to a nanopore via interaction between the target molecule and the lipid membrane. This includes conventional nanopore-based sequencing chips in which the target molecule (e.g., DNA to be sequenced) flows over a lipid-based membrane. For example, while we do not wish to be bound by any particular theory, it has been found that when target molecules are applied to a flow cell, those having affinity for lipids accumulate on the lipid membrane in the well closest to the flow cell inlet, presumably due to the membrane affinity of the target molecule to the lipid bilayer. This is shown in Figure 1A, where the target molecule interacts with the lipid membrane in the flow cell well. This reduces the number of target molecules that can flow across the cell (Figure 1A). And as more target molecules accumulate, the free flow of target molecules across the membrane is hindered, thereby reducing the uniform movement and distribution of target molecules across the membrane. As a result, the distribution of target molecules trapped in the nanopores is not uniform across the entire membrane, leading to lower sequencing efficiency and reduced throughput (Figure 1A).

[0023] To address these and other problems and inefficiencies associated with nanopore-based sequencing methods, as described herein, the target molecule is combined with a solution containing mispids, i.e., a polydisperse mixture of lipid-based complexes, each complex containing one or more lipid / lipid bilayers and one or more lipid-binding proteins. The target molecule / mispid mixture is then applied to the inlet of a nanopore-based sequencing chip, for example, so that it flows across the chip together with the target molecule. In other examples, the target molecule is mixed only with a solution of lipid-binding proteins, for example, and then flowed over the lipid membrane of the nanopore-based sequencing chip. In other examples, the target molecule is mixed with a nanodisk solution, i.e., a monodisperse and stable mixture of lipid bilayers contained within lipid-binding proteins.

[0024] While we do not wish to be bound to any particular theory, it is thought that lipid-binding molecules and / or mispids bind to the lipid membrane of the nanopore-based tip, thereby preventing target molecules from binding prematurely to the lipid membrane in the well closest to the flow cell channel inlet. This is illustrated in Figure 1B, where, for example, mispids are depicted as binding to the lipid membrane and consequently competing with target molecules for binding of the flow cell to the lipid membrane. As a result, the number of target molecules that can flow freely across the flow cell increases (Figure 1B). In some examples, it is further conceivable that target molecules interact with, for example, lipid-binding molecules and / or mispids, thereby further reducing undesirable interactions between the target molecules and the lipid bilayer of the tip.

[0025] Nevertheless, as described herein, the use of lipid-binding molecules and / or mispids allows for a more uniform distribution of target molecules across the membrane (and consequently, more uniform target molecule capture by a greater number of nanopores). Indeed, by applying lipid-binding proteins and / or mispids to the membrane, an improvement in the flow of target molecules on the membrane of a nanopore-based sequencing chip is achieved. This includes an improved rate of target molecule arrival at the nanopores and improved capture uniformity. Furthermore, at low concentrations of target molecules, the use of lipid-binding proteins and / or mispids described herein can further improve the uniformity of the flow of even small amounts of target molecules, thereby increasing the sequencing rate of low-concentration samples, which would otherwise normally decrease.

[0026] Additionally or alternatively, in certain examples, lipid-binding molecules and / or mispids can be applied to a nanopore-based tip before the application of the target molecule. In such examples, the lipid-binding molecules and / or mispids are thought to bind to the lipid membrane of the nanopore-based tip, thereby preventing the target molecule from binding prematurely to the lipid membrane at the well or channel inlet as described above. Subsequently, when applied to the tip, fewer target molecules interact with the lipid bilayer of the tip. This similarly results in the above-mentioned improvement, namely a more uniform distribution of target molecules across the membrane (and thus more uniform target molecule capture by a larger number of nanopores).

[0027] In certain cases, the target molecule is a nucleic acid, such as a DNA molecule to be sequenced. In certain cases, the target molecule is a modified nucleic acid sequence, such as a modified DNA sequence. In certain cases, the target molecule is an alternative nucleic acid polymer, and its sequencing determines the sequence of the underlying target nucleic acid sequence of interest. For example, the alternative nucleic acid polymer is Xpandomers (商標)Sequencing by expansion (SBX) techniques may be relied upon, including the fabrication of alternative nucleic acid polymers using Xpandomer("XP") sequences. However, while Xpandomer("XP") sequences are ideally suited for nanopore-based sequencing, the size of these large molecules and / or other modifications to Xpandomer sequences are thought to increase their affinity to the lipid bilayer of the chip wells. Therefore, as described herein, lipid-binding peptides and / or mispids are particularly useful for improving Xpandomer(SBX)-based sequencing.

[0028] In certain cases, the lipid-binding molecule is a lipid-binding protein such as a membrane scaffold protein (MSP) that has affinity for lipid membranes. For example, the scaffold protein may be an apolipoprotein or its derivatives or variants (such as apolipoprotein AI (Apo-AI) or its derivatives or variants). Such apolipoproteins and other nanodisc-forming peptides are amphiphilic molecules that form bands around lipid bilayers to form nanodiscs. That is, the mispids described herein can be made using the same MSPs that are used to make nanodiscs.

[0029] In certain cases, the lipid-binding capacity of MSPs can be altered by changing the amino acid sequence and / or length of the MSP, with shorter proteins generally having a lower affinity for lipids (and thus forming smaller lipid nanoparticles). For example, the MSP sequence may be tuned so that the MSP contains a predetermined secondary structure that optimizes its interaction with the lipid components of the mispid. The lipid-binding capacity of MSPs can also be altered by adjusting other parameters such as the pH and / or salt concentration and / or flow rate of any MSP-lipid-containing solution. In certain cases, the lipid-binding capacity of MSPs is tuned so that they form mispids but do not bind lipids so strongly that they disrupt the lipid bilayer membrane of the tip.

[0030] The lipids used in the preparation of the mispids described herein may be any lipids commonly used in the preparation of mispids. For example, the lipids may be phosphatidylcholine lipids, phosphoethanolamine lipids, or synthetic derivatives or variants thereof. In certain examples, different lipid types may be used to form solutions of mispids containing different lipid types. These solutions of different types of mispids may then be mixed with the target molecule, for example, as described herein. In certain examples, the mispids may be stabilized with one or more non-lipid components, such as cholesterol. In certain examples, synthetic materials such as polymers (e.g., triblock copolymers (TBCs)) may be used instead of the lipids described herein.

[0031] In certain cases, mispids can be fabricated using commercially available kits for preparing nanodiscs and / or their components. For example, instead of using the ratio of MSP to lipids required to produce a homogeneous, water-soluble lipid bilayer (e.g., the ratio commonly used when fabricating nanodiscs), the amount of lipids is substantially increased for mispids beyond these well-described nanodisc ratios. For example, MSPs can be mixed with lipid components in an MSP-to-lipid ratio (MSP:lipid) of about 1:200 to about 1:1300, for example, an MSP-to-lipid ratio of about 1:1200. For example, such a ratio of MSP to lipids results in a heterogeneous (polydisperse) mixture of mispids. This is in contrast to nanodisc mixtures where the MSP-to-lipid ratio is adjusted and controlled to produce a monodisperse mixture of nanodiscs.

[0032] Terms and technical terms The present invention will be described in detail by reference only, using the following definitions and examples. All patents and publications referred to herein, including all sequences disclosed in such patents and publications, are expressly incorporated in their entirety by reference.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which this invention pertains. Such common techniques and methods are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Volumes 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2012 (hereinafter "Sambrook"), and in Current Protocols in Molecular Biology, edited by FMAusubel et al., which was first published in book form in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., regularly supplemented until 2011, and is now published by Wiley & Sons, Inc. and available online in journal form as Current Protocols in Molecular Biology, Volumes 00-130 (1987-2020) in the Wiley Online Library. Each of these documents provides a general dictionary of many of the terms used in this invention. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein. It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them. For the purposes of interpreting this disclosure, the following definitions of terms apply, and where appropriate, terms used in the singular form also include the plural form, and vice versa.

[0034] Unless otherwise specified, nucleic acids are written from left to right in the 5' to 3' direction. Amino acid sequences are written from left to right in the amino to carboxyl direction.

[0035] The headings provided herein are not limitations on the various aspects or embodiments of the invention that can be obtained by referring to the entire specification. Accordingly, the terms defined below are further defined by referring to the entire specification.

[0036] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise.

[0037] In this specification, a range can be expressed as "approximately" from a certain value and / or to another specific value of "approximately". When such a range is expressed, the other aspect includes that certain value and / or that other specific value. It will be further understood that the endpoint of each range is significant both in relation to the other endpoint and independently of the other endpoint. Similarly, it will be understood that when a value is expressed as an approximation by the use of the antecedent "approximately", the specific value forms the other aspect.

[0038] In certain exemplary embodiments, the term “approximately” is understood as within the normal tolerance range in the art, for example, within two standard deviations of the mean. “Approximately” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all numerical values ​​provided herein can be modified by the term “approximately.” Furthermore, terms used herein such as “example,” “exemplary,” or “exemplified” are not intended to indicate priority, but rather to indicate that the embodiments described later are merely examples of the embodiments proposed.

[0039] As used herein, “membrane” refers to a component of a sensor or detection system or device. As those skilled in the art will understand, a membrane (e.g., a lipid membrane or lipid bilayer) is a thin film that separates two compartments or reservoirs (e.g., a cis chamber and a trans chamber) and prevents the free diffusion of ions and other molecules between them. In certain exemplary embodiments, the membrane is preferably a lipid bilayer. Lipid bilayers are a model of cell membranes and are widely used for experimental purposes.

