Detergent-free nanopore delivery

Abstract

The disclosure relates to nanopore preparations with reduced concentrations of detergents including detergent-free preparations. The disclosure provides lipid nanoparticles (LNPs) comprising a lipid, saposin, and a nanopore, methods for producing such LNPs, as well as systems and methods for nanopore-based sequencing using such LNPs. The new nanopore preparation avoids the pitfalls of using detergent-based compositions, such as disrupting the interactions between a target molecule and a nanopore during sequencing, disruption of the lipid bilayer, and batch-to-batch variability in nanopore preparations.

Description

DETERGENT-FREE NANOPORE DELIVERYRELATED APPLICATIONSThis application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 63 / 639,470, filed April 26, 2024, and titled ‘ DETERGENT-FREE NANOPORE DELIVERY,” the disclosure of which is hereby incorporated by reference in its entirety.SEQUENCE LISTINGThe official copy of the sequence listing is submitted electronically via EFS-Web with a file named, “P38601WO_SeqList_ST26.xml,” created on 17 March 2025, and having a size of 6.28 kilobytes, and is filed concurrently with the specification. The sequence listing contained in the XML formatted document is part of the specification and is herein incorporated by reference in its entirety.FIELD OF THE DISCLOSUREThe present disclosure relates generally to the preparation of biological nanopores, and in particular to the preparation of a nanopore in a lipid nanoparticle, to improve nanopore deliver}' to a barrier in a nanopore-based sequencing chip and to improve sequencing accuracy.BACKGROUNDOver the last two decades, biological membranes have emerged as an important tool in a variety of biomedical applications. This includes the use of lipid bilayer membranes in nanopore based sequencing applications, where nanopores provide a constant and reproducible physical aperture, through which a target molecule can be directed and sequenced.Generally, nanopores are associated with a chip-based flow cell, the flow cell having an inlet to which a solution comprising the target molecules is added. The flow cells also include multiple wells, each well having a barrier, such as a lipid bilayer membrane, and a single nanopore that is embedded within the barrier. Also associated with each well is a sensing electrode that can detect changes in current and / or voltage across the barrier of the well.As the solution comprising a target molecule is introduced into the flow cell sample port, the solution flows across the barrier of the wells, allowing nanopores within the barrier to capture the target molecules. The barrier exhibits a high electrical resistance. The nanopore allows ions to pass through when a suitable potential is applied across the barrier, thus generating a current. Theionic current through the nanopore also drives a target molecule through the nanopore. As the target molecule passes through the nanopore, it causes changes in the ionic current, for example, which can be detected via the sensing electrodes associated with the wells of the flow cells. Each base of a nucleic acid that passes through the nanopore, for example, can then be identified through the characteristic disruption it causes to the current in real-time. An overview of nanopore-based sequencing systems can be found at Wang I and Feng.The nanopores used in these sequencing systems ty pically come in one of three flavors: biological nanopores, solid state nanopores, and hybrid nanopores. Biological nanopores are formed by nanopore-forming molecules, especially proteins such as porins, hemolysins, and the like. Commonly used pore-forming proteins include a-hemolysin (aHL) protein from Staphylococcus aureus, outer membrane protein G (ompG) from Escherichia coli, and porin MspA (MspA) from Mycobacterium smegmatis. In some cases, like in the case of ompG, the pore is formed from a single subunit of the protein. In other cases, like with aHL and MspA, the pore is formed by a multi-subunit assembly of the nanopore-forming protein. For example, aHL forms a heptameric nanopore structure and MspA forms an octameric nanopore structure. Exemplary engineered nanopores based on these proteins can be found at, for example, WO 2016 / 069806 (aHL), WO 2017 / 050728 (aHL), WO 2017 / 184866 (aHL), WO 2018 / 002125 (aHL), WO 2012 / 178097 (aHL), Gari (ompG), WO 2017 / 050722 (ompG), US 2015-0080242 (ompG), Manrao (MspA), Pavlenok (MspA), WO 2013 / 098562 (MspA), US 2014-0309402 (MspA), US 2013-0146457 (MspA), and Wang II (various).Solid state nanopores are nanopore structures fabricated from synthetic materials, for example, by forming nanometer-sized holes in synthetic membranes. Exemplary’ materials from which solid state nanopores can be formed include silicon nitrides, silica, alumina, graphene, boron nitride, and molybdenum disulfide. Solid state nanopores are reviewed by Chen, Lee, Wasfi, Wang I, and Feng.Hybrid nanopores incorporate both biological nanopores and solid state nanopores. For example, a biological nanopore (such as an aHL nanopore) can be inserted into a solid state nanopore. Hybrid nanopores are reviewed by Lee, Wasfi, and Feng.One approach for nanopore-based sequencing of, for example, nucleic acids involves threading single stranded nucleic acids directly through the pore (so-called “direct sequencing”). Each nucleotide (or unique combination of nucleotides) generates a unique change in an electrochemical characteristic of the nanopore. These systems frequently use means to control the rate at which the nucleic acid translocates through the nanopore, such as tethering enzy mes to the nanopore (including polymerases and helicases), removing negatively charged residues from and adding positively charged residues to the surface forming the nanopore channel, and adding doublestranded regions to the single stranded nucleic acid. Exemplary direct sequencing approaches are discussed by, for example, Feng, Manrao, and Wang I.Another method involves a sequencing-by-tagging (SBT) approach by performing a polymerase-catalyzed amplification reaction near an opening of the nanopore with tagged nucleotide polyphosphate molecules. Each tagged nucleotide polyphosphate includes a distinct tag moiety that generates a unique electrochemical signature when it resides in or near the nanopore. As the tagged nucleotide polyphosphates are incorporated into the amplicon, the tag is passed into or near the nanopore, and the electrochemical signature of the tag is recorded. The sequence of the amplicon is derived from the order in which tag moieties enter into the nanopore. Exemplary SBT approaches and materials for performing such methods are described at, for example, WO 2012- 083249, WO 2013 / 154999, US 2014 / 0309144, US 9,017,937, WO 2015 / 148402, WO 2016 / 069806, WO 2016 / 144973, US 2016 / 0222363, US 2016 / 0333327, WO 2017 / 050728, WO 2017 / 184866, WO 2017 / 050722, US 2017 / 0267983, US 2018 / 0245147, US 2018 / 0094249, WO 2018 / 002125, and Kumar. Various tags have been proposed for use in such systems, including tags based on polypeptides (such as polylysine tags) and polynucleotides. See, e.g, US 8,652,779 and WO2017042038A1.A further method involves a sequencing-by-expansion approach by transcribing the sequence of DNA into a simple to measure polymer molecule called an Xpandomer (XP). Much like with polymerase chain reaction (PCR), Xpandomer synthesis is based on the natural function of DNA replication where expandable nucleoside triphosphates (XNTPs) act as substrates for replication.Xpandomer synthesis is based on four easily differentiated XNTPs that include High Signal-to-Noise Reporters, one for each DNA base. Engineered polymerases incorporate these modified nucleotides into Xpandomers, producing a copy of the target nucleic acid template from the library. As the Xpandomer molecule transits through the nanopore, the distinct electrical signal of each base reporter is easily identifiable to enable highly accurate and high throughput nanoporebased nucleic acid sequencing. See, e.g., U.S. Pat. No. 7.939,259, titled "‘High Throughput Nucleic Acid Sequencing by Expansion;" and PCT publication WO 2020 / 236526 Al, titled ‘'Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing”, both of which are hereby incorporated herein in their entirety.Nanopore-forming proteins that form biological nanopores, such as a-hemolysin, are membrane proteins that are usually prepared and delivered onto a sequencing chip in an aqueous buffer solution in the presence of a detergent in order to solubilize the nanopore-forming proteins. However, there are some disadvantages associated with detergent in the nanopore preparation: First, the detergent will also be concentrated when the nanopore is concentrated during thepreparation, which will cause variations from batch-to-batch preparation. Moreover, the detergent in the buffer will have an impact on the interaction between a target molecule to be sequenced, such as an Xpandomer, and the nanopore, which may affect the sequencing accuracy. In addition, if there is too much detergent in the buffer, it may destabilize a lipid bilayer membrane which also affects sequencing accuracy. In some cases, the detergent may even break the lipid bilayer, resulting in low yield of functional nanopore, which may significantly reduce the overall performance of the sequencer.Thus, there remains a need for improved delivery of biological nanopores