[0040] A suitable membrane is an amphiphilic layer formed of amphiphilic molecules, i.e., molecules possessing both hydrophilic and lipophilic properties. Such amphiphilic molecules may be naturally occurring, such as phospholipids, or synthetic. Exemplary amphiphilic substances include various phospholipids, 1,2-difitanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), palmitoyl-oleoyl-phosphatidylcholine (POPC), dioleoyl-phosphatidylmethyl ester (DOPME), 1,2-difitanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin. Examples of synthetic amphiphilic molecules include poly(n-butyl methacrylate-phosphorylcholine), poly(esteramide)-phosphorylcholine, polylactide-phosphorylcholine, polyethylene glycol-poly(caprolactone)-di- or tri-block, polyethylene glycol-polylactide-di- or tri-block, and polyethylene glycol-poly(lactide-glycolide)-di- or tri-block.

[0041] As used herein, “lipid component” refers to the lipid portion of the mispid contained within the mispid's stabilizing protein belt.

[0042] As used herein, the term “mispid” refers to a heterogeneous mixture of lipids and lipid-binding protein complexes of varying sizes and stoichiometric concentrations, which may include lipids available in different layers and structures. In certain exemplary embodiments, the lipid-binding protein comprises one or more alpha-helices.

[0043] As used herein, the term “nanodisc” refers to at least one phospholipid bilayer stabilized by at least one lipid-binding molecule, such as a membrane scaffold protein. See Bayburt, T. et al., Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer. J. Struct. Biol. (1998), 123(1):37-44, and Civjan, N. et al. Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers. BioTechniques (2003) 35:556-563, both of which are expressly incorporated herein by reference in their entirety. For example, the lipid-binding molecule may be a lipid-binding protein (e.g., an apolipoprotein or a portion thereof), such as a membrane scaffold protein. Generally, nanodiscs have a diameter of less than 1 micron. Also, when used in relation to sequencing reactions, nanodiscs contain nanopores. Furthermore, a solution or mixture of nanodiscs generally refers to a monodisperse and stable lipid bilayer having approximately the same amount of lipid per belt of MSP.

[0044] As used herein, the term “nanopore protein” refers to polypeptide subunits and multimers of subunits that can create membrane-penetrating openings when appropriate higher-order structures are formed. Nanopore protein may also refer to a single polypeptide subunit of a multimer nanopore protein or different oligomeric forms of a single polypeptide subunit. “Mixture of nanopore proteins” refers to a solution that may contain heterogeneous combinations of single and / or oligomeric forms of nanopore proteins. “Natural nanopore protein” refers to the native higher-order state of subunit oligomerization that can form functional nanopores within a membrane. Examples of nanopore proteins, or biological nanopores, include α-hemolysin, Mycobacterium smegmatis porin A (MspA), aerolysin, phi29, gramicidin A, maltoporin, OmpG, OmpF, OmpC, Vibrio cholerae cytolysin, PhoE, Tsx, and F-pilus.

[0045] The preferred nanopore protein is α-hemolysin (α-HL). α-HL is the primary cytotoxic substance released by the bacterium Staphylococcus aureus and is the first identified member of the pores that form the beta-barrel toxin family. This toxin consists mostly of beta-sheets (68%) with only about 10% α-helices. The hla gene on the S. aureus chromosome encodes a 293-residue protein monomer that forms a heptameric oligomer in the cell membrane to form a complete beta-barrel pore. Therefore, the native α-HL nanopore protein is an assembly, i.e., an oligomer, of seven α-HL protein monomers.

[0046] As used herein, the term “nanopore” generally refers to a pore, channel, opening, or passage of a specific size that is formed in or otherwise provided in a membrane composed of nanopore proteins. The size of a nanopore is such that, for example, when a molecule of interest passes through the opening, its passage can be detected by a signal, such as an electrical signal, such as a change in electric current. A nanopore includes, for example, a nanopore protein or a group of nanopore proteins. For example, a nanopore may include seven α-HL proteins for forming an α-HL nanopore.

[0047] As used herein, the term “membrane scaffolding protein” refers to a protein that can stabilize the phospholipid bilayer within a Missipid by binding to the bilayer periphery. Generally, membrane scaffolding proteins have a hydrophobic surface that can associate with the nonpolar interior of the phospholipid bilayer and a hydrophilic surface that interacts favorably with polar solvents such as aqueous buffers. Membrane scaffolding protein sequences may be naturally occurring, manipulated using recombinant techniques, or constructed de novo. Naturally occurring membrane scaffolding proteins include apolipoproteins, such as cleaved apolipoproteins that are components of lipoproteins. Known classes of apolipoproteins include A (including, e.g., apoA-I and apoA-II), B, C, D, E, and H. Non-natural membrane scaffolding proteins include MSP1 and MSP2, described in U.S. Patent No. 7,691,414, which is incorporated herein by reference in whole. An example of a commercially available, non-naturally occurring MSP is, for example, Sigma (商標) MSP1D1 is available from [source]. Membrane scaffold proteins can be full-length proteins or cleaved proteins. Membrane scaffold proteins are not intended to encompass a wide range of functional membrane proteins, including but not limited to ion channels and other transmembrane receptors, porins, certain cell adhesion molecules, and electron transport proteins such as NADH dehydrogenase and ATP synthase.

[0048] As used herein, the term "DNA" refers to a molecule containing at least one deoxyribonucleotide residue. A "deoxyribonucleotide" is a nucleotide that does not have a hydroxyl group, but instead has a hydrogen atom at the 2' position of the β-D-deoxyribofuranose moiety. The term encompasses double-stranded DNA, single-stranded DNA, DNA having both double-stranded and single-stranded regions, isolated DNA, such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinant DNA, and modified or analog DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides.

[0049] An "isolated" molecule is a nucleic acid molecule that has been separated from at least one other molecule with which it normally associates, for example, in its natural environment. Isolated nucleic acid molecules include those normally found in cells that express nucleic acid molecules, but which are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0050] The term "nucleic acid molecule" includes RNA, DNA, and cDNA molecules. It will be understood that, as a result of the degeneracy of the genetic code, a large number of nucleotide sequences encoding a given protein, such as alpha-hemolysin and / or its variants, can be produced. This invention intends all possible variant nucleotide sequences encoding variant alpha-hemolysin, all of which are possible considering the degeneracy of the genetic code.

[0051] The term "nucleotide" is used herein as is recognized in the art to include natural bases (standards) and modified bases well known in the art. Such bases are generally located at the 1' position of the nucleotide sugar moiety. Nucleotides generally consist of a base, a sugar, and a phosphate group.

[0052] As used herein, the term “lipid” refers to lipid molecules, which may include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, and derivatized lipids, as described in detail below. Lipids can form micelles, monolayers, and dilayers. Lipids can self-assemble with other components to form mispipes. As used herein, the term “phospholipid” refers to a hydrophobic molecule containing at least one phosphorus group. For example, phospholipids may contain a phosphorus-containing group and optionally saturated or unsaturated alkyl groups substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.

[0053] As used herein, the term “apolipoprotein” refers to an amphiphilic protein that binds to a lipid to form a lipoprotein. The term “amphiphilic” refers to a molecule that possesses both hydrophilic and hydrophobic properties. Exemplary amphiphilic molecules include molecules that have hydrophobic and hydrophilic regions / parts in their structure. Examples of amphiphilic biomolecules include, but are not limited to, phospholipids, cholesterol, glycolipids, fatty acids, bile acids, saponins, and further lipids that can be identified by those skilled in the art.

[0054] As used herein, “lipoprotein” refers to a biomolecular assembly containing both proteins and lipids. More specifically, in lipoproteins, the protein component surrounds or solubilizes lipid molecules, enabling particle formation. Exemplary lipoproteins include plasma lipoprotein particles, classified as high-density (HDL) and low-density (LDL) lipoproteins that enable the transport of fats in the bloodstream, mitochondrial and chloroplast transmembrane proteins, and bacterial lipoproteins. In particular, although the lipid components of lipoproteins are insoluble in water, their amphiphilicity allows apolipoproteins such as certain apolipoprotein A and apolipoprotein B, as well as other amphiphilic protein molecules, to surround the lipids, creating lipoprotein particles that are themselves water-soluble and can therefore be transported via the aqueous cycle (e.g., blood, lymph in vivo or in vitro).

[0055] Apolipoproteins, known to provide the protein components of lipoproteins, can be divided into six classes and several subclasses based on their different structures and functions. Exemplary apolipoproteins known to be able to form lipoproteins include apolipoprotein A (apoA-I, apoA-II, apoA-IV, and apoA-V), apolipoprotein B (apoB48 and apoB100), apolipoprotein C (apoC-I, apoC-II, apoC-II, and apoC-IV), apolipoprotein D, apolipoprotein E, and apolipoprotein H. For example, apolipoprotein B can form low-density lipoprotein particles, mostly having a beta-sheet structure, and can irreversibly associate with lipid droplets, while apolipoprotein A1 contains an alpha-helix and can reversibly associate with lipid droplets to form high-density lipoprotein particles.

[0056] The term "alpha-helix" or "α-helix" refers to a right-handed or helical conformation (helix) of a polypeptide in which all NH groups in the backbone donate hydrogen bonds to the C=O groups of the amino acid backbone four residues prior, thereby enabling hydrogen bonding. The alpha-helix is ​​a common secondary structure of proteins and is sometimes called the classic Pauling-Corey-Branson alpha-helix. The name 3.613-helix is ​​also used for this type of helix, indicating the number of residues per helical turn, with 13 atoms involved in the ring formed by hydrogen bonding. An "amphiphilic helix" refers to an alpha-helix characterized by the spatial separation of hydrophobic and hydrophilic amino acid residues in hydrophobic and hydrophilic regions typically located on the opposite side of the helix, which makes the alpha-helix amphiphilic. The clustered nonpolar residues can then stabilize lipid molecules and promote the formation of lipoprotein complexes, thereby stabilizing the lipid bilayer conformation that supports the mispid construct.

[0057] As used herein, “polymerase” refers to an enzyme that performs template-directed synthesis of polynucleotides. As used herein, the term also refers to a catalytically active polymerase domain. Generally, this enzyme initiates synthesis at the 3' end of a primer annealed to a polynucleotide template sequence and proceeds toward the 5' end of the template strand. “DNA polymerase” catalyzes the polymerization of deoxynucleotides.

[0058] As used herein, the term "processing capacity" refers to the ability of a nucleic acid modifying enzyme to perform multiple modification reactions while bound to a template or substrate. Processing capacity is generally measured by the number of catalytic events that occur for each binding event.

[0059] As used herein, “purified” means that the molecule is present in the sample at a concentration of at least 95% by weight or at least 98% by weight of the sample containing it. The term “purified” generally refers to subjecting transgenic nucleic acid or protein-containing cells to biochemical purification and / or column chromatography. The term “purified” does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified or “substantially pure” protein preparation is one in which the protein referred to is purer than the protein in its natural environment within the cell or (where appropriate) the production reaction chamber.

[0060] As used herein, “sequence identity” refers to the similarity between two nucleic acid sequences or two amino acid sequences, expressed in units of similarity between sequences, otherwise called sequence identity. Sequence identity is often measured in units of identity (or similarity or homology) percentage, with higher percentages indicating greater similarity between the two sequences. For example, 80% homology means the same as 80% sequence identity determined by a defined algorithm, and therefore homologs of a given sequence have more than 80% sequence identity over a given length of the sequence. Exemplary levels of sequence identity include, for example, 80%, 85%, 90%, 95%, 98%, or more sequence identity to a given sequence, e.g., the coding sequence of any one of the polypeptides of the present invention described herein.

[0061] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith & Waterman Adv.Appl.Math.2:482,1981, Needleman & Wunsch J.Mol.Biol.48:443,1970, Pearson & Lipman Proc.Natl.Acad.Sci.USA 85:2444,1988, Higgins & Sharp Gene 73:237-244,1988, Higgins & Sharp CABIOS 5:151-153,1989, Corpet et al.Nuc.Acids Res.16,10881-90,1988, Huang et al.Computer Appls.In the Biosciences 8,155-65,1992, and Pearson et al.Meth.Mol.Bio.24,307-31,1994.Altschul This is described in et al. (J.Mol.Biol.215:403-410, 1990), and presents a detailed discussion of sequence alignment methods and homology calculations.

[0062] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al. J.Mol.Biol.215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland) and the Internet, for use in conjunction with sequence analysis programs, such as the complete BLAST program suite, including BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN.

[0063] Sequence searches are typically performed using the BLASTN program when evaluating a given nucleic acid sequence by comparing it to GenBank DNA sequences and nucleic acid sequences in other public databases. The BLASTX program is preferred for searching for nucleic acid sequences translated in all reading frames against GenBank protein sequences and amino acid sequences in other public databases. Both BLASTN and BLASTX are run with default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix (see, for example, Altschul, SF, et al., Nucleic Acids Res. 25:3389-3402, 1997).

[0064] In certain exemplary embodiments, the alignment of selected sequences to determine the "% identity" between two or more sequences is performed using the CLUSTAL-W program in MacVector version 13.0.7, which operates with default parameters including, for example, an open-gap penalty of 10.0, an extended-gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0065] Methods, systems, and compositions This specification provides methods, systems, and compositions for improving the sequencing efficiency and throughput of nanopore-based sequencing systems. As described herein, for example, such methods, systems, and compositions can be used to improve the sequencing efficiency and throughput of any nanopore-based sequencing system in which a target molecule can interact with a lipid, for example, a lipid bilayer membrane of the nanopore-based sequencing system, and / or otherwise have an affinity for that lipid.

[0066] Referring to the drawings, throughout the drawings, similar reference numerals indicate similar but not necessarily identical elements, and exemplary embodiments are described in detail. More specifically, Figure 2 is a block flow diagram showing a method for sequencing a target molecule in the presence of a mispid according to a particular exemplary embodiment. However, while Figure 2 describes the use of a mispid, it should be understood that in certain exemplary embodiments, the lipid-binding proteins described herein can be used alone or in combination with a mispid to achieve the improved target molecule sequencing described herein.

[0067] Referring to Figure 2, in block 105, a mispid composition is prepared for use in the method described herein. Mispids can be prepared according to methods known in the art for preparing nanodiscs, except that a larger amount of lipid is used instead of the smaller amount of lipid typically used when preparing nanodiscs. That is, known methods for preparing nanodiscs can be readily used and adapted to prepare the mispids described herein, for example, by using commercially available components and kits for preparing nanodiscs (but containing larger amounts of lipid). As those skilled in the art will understand, preparing nanodiscs generally involves forming vesicles of lipid components (e.g., vesicles or surfactant-solubilized lipid complexes), followed by the addition of lipid-binding molecules and a solubilizing surfactant, typically sodium cholate. Nanodiscs are formed when the surfactant is removed by either dialysis or size exclusion chromatography, where the lipid-binding molecules act as a "belt" around the lipid bilayer of the lipid components, for example, thereby stabilizing the entire nanodisc structure. Using these same techniques, the mispids described herein can be prepared by modifying the mixture to be a polydisperse mixture of lipid-based complexes, where each complex contains one or more lipids and one or more lipid-binding proteins.

[0068] For example, the lipid-binding molecule may be any naturally occurring or synthetic molecule (e.g., DNA, peptides, or other compounds) that adopts an alpha-helical structure and interacts with or inserts into the lipid bilayer. If the lipid-binding molecule is a protein, it can be synthesized, for example, by methods known in the art. If the lipid-binding molecule is DNA, such a molecule can be prepared, for example, by methods known in the art, as described in Zhao et al., J.Am.Chem.Soc.2018, 140, 34, 10639-10643, which is incorporated herein by reference in whole.

[0069] In certain exemplary embodiments, the lipid-binding molecule is a membrane scaffolding protein (MSP) such as an apolipoprotein or its derivatives or variants (e.g., apolipoprotein AI (Apo-AI) or a variant of its derivative). Exemplary MSPs include, for example, membrane scaffolding protein 1D1 (MSP1D1), membrane scaffolding protein 2N2 (MSP2N2), and membrane scaffolding protein 1E3D1 (MSP1E3), each being Sigma-Aldrich. (商標) It is commercially available from [company name]. Additionally or alternatively, lipid-binding molecules may include derivatives of saposin A, amyloid-beta peptides, α-synuclein derivatives, styrene-maleic anhydride (SMA) acid derivatives that solubilize lipid bilayers, diisobutylene-malate (DIBMA) derivatives, and polyacrylate-co-styrene derivatives. In certain exemplary embodiments, the lipid-binding molecule is a synthetic apolipoprotein, i.e., an apolipoprotein that does not exist in nature, for example, one of those described in U.S. Patent No. 11,279,749, which is incorporated herein by reference in whole.

[0070] In certain exemplary embodiments, the lipid-binding molecule comprises a protein having a predetermined number of alpha helices. As those skilled in the art will understand, such an alpha helix comprises a right-handed or helical conformation (helix) of a polypeptide in which all NH groups of the backbone donate hydrogen bonds to the C=O groups of the amino acid backbone four residues prior, thereby enabling hydrogen bonding. The alpha helix is ​​a common secondary structure of proteins and is sometimes called the classic Pauling-Corey-Branson alpha helix. 3.6 13 The name "helix" is also used for this type of helix, indicating the number of residues per helical turn, with 13 atoms involved in a ring formed by hydrogen bonds.

[0071] In certain exemplary embodiments, the lipid-binding protein comprises 3 to 10 alpha helices, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 alpha helices. Shorter proteins, such as the shorter MSP1D1 protein, can be produced by deleting or partially deleting one or more MSP1D1 alpha helices, as described in Hagn et al., Nat Protoc. 2018. Jan;13(1):79-98, which is incorporated entirely herein. That is, the size of the mispid can be determined by modifying the number of alpha helices. For example, with respect to MSP1D1, the protein may include ΔH4 / 2 deletion, ΔH4 deletion, ΔH5 deletion, ΔH4 / 2ΔH5 deletion, ΔH4H5 deletion, and / or ΔH4-H6 deletion. See Hagn et al., Nat Protoc. 2018 Jan;13(1):79-98. Mispids of different sizes produced from such modified MSP1D1 proteins may, for example, have altered lipid-binding ability to the lipid bilayer.

[0072] While we do not wish to be bound by any particular theory, smaller lipid-binding proteins and the mispids produced therefrom are thought to have lower affinity for nanopore bilayers than those produced using larger lipid-binding proteins. Therefore, in certain exemplary embodiments, the size of lipid-binding proteins and / or mispids can be used to adjust and regulate, for example, the capture and distribution of target molecules flowing on a membrane, such as the membrane of a flow cell. In other words, for example, controlling and managing the size of mispids produced therefrom using lipid-binding proteins can ultimately be used to adjust the flow rate of the membrane in a well, thereby adapting (or regulating) the flow rate of the mispids, and consequently the target molecules, to a given application.

[0073] In certain exemplary embodiments, the flow rate of target molecules on the lipid membrane can be determined by adjusting (i) the length of the lipid-binding protein and / or (ii) the size of the mispids produced therefrom, and / or (iii) the flow rate at which the lipid-binding protein and / or mispids are delivered. That is, each of these parameters can be adjusted to control the distribution of target molecule capture on the surface across the entire flow cell surface. In other words, by manipulating both the length of the lipid-binding protein / mispids and the flow rate at which they are delivered to the bilayer along with the target molecules, the sequencing efficiency across the entire flow cell can be adjusted with respect to the target molecule affinity to the bilayer and improved capture across the flow cell.

[0074] As described herein, a method for preparing nanodiscs that can be readily modified and adapted to form missypids is outlined in its entirety in Li et al., Preparation of Lipid Nanodiscs with Lipid Mixtures, Curr Protoc Protein Sci. 2019 Dec;98(1):e100, which is hereby expressly incorporated by reference, and see also Hagn et al., Nat Protoc. 2018 Jan;13(1):79-98. In certain exemplary embodiments, a lipid-binding protein (MSP) is mixed with lipids selected at a specific ratio in the presence of a solubilizing surfactant (sodium cholate), followed by removal of the surfactant either by dialysis or by use of Bio-Beads to prepare missypids. Additionally or alternatively, the preparation of missypids can include cell-free methods such as those described in U.S. Patent No. 11,053,322 (for nanodiscs), which is hereby incorporated by reference in its entirety.

[0075] In certain exemplary embodiments, the components for making nanodiscs, and thus missypids, are available from Merck KGaA (商標) , Cube Biotech (商標) , Anatrace Products, LLC (商標) , Mempro (商標) , and Avanti Lipids (商標) . Additionally or alternatively, customizable kits for nanodisc assemblies, such as those from Cube Biotech (商標) and Mempro (商標) that use membrane scaffold protein MSP2N2 and phospholipid DMPC as the major components of the lipid bilayer of missypids, are generally available. Mempro (商標)In the kit, for example, self-assembly is used in a two-step process, where phospholipids are first dissolved in mixed lipid-surfactant micelles by a surfactant. The mixed micelles are then converted into phospholipid bilayers by incubation with MSP, after which the surfactant is removed using beads. Upon removal of the surfactant, the effective packing parameter of the surfactant-lipid micelles changes from spherical to planar bilayer shape, and the planar bilayer is stabilized by the addition of MSP.

[0076] Conventional nanodisk formation relies on the optimal molar ratio of lipid-binding molecules (e.g., MSPs) to lipids, which is the primary factor in forming a stable lipid bilayer, lipid type, lipid phase behavior, and lipid phase temperature. However, the mispids described herein increase the amount of lipid relative to the amount of lipid-binding molecules, thereby forming various lipid-MSP complexes that may or may not contain a stable lipid bilayer. Accordingly, in certain exemplary embodiments, mispids for use in the methods and systems described herein have a lipid-binding molecule (e.g., MSP) to lipid ratio of approximately 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000; 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, or 1:2200. This is in contrast to nanodiscs where the ratio of lipid-binding protein to lipid is 1:100, for example, 1:50, 1:60, 1:70, 1:80, or 1:90.

[0077] In certain exemplary embodiments, the molar ratio of lipid-binding molecules (e.g., MSPs) to lipids is 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, for example, about 1:1200. In certain exemplary embodiments, the mispid is a heterogeneous mixture of lipid-binding molecule to lipid component ratios (i.e., lipid-binding molecule:lipid component), such as mixtures having ratios of about 1:500 to about 1:2200, or about 1:1000 to 1:1400, for example, mixtures having ratios of 1:1100, 1:1200, 1:1300, and / or 1:1400, as described herein. In other words, mispids for use in the method described herein may include mixtures having different lipid-binding component to lipid ratios.

[0078] In certain exemplary embodiments, the lipid-binding molecule is the MSP2N2 protein, while the lipid is DPhPE. In such exemplary embodiments, the molar ratio of MSP2N2:DPhPE is 1:200, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000; 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2000, 1:2100, or 1:2200. In certain exemplary embodiments, the molar ratio of MSP2N2:DPhPE is 1:1000, 1:1100, 1:1200, 1:1300, 1:1400, for example, about 1:1200. In certain exemplary embodiments, the mispid is a heterogeneous mixture of MSP2N2:DPhPE ratios, for example, a mixture having a ratio of about 1:500 to about 1:1700 or about 1:1000 to 1:1400, for example, a mixture having a ratio of 1:1100, 1:1200, 1:1300 and / or 1:1400. Needless to say, as described herein, other MSP proteins and / or their variants, such as MSP1D1, MSP1E3D1 and / or MSP1D1ΔH5, can be used in these ratios together with lipid components, such as DPhPE, POPC, DOPME, DPhPC and / or DPPC.

[0079] In certain exemplary embodiments, the size of mispids can be manipulated by adjusting the ratio of lipid-binding protein to lipid component. While not bound by any particular theory, it is thought that a higher ratio of lipid-binding protein to lipid component (e.g., a decrease in the amount of lipid relative to the amount of lipid-binding protein) results in smaller mispids with reduced affinity to a given lipid bilayer. This allows for a greater flow of smaller mispids across the membrane, enabling more uniform (and rapid) distribution of the mispids across the membrane, thereby increasing the capture of target molecules. Conversely, by increasing the amount of lipid component (i.e., by decreasing the ratio of lipid-binding protein to lipid), the resulting larger mispids are thought to have increased affinity to a given lipid bilayer and therefore move more slowly across the flow cell, but nevertheless interact more with the membrane, resulting in the release of more target molecules moving across the cell. Therefore, the ratio of lipid-binding protein to lipid component can be adjusted depending on the application, the properties of the target molecule, the desired flow rate, and the desired target molecule nanopore capture rate. In certain exemplary embodiments, for example, a mixture of mispids with different ratios of lipid-binding protein to lipid component may be preferred. In certain exemplary embodiments, nanodiscs may be used instead of mispids, as described herein.

[0080] In block 110, the mispid is mixed with the target molecule. That is, for example, the mispid is combined with the target molecule before the target molecule is added to the nanopore-based sequencing chip. For example, the mispid described herein can be mixed with the target molecule in any suitable buffer. Non-limiting examples of buffers include phosphates (e.g., PBS), citrates, acetates, glutamates, carbonates, tartrates, triethanolamine (TRIS), glycylglycine, histidine, glycine, lysine, arginine, and other organic acids. More specifically, non-limiting examples of buffers include sodium HEPES, MES, potassium phosphate, potassium thiocyanate, sterilizers, TAE, TBE, ammonium sulfate / HEPES, BuffAR, sodium acetate, sodium carbonate, sodium citrate, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium phosphate. In certain exemplary embodiments, the buffer solutions are 20 mM Tris HCl (pH 8) and 200 mM NaCl, or 100 mM PBS (pH 7.4), 50 mM MES (pH 5.5), and 200 mM NaCl, etc.

[0081] Additionally or alternatively, in certain exemplary embodiments, the mispipd and target molecule can be combined at the flow cell inlet of the sequencing chip without mixing the target molecule and mispipd before applying them to the sequencing chip. That is, the mispipd and target molecule can be added separately to the flow cell inlet of the sequencing chip, and the mispipd and target molecule can be mixed together at the flow cell inlet. In such exemplary embodiments, a flow cell buffer can be used to suspend the target molecule and mispipd. Additionally or alternatively, the mispipd can be applied first to the flow cell inlet and allowed to flow across the flow cell, followed by the target molecule (to flow across the flow cell) at the flow cell inlet. That is, the mispipd and target molecule can be added sequentially.

[0082] The target molecule may be, for example, any nucleic acid sequence, modified nucleic acid sequence, and / or nucleic acid substitute for which detection and / or determination of the target molecule sequence is desired. For example, the target molecule may be a DNA sequence or an RNA sequence, or in some examples, an improved DNA sequence or RNA sequence for improved sequencing or other applications. For example, U.S. Patent No. 10,851,405 describes a modified target molecule containing a hydrophobic capture element. The hydrophobic capture element increases the affinity of the target molecule to a lipid membrane, for example, and thereby improves nanopore capture of the target molecule. See U.S. Patent No. 10,851,405, which is incorporated in its entirety herein. Thus, the target molecule may be a modified target molecule, such as a target molecule containing a hydrophobic capture element or other features that modulate the affinity of the target molecule to a membrane. In other exemplary embodiments, the target molecule may include, for example, a label, tag, or other molecule bound thereto that assists and / or aids sequencing.

[0083] In certain exemplary embodiments, the target molecule may be a nucleic acid substitute whose decoded sequence detects and / or represents the target nucleic acid sequence of interest. That is, if the sequence of the nucleic acid substitute is determined by decoding the substitute's sequence, the determined sequence identifies and provides a nucleic acid sequence corresponding to the target nucleic acid sequence of interest. In other words, the nucleic acid substitute functions, for example, as a substitute sequence for the target sequence of interest.

[0084] In certain exemplary embodiments, the nucleic acid substitute is based on Xpandomer technology, which relies on a nanopore-based sequencing-by-expansion (Nano-SBX) method for decoding the Xpandomer sequence. See, for example, U.S. Patent No. 7,939,259 entitled "High Throughput Nucleic Acid Sequencing by Expansion" and PCT International Publication No. 2020236526 entitled "Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing," both of which are incorporated herein in their entirety. Generally, SBX uses biochemical polymerization to transfer the DNA template sequence onto a measurable polymer called "Xpandomer."

[0085] More specifically, the SBX technology is based on the polymerization of highly modified non-natural nucleotide analogs called "XNTPs." The transcribed sequence is encoded along the Xpandomer backbone in a high-signal-versus-noise reporter approximately 10 nm away using XNTPs, designed for a high-signal-versus-noise, clearly discriminable response. These differences result in a significant improvement in the sequence read efficiency and accuracy of Xpandomer compared to natural DNA. XNTPs are, for example, template-dependent enzymatic polymerization-compatible and expandable 5'-triphosphate modified non-natural nucleotide analogs. Each XNTP has two distinct functional regions: a selectively cleavable phosphoramide bond that binds the 5'-α-phosphate to the nucleic acid base, and a symmetrically synthesized reporter tether (SSRT) bound within a nucleoside triphosphoramide at a position that allows for controlled expansion by cleavage of the phosphoramide bond. The SSRT contains a linker separated by the selectively cleavable phosphoramide bond. Each linker is attached to one end of the reporter code. XNTP substrates are bound to the daughter chain via template-dependent polymerization, but they exist in a "constrained configuration" due to their size. The constrained configuration of polymerized XNTPs is a precursor to the extended configuration, as seen in the Xpandomer product. The transition from the constrained to the extended configuration occurs when the PN bond of the phosphoramide in the primary skeleton of the daughter chain is cleaved.

[0086] During Xpandomer assembly, monomer XNTP substrates (XATP, XCTP, XGTP, and XTTP) are polymerized on the elongable ends of the nascent daughter strands by a template-directed polymerization process using a single-stranded template as a guide. This process, for example, starts from a primer and proceeds in the 5' to 3' direction. Generally, DNA polymerase or other polymerases are used to form the daughter strands, and conditions are selected to obtain complementary copies of the template strand. After the daughter strands are synthesized, the coupled SSRTs form constrained Xpandomers that further form the daughter strands. The SSRTs in the daughter strands have a "constrained configuration" of the XNTP substrates. The constrained configuration of the SSRTs is a precursor to the extended configuration, as seen in the Xpandomer product.

[0087] The transition from a constrained configuration to an extended configuration is due to the cleavage of selectively cleavable phosphoramide bonds (shown by unshaded ellipses for simplification) within the primary backbone of the daughter strands. In this embodiment, the SSRT includes one or more reporters or reporter codes specific to the nucleic acid bases to which they are linked, i.e., A, C, G, or T, thereby encoding (as a substitute) the sequence information of the template. In this way, the SSRT provides a means to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand.

[0088] In certain exemplary embodiments, the target molecule may be a nucleic acid-based reporter region that facilitates the identification of the target molecule. That is, the target molecule may be part of a larger sequence, and sequencing of it detects the presence of the target molecule or identifies it in other ways. For example, the target molecule may include an address region and a probe region as the reporter region. See, for example, U.S. Patent No. 9,850,534, which is incorporated herein in its entirety. In such exemplary embodiments, the target molecule is identified based on the nucleic acid sequence of the address region by sequencing the address region while the target molecule is being guided through a nanopore to determine the nucleic acid sequence of the address region.

[0089] In block 115, the nanopore-based sequencing tip is brought into contact with a mixture of mispid and target molecules. That is, the mixture of mispid and target molecules to be sequenced is applied to the flow cell inlet of the tip (or another port or area of ​​the tip where the sample is placed), thereby bringing the tip into contact with the mixture. For example, if the mispid and target molecules are mixed in a solution, a portion of the solution is applied to the flow cell inlet of the tip, thereby allowing the mispid and target molecules to flow across the tip, for example, in the sequencing reaction.

[0090] Furthermore, as described herein, Mispid improves the capture, arrival time, and effective concentration of target molecules in nanopore-based sequencing chips containing lipid membranes, so it is considered that any chip having such a lipid membrane can be used with the method and system described herein. In other words, the method and system described herein can be usefully used with hydrophobic bilayers, particularly lipid bilayers. This includes, for example, exemplary chips having CMOS from TSMC and microwells from TSI (Roseville).

[0091] As those skilled in the art will understand, nanopore-based chip devices for detecting nucleic acids have been developed for rapid sequencing, and various designs and uses are known in the art. See, for example, U.S. Patents 9,494,554, 9,567,630, 9,557,294, and 9,605,309, each of which is incorporated herein by reference in whole. These devices generally include an electrochemical cell having a chamber containing nanopores embedded in a membrane. The membrane acts to separate the cell chamber into two sub-chambers called the cis and transform sides of the cell, each containing electrodes. The membrane may be an organic membrane such as a lipid bilayer, or a synthetic membrane made of a polymer material that does not exist naturally.

[0092] The pores of the nanopores act as channels (or passages) within the membrane between the cis and trans sides of the cell. Depending on the nanopore used, the pore may have a width or diameter ranging from about 1 angstrom to about 10,000 angstroms. The nanopores may be naturally occurring pore-forming proteins such as α-hemolysin from Staphylococcus aureus (S. aureus), or non-naturally occurring variants or variants of wild-type pore-forming proteins. A range of naturally occurring and non-naturally occurring nanopores with various pore sizes and properties is known in the art. See, for example, U.S. Patents 1,0351908, 1,0934582, and 1,0227645, each of which is incorporated herein by reference in whole. Within an electrochemical cell, the membrane-embedded nanopores are positioned in close proximity to electrodes coupled to a detection circuit, such as a complementary metal-oxide-semiconductor (CMOS) or field-effect transistor (FET) circuit. The detection electrode determines the signal generated from the nanopore when, for example, the target molecule traverses the nanopore from the cis side to the trans side.

[0093] Electrochemical cells for nanopore-based sequencing of nucleic acids are typically used in a large-scale parallel configuration, where thousands of such cells are configured as an array within a single device, often referred to as a chip (or biochip). Indeed, the use of mispids, as described herein, to improve sequencing efficiency and throughput is considered applicable to synthetic nanopore-based sequencing (Nano-SBS). A typical nanopore-based sequencing chip device may incorporate an array of more than one million electrochemical cells, containing 1000 rows x 1000 columns of such cells (see, for example, a chip manufactured by Roche Sequencing Solutions in Santa Clara, California, USA). Methods for manufacturing and using such nanopore array microchips (Nano-SBS) can also be found in U.S. Patent Applications Publications 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870, 20190085386, as well as International Publication 2019 / 166457, which are incorporated herein by reference in their entirety.

[0094] Each well in the array can be manufactured using a standard CMOS process with surface modifications that allow constant contact between the bioreagent and the conductive salt. Each well can support a phospholipid bilayer film embedded with a nanopore conjugate as part of an integrated circuit. The integrated circuit may be an application-specific integrated circuit (ASIC). In certain exemplary embodiments, the integrated circuit is a field-effect transistor or a CMOS device. The detection circuit may be located within or outside the chip or other device having nanopores, for example, in an off-chip configuration. The semiconductor may be any semiconductor, including but not limited to Group IV (e.g., silicon) and Group III-V semiconductors (e.g., gallium arsenide) (see, for example, International Publication 2013 / 123450). Furthermore, the electrodes in each well are individually addressable by a computer interface. All reagents used are generally introduced into a simple flow cell above the array microchip using a computer-controlled syringe pump. The chip supports analog-to-digital conversion and reports electrical measurements independently from all electrodes at a rate exceeding 1000 points / second. Nanopore measurements can be performed asynchronously at least once every 1 millisecond (msec) on each of the 8M addressable nanopore-containing films in the array and recorded on an interfaced computer.

[0095] In block 120, the target molecule is sequenced. That is, the sequencing reaction is performed on the chip via the chip manufacturer's protocol or other protocols known in the art. For example, when a solution containing a mixture of mispid and the target molecule is applied to the chip, the solution containing the mixture of mispid and the target molecule moves or "flows" across the chip, bringing the target molecule and mispid into contact with the lipid membrane of the chip's wells. Subsequently, when a potential is applied (via electrodes) across a nanopore immersed in a conductive fluid, a small current can be observed due to the flow of ions through the nanopore. This ion flow is sensitive to the pore size, and therefore, molecules entering the pore affect the ion flow and voltage measured through this sensor circuit. If the target molecule is Xpandomer, for example, as the Xpandomer product extends and passes through the nanopore, the excited fluorophore associated with the Xpandomer emits a detectable signal. This signal is temporally discrete as a function of the tether length and the velocity of the Xpandomer passing through the nanopore. For example, see U.S. Patent No. 7,939,259.

[0096] In block 125, the target nucleic acid sequence is determined. That is, the nucleotide sequence of the underlying target nucleic acid is determined (e.g., decoded) from signals associated with the sequencing reaction, for example, by using techniques known in the art. See, for example, U.S. Patent Application Publication 7,939,259, U.S. Patent Application Publications 2013 / 0244340, 2013 / 0264207, 2014 / 0134616, 2015 / 0368710, and 2018 / 0057870, 20190085386, and International Publication 2019 / 166457. Furthermore, if the target molecule is a DNA strand and the non-coding strand is sequenced, the coding strand sequence may be determined, thereby providing the nucleic acid sequence of the target nucleic acid sequence. Similarly, if the target molecule is Xpandomer, the Xpandomer sequence is used to determine the underlying nucleic acid sequence.

[0097] The exemplary embodiments described above relate to the preparation and use of mispids for use in the methods provided herein, but in certain exemplary embodiments, lipid-binding molecules can be mixed with target molecules without preparing mispids as described in Block 105 to improve the sequencing efficiency and throughput of nanopore-based sequencing systems. That is, in certain exemplary embodiments, the lipid-binding molecules described herein are used alone with target molecules without the need to use mispids. In such exemplary embodiments, the lipid-binding molecules may be any of the lipid-binding molecules described herein, for example, any of the MSPs described herein.

[0098] Furthermore, in such exemplary embodiments, the methods described herein with respect to blocks 110-115 are modified to illustrate the use of lipid-binding molecules. For example, in block 110, the lipid-binding molecule is combined with the target molecule in a buffer suitable for both the lipid-binding molecule and the target molecule. Then, in block 115, the mixture of lipid-binding molecule and target molecule is applied to a sequencing chip. That is, the mixture is introduced, for example, into the flow cell inlet of the sequencing chip so that the mixture can come into contact with the chip and flow on the flow cell. In certain exemplary embodiments, the lipid-binding molecule and target molecule may be mixed first at the flow cell inlet, i.e., there is no mixing of the lipid-binding molecule and target molecule before they are applied to the chip (rather, they are mixed, for example, at the flow cell inlet of the chip). Nevertheless, once the chip comes into contact with the mixture of lipid-binding molecule and target molecule, the target molecule can be sequenced as shown in block 120.

[0099] While we do not wish to be bound by any particular theory, it has been found that during target molecule sequencing reactions, for example, when sequencing Xpandomer, target molecules with affinity for lipids, such as the lipid bilayer of the sequencing chip, tend to accumulate in the wells closest to the flow cell inlet. These wells are, for example, closest to the flow cell inlet and are therefore the first wells to be exposed to such target molecules, thereby providing the first opportunity for the target molecules to interact with their lipid bilayer. As more target molecules accumulate, the free flow of target molecules across the membrane is hindered. That is, during the sequencing reaction, the free flow of target molecules slows down and decreases, thereby reducing the uniform movement and distribution of target molecules across the membrane. As a result, the distribution of target molecules trapped in the nanopores is less uniform across the membrane, resulting in lower sequencing efficiency and reduced throughput. And needless to say, in certain exemplary embodiments, both lipid-binding molecules and mispids can be mixed with the target molecules, the mixture applied to the chip, and then sequenced as described herein.

[0100] By bringing the tip into contact with lipid-binding molecules and / or mispipes and performing a sequencing reaction in the presence of the lipid-binding molecules and / or mispipes, the lipid-binding molecules and / or mispipes flow across the tip along with the target molecules. Furthermore, although not bound by any particular theory, it is thought that the lipid-binding molecules and / or mispipes bind to the lipid membrane of the nanopore-based tip, thereby preventing the target molecules from prematurely accumulating in the wells at the inlet of the flow cell channel. For example, the lipid-binding molecules and / or mispipes may compete with the target molecules for interaction with the well lipid bilayer, thereby reducing the interaction between the target molecules and the lipid bilayer. Additionally or alternatively, it is further conceivable that the target molecules interact with, for example, the lipid-binding molecules and / or mispipes, thereby further reducing undesirable interactions between the target molecules and the lipid bilayer of the tip.

[0101] While the embodiments provided herein include those relating to nanopore-based sequencing applications, such as the modification of interactions between a target molecule to be sequenced and a lipid membrane of a nanopore-based sequencing chip, it should be understood that the methods, systems, and compositions provided herein can be broadly used to modify interactions between target molecules and lipid membranes. For example, if a target molecule is modified to include a hydrophobic capture element, as described in U.S. Patent No. 10,851,405, which is entirely incorporated herein, the interaction between the modified target molecule and the lipid membrane can be manipulated using the lipid-binding proteins and / or mispids provided herein. That is, the mispids compete with the target molecule for space on the lipid bilayer, and if the target molecule distribution across the flow cell is insufficient, the mispids can be used to manipulate / improve the capture distribution, thereby shortening the sequencing time. In other exemplary embodiments, for example, by delivering membrane proteins and / or specific lipid components to a general lipid bilayer (lipidomics), the mispids can be used to directly modify components of the lipid bilayer, thereby modifying the pore behavior.

[0102] As is evident from this disclosure, the methods and systems described herein are particularly useful when the concentration of the target molecule is low. For example, by sequencing the target molecule as described herein in the presence of a lipid-binding protein and / or mispid as described herein, more target molecules in a given amount of solution applied to the sequencing chip are allowed to flow freely across the chip. In other words, fewer of the originally small amount of target molecules bind to the lipid membrane of the chip's wells, allowing more target molecules to flow down the chip and interact with the nanopores. Thus, especially when the concentration of the target molecule is low, the methods and systems described herein can improve the uniformity of the flow of the small amount of target molecules, thereby increasing the sequencing rate of low-concentration samples, which would normally decrease.

[0103] Those skilled in the art will likely consider these and other related embodiments in light of this disclosure. [Examples]

[0104] The following embodiments further illustrate the present invention but should not be construed as limiting its scope. In light of this disclosure and the general level of those skilled in the art, those skilled in the art will understand that the following embodiments are for illustrative purposes only and that numerous changes, modifications, and alterations can be made without departing from the subject matter of this disclosure.

[0105] As used herein, the following abbreviations apply: eq (equation), M (mol), μM (micromol), N (standard), mol (mol), mmol (millimole), μmol (micromol), nmol (nanomole), g (gram), mg (milligram), kg (kilogram), μg (microgram), L (liter), ml (milliliters), μl (microliters), cm (centimeter), mm (millimeter), μm (micrometer), nm (nanometer), °C (degrees Celsius), h (hour), min (minute), sec (second), msec (millisecond).

[0106] Example 1 -- Preparation of mispip This example describes the preparation of mispids. Briefly, membrane scaffold proteins (MSPs) and MSP variants were selected to cover a wide range of lipid capacities for testing, i.e., to cover MSP2N2 with a higher lipid binding capacity compared to MSPΔH5 with a lower lipid capacity.

[0107] To prepare the mispid, purified MSP protein suspended in 20 mM TriHCl (pH 8) / 200 mM NaCl buffer was directly mixed with lipids solubilized in sodium cholate in a 1:200 ratio. The mixture of MSP, lipids, and sodium cholate was dialyzed overnight in 20 mM TriHCl (pH 8) / 200 mM NaCl buffer at temperatures below the phase transition temperatures of the individual lipids. Upon removal of the surfactant from the mixture, the mispid spontaneously formed.

[0108] Next, the MSP2N2:DPhPE (1:200 molar ratio) mispid was dialyzed overnight and further purified by size exclusion chromatography using a Superdex 200 column (20 mM Tris, pH 8, in 200 mM NaCl) to obtain fractions of varying purities (Figure 3A). As shown, the main peak of the mispid preparation roughly corresponds to elution at pore volume and exhibits a very large and heterogeneous distribution of particles.

[0109] Figure 3B shows gel electrophoresis of fractions eluted from size exclusion chromatography purification of a 1:200 MSP2N2:DPhPE mispit preparation. All lanes contain approximately 40 kDa MSP2N2 protein. Monodisperse and highly homogeneous nanodisk fractions, where the majority of lipids and MSPs are in a specified stable ratio, elute from the column in a clearly defined retention volume. However, less pure fractions may also be observed, and in this application, these less pure fractions can be successfully used as demonstrated by the mispit MSP2N2 preparation. Optimal fractions can be tested directly during sequencing and stored at 4°C for two weeks. Higher lipid content in the mixture significantly reduces the formation of stable nanodisks.

[0110] Example 2 -- Target molecule sequencing with and without mispids This example describes the sequencing reaction of the Xpandomer sequence with and without the inclusion of a mispid in the sequencing reaction.

[0111] For the sequencing reaction, a target molecule / mispid mixture was prepared using the mispid from Example 1. Briefly, the size-excluded and purified mispid from Example 1 (20 mM TriHCl (pH 8) / 200 mM NaCl) was directly mixed with Xpandomer stored in 10–40% acetonitrile / 1–10% trehalose, and then diluted with Xpandomer dilution buffer (100 mM MES, pH 6.2, 842 mM urea, 5.26% PEG8k, 0.16% trehalose, 1.05 M NH4Cl, 158 mM K3Fe(CN)6, 158 mM K4Fe(CN)6). The optimal mispid:Xpandomer ratio depends on the total lipid and MSP2N2 concentrations, but a typical amount is used: 1–2 μL per 10 μL of Xpandomer.

[0112] For Xpandomer sequencing, sequencing was performed using a mixture of Xpandomer / Mispid mixed according to Example 2, in accordance with International Publication No. 2020236526, in a sequencing reaction containing Mispid. Sequencing was performed and tested with or without Mispid, or with various concentrations of Mispid at 1.4%, 2.4%, and 4.8% of the total volume of Xpandomer used. Mispid was found to improve sequencing performance without affecting sequencing accuracy. For example, a concentration of 1.4% of the total Xpandomer volume performed better than Xpandomer sequencing without Mispid, with the following median metrics. num_hqmt_count is 76 when no mispids are used and 140 when 1.4% mispids are used; num_ahqmt_per_functional_min_per_cel is 2.55 when no mispids are used and 4.72 when 1.4% mispids are used; extended_arrival_rate is 0.29 when no mispids are used and 0.53 when 1.4% mispids are used; align_edit_pct_identical is 97.7 when no mispids are used and 97.7 when 1.4% mispids are used.

[0113] The improved Xpandomer sequencing performance in the presence of mispids is shown in Figures 4A-4C and 5A-5C. For example, a 1.4% mispid (orange) with Xpandomer results in nearly a twofold improvement in throughput compared to Xpandomer alone (shown in blue, throughput (1) on the left) (Figure 4A). In terms of reach, a 1.4% mispid concentration with Xpandomer (throughput (2) shown in orange) is nearly twice as fast as Xpandomer alone (throughput (1) shown in blue on the left) (Figure 4B). Furthermore, it is noteworthy that the addition of mispids (1.4%) does not affect sequencing accuracy (Figure 4C).

[0114] Referring to Figures 5A–5C, a series of heatmaps are shown illustrating the spatial distribution of different metrics representing Xpandomer capture along the entire flow cell according to a particular exemplary embodiment. More specifically, Figure 5A shows four heatmaps (1, 2, 3, and 4) illustrating capture across the entire surface of a sequencer flow cell control Xpandomer molecular flow (i.e., 95% Xpandomer diluted buffer and 5% concentrated Xpandomer) without mispiping. The physical location of each cell in the flow cell is shown by plotting cell_id_row on the y axis against columns on the x axis. The two heatmaps below (3 and 4) show the capture metrics, clearly illustrating a concentration gradient where Xpandomer is highest (red) on the left side (columns 0–1000) where the inlet is located, and decreases (blue) towards the outlet (right).

[0115] Figure 5B shows four heatmaps (1-4) similar to Figure 5A, but with mispid present in the flowing solution (i.e., 90.48% Xpandomer diluted buffer, 4.93% concentrated Xpandomer, and 1.41% 1:200 MSP2N2:DPhPE mispid preparation). Figure 5C also shows four heatmaps (1-4), but with a higher concentration of mispid (i.e., 90.48% Xpandomer diluted buffer, 4.76% concentrated Xpandomer, and 4.76% 1:200 MSP2N2:DPhPE mispid preparation). As can be seen, the two lower heatmaps in Figure 5B (i.e., heatmaps 3 and 4) show a substantial change in the capture distribution in the presence of mispid. The capture of Xpandomer is also more uniformly distributed along the flow cell, as indicated by the red cells in columns 3000-4000.

[0116] Regarding Figure 5C, four heatmaps (1-4) are again shown, but with higher concentrations of mispid (i.e., 90.48% Xpandomer diluted buffer, 4.76% concentrated Xpandomer, and 4.76% 1:200 MSP2N2:DPhPE mispid preparation). As seen in the two heatmaps below (i.e., heatmaps 3 and 4), when higher levels of mispid are mixed with Xpandomer, the capture distribution shifts to the right. That is, the capture distribution is pushed towards the exit, as indicated by the red cells in columns 2500-4000. For example, as indicated by the lower capture cells in columns 0-2000 (blue), it is thought that higher mispid concentrations outperform Xpandomer for space on the bilayer. However, overall, as can be seen by comparing heatmaps 3 and 4 in Figures 5A and 5B, the addition of a 1.41% mispid preparation (Figure 5B) resulted in more uniform capture of Xpandomer molecules across the flow cell compared to the control (Figure 5A), while a larger amount of mispid (4.76%, Figure 5C) resulted in reduced capture compared to the control (Figure 5A).

[0117] Sequencing was performed according to the following parameters and settings: Pore: P-0445 loading concentration 0.4 nM, Xpandomer concentration approximately 1.25 nM, Waveform: SBX3a, Sequencing time: 30 minutes, Labcodes branch: PEG_6.0.1_SBX, Labcodes distribution / release: rel / 5.4.2, NS parameters: 6.0.1 / SBX_PEG, ACAP: Docker 16 (sbx-dev-ms2-conda-fc6514f) 1% subsampling, run, cycle: 4 stations x 4 cycles, single lane multicycle Xpandomer Manual Mix, N=4. High quality: Xpandomer molecules with sequence accuracy exceeding 90% and length exceeding 40 nucleotides. Molecular trace (mt): Historical representation of the SBX signal showing Xpandomer nucleotides passing through the pore. num_hqmt_count: Number of high-quality molecular traces (mt), num_ahqmt_per_functional_min_per_cell: Number of high-quality molecular traces in a single pore / cell during the observed functional lifetime, extended_arrival_rate: Time between the start of Xpandomer captured by a pore and the time until the next Xpandomer is captured by the same pore.

[0118] Example 3 - Target molecule sequencing with and without lipid-binding proteins This example describes the sequencing reaction of a target molecule in the presence of lipid-binding proteins, i.e., lipid-binding proteins that are not associated with lipid components that form mispids. That is, to demonstrate that improvements in sequencing and capture can be achieved using lipid-binding proteins, and to evaluate the sequencing metrics of Example 2, which does not contain lipid components, the experiment was conducted in the same manner as in Example 2, but using only the lipid-binding protein saposin A. In short, Xpandomer sequencing was performed using a 1 μL Xpandomer flow, without saposin, and with saposin at various concentrations of 1.25, 2.5, 5, 10, and 20 μM, according to International Publication No. 2020236526 (see Example 2), and the following sequencing conditions were used. Pore: P-0445 load concentration 0.4nM, Xpandomer concentration approx. 1.25nM, Waveform: SBX3a, Sequencing time: 30 minutes, Labcodes branch: PEG_6.0.1_SBX, Labcodes distribution / release: rel / 5.4.2, NS parameters: 6.0.1 / SBX_PEG, ACAP: Docker 16 (sbx-dev-ms2-conda-fc6514f) 1% subsampling, Execution, Cycle: 4 stations x 4 cycles, Single lane multicycle Xpandomer Manual Mix, N=4.

[0119] Figures 6A–6C include a heatmap (Figure 6A) showing the spatial distribution of Xpandomer molecule capture frequencies along a flow cell for a specific exemplary embodiment, and histograms (Figures 6B–6C) of Xpandomer capture metrics in the presence of increasing concentrations of saposin (lipid-binding protein only). More specifically, Figure 6A shows the Xpandomer capture distribution along flow cells from column 0 to 4000 when treated with increasing concentrations of saposin. All conditions performed better than Xpandomer alone (lane 1), but the 20 μM concentration performed best (lane 6). Figure 6B shows the overall Xpandomer capture for each Xpandomer and saposin condition, with the following metrics detailing the highest improvement of 2.97 for the 20 μM saposin condition compared to 2.04 for the control without saposin. Figure 6C shows the overall Xpandomer capture rate for each Xpandomer condition and increasing saposin concentration with the following metrics, detailing the highest improvement of 0.929 for the 20uM saposin condition compared to 0.397 for the saposin-free control. As shown, a higher and more uniform capture distribution correlates with saposin concentration, with the 20uM concentration exhibiting the highest capture rate and a more uniform flow cell distribution.

[0120] Example 4 -- Sequencing of target molecule (FauXmer) This example describes a sequencing reaction of a target molecule that is not an Xpandomer. That is, to demonstrate the usefulness of mispids for improving the capture of non-Xpandomer molecules, the inventors used a fauXmer molecule. For example, fauXmer contains an enrichment element that plays a role in capturing the Xpandomer molecule in a bilayer. fauXmer has the following sequence (SEQ ID NO: 1). [ka] In experiments using fauXmer, fauXmer is used instead of Xpandomer, and the performance metrics focus primarily on capture rate and fauXmer number, as opposed to precision and mts (molecular traces).

[0121] In short, sequencing was performed using fauXmer and various amounts of mispid (0.5, 1, 2, 4, and 8 μL) added to fauXmer diluted to 25 nM fauXmer in Xpandomer dilution buffer. Samples were tested using the following sequencing conditions and settings: Rev.5 baseline buffer pH 7.4 HEPES, waveform: SBX3a (santa clara baseline), membrane: PE:Span80 lipid, sequencing time: 30 min, short controller, Xpandomer lot: FauXmer, ACAP analysis: fauXmer, 1% subsampling used, 4 stations × 6 cycles, N=6. In the following metrics used to define yield and efficiency, the 0.5 μL mispid spike-in showed superior performance compared to the control. The mt_count was 20 for the fauXmer control and 40 for the 0.5uL spike-in, and the throughput was 1.97 for the fauXmer control and 3.78 for the 0.5uL spike-in. The sequencing conditions and parameters were as described in Examples 1 and 2.

[0122] Figures 7A and 7B include a heatmap (Figure 7A) showing the spatial capture distribution of the alternative molecule fauXmer in the presence of increasing mispid concentrations according to a particular exemplary embodiment, and a histogram (Figure 7B) showing the total number of fauXmers (mt_count). More specifically, Figure 7A shows a fauXmer capture heatmap (lanes 1-6) showing the distribution of captures across the flow cell (columns 0-4000) when fauXmer is mixed with increasing concentrations of mispid. The maximum amount of capture occurs from 0 to 3000 with a spike in 0.5 μL of mispid, compared to the capture distributed from column 0 to 1000 for the fauXmer control. Figure 7B shows a histogram of the total number of fauXmers (mt_count) for five conditions in which fauXmer was mixed with a fauXmer control and mispid, where the number of fauXmers was highest at 40 under the 0.5 μL mispid condition compared to 20 under the fauXmer control without mispid. Mispid dramatically improves the fauXmer capture distribution across the flow cell. Higher concentrations of mispid reduce fauXmer capture.

[0123] Example 5 -- A sequencing reaction showing no mispipe flow across the cell following the target molecule. This example describes experimental setups and results demonstrating that the mispid / saposin mechanism is due to interaction with the bilayer. Theoretically, if the mispid and target molecule are not mixed together, the bilayer-mediated mechanism has the same improvement in Xpandomer capture. To test this theory, these experiments required the use of a “two-flow” lab code, which allowed for two consecutive flows: a first flow of other molecules that could affect sequencing, followed by a second flow of Xpandomer (with or without additives). Conditions 3, 4, and 5, performed with the two-flow lab code, are compared to standard single-flow experiments (conditions 1 and 2) and are detailed in Table 1. [Table 1]

[0124] This experiment demonstrates that the mispid effect is primarily due to interaction with the bilayer.

[0125] Figures 8A and 8B include a histogram (Figure 8A) showing the total number of high-quality Xpandomer captures (num_ahqmt_per_functional_min_per_cell) and a heatmap (Figure 8B) showing Xpandomer captures across the entire flow cell, according to a specific exemplary embodiment. More specifically, Figure 8A shows a histogram of the total number of high-quality Xpandomer captures (num_ahqmt_per_functional_min_per_cell) for each condition tested, where throughput is improved compared to Xpandomer control alone when mispid is flowed first and Xpandomer is flowed second (red and purple histograms compared to green histogram). As shown, Xpandomer throughput does not change when the two flow settings (blue and green) and flow rates are changed (red and purple). Although throughput improvement over the control is seen in all mispid conditions, mispid directly added to Xpandomer continues to perform better than directly modifying the bilayer (orange vs red / green). Figure 8B demonstrates Xpandomer capture across flow cells 0-4000 in columns 1-5 in lanes 1-5 for each condition listed in Table 1. The comparison of lanes 3, 4, and 5 shows that the Xpandomer capture location across the flow cells can be manipulated by changing the flow rate. Heatmaps 1 and 3 in Figure 8B show that the Xpandomer capture distribution does not change in the two flow settings, while heatmaps 4 and 5 show that the capture distribution changes dramatically as the flow rate changes, demonstrating the ability to control the Xpandomer capture location along the flow cells.

[0126] This experiment clearly demonstrates the bilayer-mediated mechanism of the mispid / saposin effect, but the improved throughput of directly mixing Xpandomer with mispid (orange histogram in Figure 8A and lane 2 in Figure 8B) remains the best result and is the subject of ongoing experiments. The sequencing conditions for this experiment were as follows: Rev.5 baseline buffer: pH 7.4 HEPES, waveform: SBX3a (Santa Clara Baseline), sequencing time: 30 minutes, short controller, Xpandomer lot: PXPC00043, ACAP analysis: Docker 19, using 1% subsampling, 4 stations x 5 cycles.

[0127] Considering the many possible embodiments to which the principles of the invention of this disclosure may be applied, it should be understood that the illustrated exemplary embodiments are merely preferred examples of the invention and should not be construed as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Accordingly, the inventors claim as their invention everything that falls within the scope of these claims and spirit.

Claims

1. A method for sequencing target molecules, The present invention provides a chip comprising a plurality of wells, each well comprising a detection electrode, a lipid membrane positioned adjacent to or near the detection electrode, and a nanopore positioned within the lipid membrane. The chip is brought into contact with multiple mispipes and target molecules, Applying a voltage to both ends of the film of the chip, One or more of the aforementioned detection electrodes are used to measure one or more changes in current or voltage related to the nanopore, With the help of a computer processor, the arrangement of the target molecule is determined based on one or more of the measured changes in current or voltage associated with the nanopore, Methods that include...

2. The method according to claim 1, wherein the target molecule is a nucleic acid, a modified nucleic acid, or a nucleic acid substitute.

3. The method according to claim 2, wherein the nucleic acid substitute comprises Xpandomer.

4. The method according to any one of claims 1 to 3, wherein the determined sequence of the target molecule determines the sequence of the target nucleic acid sequence.

5. The method according to any one of claims 1 to 4, wherein the plurality of mispids are prepared by contacting a plurality of lipid-binding proteins with a lipid component to form the plurality of mispids.

6. The method according to claim 5, wherein the lipid-binding protein is a membrane scaffold protein (MSP).

7. The method according to claim 6, wherein the MSP is an apolipoprotein or a derivative thereof.

8. The method according to claim 7, wherein the MSP is apolipoprotein A1 or a derivative thereof.

9. The method according to any one of claims 5 to 8, wherein the lipid-binding protein comprises a predetermined number of alpha helices.

10. The method according to claim 9, wherein the predetermined number of alpha helices is 3 to 10.

11. The method according to any one of claims 5 to 10, wherein the ratio of the lipid-binding protein to the lipid component is about 1:200 to 1:1500.

12. The method according to claim 11, wherein the ratio of the lipid-binding protein to the lipid component is approximately 1:1200.

13. The method according to any one of claims 5 to 12, wherein the lipid component comprises a phosphatidylcholine-based lipid, a phosphoethanolamine-based lipid, a derivative thereof, or a combination thereof.

14. The method according to claim 13, wherein the lipid component comprises 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DphPE), derivatives thereof, or combinations thereof.

15. The method according to any one of claims 1 to 14, wherein the mispipe and target molecule are mixed together before the tip is brought into contact with the mispipe and target molecule.

16. The method according to any one of claims 1 to 15, wherein the throughput related to sequencing the target molecule is improved by bringing the chip into contact with a plurality of mispipes and target molecules.

17. The method according to any one of claims 1 to 16, wherein the flow cell capture of the target molecule is improved by bringing the tip into contact with a plurality of mispipes and target molecules.

18. A method for sequencing target molecules, The present invention provides a chip comprising a plurality of detection electrodes and a lipid membrane arranged adjacent to or near the detection electrodes, wherein a plurality of nanopore assemblies are arranged within the lipid membrane. The chip is brought into contact with multiple lipid-binding proteins, The chip is brought into contact with the target molecule, Applying a voltage to both ends of the aforementioned film, One or more of the aforementioned detection electrodes are used to measure one or more changes in current or voltage related to the nanopore assembly. With the help of a computer processor, the arrangement of the target molecules is determined based on one or more of the measured current or voltage changes associated with the nanopore assembly, Methods that include...

19. The method according to claim 18, wherein at least a portion of the plurality of lipid-binding proteins form a complex with a lipid component to form a plurality of mispipes, and the tip is brought into contact with the plurality of mispipes, thereby bringing the tip into contact with the plurality of lipid-binding proteins.

20. The method according to claim 18 or 19, wherein the target molecule is a nucleic acid, a modified nucleic acid, or a nucleic acid substitute.

21. The method according to any one of claims 18 to 20, wherein the nucleic acid substitute comprises Xpandomer.

22. The method according to any one of claims 18 to 21, wherein the lipid-binding protein is a membrane scaffold protein (MSP).

23. The method according to claim 22, wherein the MSP is apolipoprotein A1 or a derivative thereof.

24. The method according to any one of claims 18 to 23, wherein the lipid-binding protein comprises a predetermined number of alpha helices.

25. The method according to any one of claims 19 to 24, wherein the ratio of the lipid-binding protein to the lipid component is about 1:

200.

26. The method according to any one of claims 19 to 25, wherein the lipid component comprises a phosphatidylcholine-based lipid, a phosphoethanolamine-based lipid, a derivative thereof, or a combination thereof.

27. The method according to claim 26, wherein the lipid component comprises 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DphPE), derivatives thereof, or combinations thereof.

28. A method for modifying the interaction between a target molecule and a lipid bilayer, To prepare a lipid membrane, The lipid membrane is brought into contact with multiple mispipes and target molecules, Includes, The target molecule has affinity for the lipid membrane, and by bringing the lipid membrane into contact with the plurality of mispids, the affinity of the target molecule for the lipid membrane is reduced. method.

29. The method according to claim 28, wherein the target molecule is a nucleic acid, a modified nucleic acid, or a nucleic acid substitute.

30. The method according to claim 29, wherein the nucleic acid substitute comprises Xpandomer.

31. The method according to any one of claims 28 to 30, wherein the plurality of mispids are prepared by contacting a plurality of lipid-binding proteins with a lipid component to form the plurality of mispids.

32. The method according to claim 31, wherein the lipid-binding protein is a membrane scaffold protein (MSP).

33. The method according to claim 32, wherein the MSP is an apolipoprotein or a derivative thereof.

34. The method according to claim 33, wherein the MSP is apolipoprotein A1 or a derivative thereof.

35. The method according to any one of claims 31 to 34, wherein the lipid-binding protein comprises a predetermined number of alpha helices.

36. The method according to claim 35, wherein the predetermined number of alpha helices is 3 to 10.

37. The method according to any one of claims 31 to 36, wherein the ratio of the lipid-binding protein to the lipid component is about 1:200 to 1:1500.

38. The method according to claim 37, wherein the ratio of the lipid-binding protein to the lipid component is approximately 1:1200.

39. The method according to any one of claims 28 to 37, wherein the lipid component comprises a phosphatidylcholine-based lipid, a phosphoethanolamine-based lipid, a derivative thereof, or a combination thereof.

40. The method according to claim 39, wherein the lipid component comprises 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DphPE), derivatives thereof, or combinations thereof.

41. The method according to any one of claims 28 to 40, wherein the mispid and the target molecule are mixed together before contact with the lipid membrane.

42. The method according to any one of claims 28 to 41, wherein the throughput related to the sequencing of the target molecule in the sequencing reaction is improved by bringing the lipid membrane into contact with the plurality of mispids and target molecules.

43. The method according to any one of claims 28 to 42, wherein the flow cell capture of the target molecule in the sequencing reaction is improved by bringing the lipid membrane into contact with a plurality of mispipes and target molecules.

44. A composition for modifying the interaction between a target molecule and a lipid membrane, comprising a heterogeneous mixture of lipids and lipid-binding proteins.

45. The composition according to claim 44, wherein the lipid-binding protein is a membrane scaffold protein (MSP).

46. The composition according to claim 45, wherein the MSP is an apolipoprotein or a derivative thereof.

47. The composition according to claim 46, wherein the MSP is apolipoprotein A1 or a derivative thereof.

48. The composition according to any one of claims 44 to 47, wherein the lipid-binding protein comprises a predetermined number of alpha helices.

49. The composition according to claim 48, wherein the predetermined number of alpha helices is 3 to 10.

50. The composition according to any one of claims 44 to 49, wherein the ratio of the lipid-binding protein to the lipid component is about 1:600 ​​to 1:1500.

51. The composition according to claim 50, wherein the ratio of the lipid-binding protein to the lipid component is about 1:1200.

52. The composition according to claim 50, wherein the ratio of the lipid-binding protein to the lipid component is about 1:

200.

53. The composition according to any one of claims 44 to 52, wherein the lipid component comprises a phosphatidylcholine-based lipid, a phosphoethanolamine-based lipid, a derivative thereof, or a combination thereof.

54. The composition according to claim 53, wherein the lipid component comprises 1,2-diphytanoyl-sn-glycero-3-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DphPE), derivatives thereof, or combinations thereof.

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