to a nanoporebased sequencing chip. In particular, there is a need for methods and systems that improve the batch-to-batch vanability of nanopore preparations and provide improved sequencing performance.SUMMARY OF THE DISCLOSUREThe present disclosure comprises a new method for nanopore preparation using lipid nanoparticles including a saposin, which does not require a detergent. This method can thus avoid the problems associated with detergents present in the nanopore preparation. It has been discovered that reducing the detergent concentration in the nanopore preparation increases the accuracy of sequencing.Saposins are small lipid binding proteins (~ lOkD) and can be used as scaffolding proteins in a variety of lipid nanoparticle (LNP) systems for membrane protein reconstitution. Compared to other types of saposin, Saposin A (SapA) has a lower lipid selectivity for LNP formation than other types of saposin, and SapA-based LNPs can adapt to membrane proteins of different sizes and architectures (Frauenfeld, Flayhan). Membrane proteins reconstituted in SapA LNPs are more stable than when reconstituted in detergent and can be treated as soluble proteins (Frauenfeld, Flayhan). Moreover, compared to other types of LNPs, such as nanodiscs, the LNPs based on saposin can be readily generated with different membrane proteins.Disclosed herein are lipid nanoparticles comprising a lipid, a saposin and a nanopore, methods of producing the same, and their use in nanopore-based sequencing chips, systems and methods.Exemplary embodiments of the disclosure are as follows:(a) A lipid nanoparticle (LNP) comprising: a lipid, a saposin, and a nanopore.(b) A method for producing a lipid nanoparticle (LNP), comprising the steps: mixing a first solution comprising a nanopore-forming protein solubilized by a detergent, a second solution comprising a lipid solubilized by a detergent, and a third solutioncomprising a saposin. and reducing the total concentration of detergents comprised in the product of the first step.(c) A chip for performing nanopore-based sequencing, comprising: (i) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side; (ii) a first electrolyte solution on the cis side of the barrier; (iii) a second electrolyte solution on the trans side of the barrier; (iv) an LNP as described herein inserted into the barrier, wherein the nanopore comprised in the LNP has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and (v) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.(d) A chip for performing nanopore-based sequencing, comprising: (i) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side; (ii) a first detergent- free electrolyte solution on the cis side of the barrier; (iii) a second detergent-free electrolyte solution on the trans side of the barrier; (iv) a nanopore inserted into the barrier, wherein the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and (v) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule, wherein the barrier is a lipid bilayer membrane that is detergent-free.(e) A method for producing a chip for performing nanopore-based sequencing, comprising the steps of: (i) providing a solid support comprising an aperture, (ii) forming an electrochemically resistive barrier over the aperture, such that the barrier separates a cis side from a trans side; (iii) providing a first electrolyte solution on the cis side of the barrier, (iv) providing a second electrolyte solution on the trans side of the barrier, (v) contacting the barrier with an LNP or a composition comprising an LNP. as described herein, to insert the nanopore comprised in the LNP into the barrier, such that the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier permitting ion exchange between the first electrolyte solution and the second electrolyte solution, and (vi) positioning a working electrode to detect changes inan electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.(f) A system for performing nanopore-based sequencing, comprising a chip as described herein and a computing system adapted to record changes in one or more electrical characteristics of the nanopore, wherein the working electrode in the chip is in electronic communication with the computing system, and wherein the electrode is positioned to transmit any detected change(s) in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule to the computing system.(g) A method for sequencing a target molecule, comprising: (i) providing a chip as described herein; (ii) contacting the chip with a target molecule; (iii) applying a voltage across the barrier of the chip; (iv) determining, by the working electrode, one or more changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by the target molecule; and (v) determining, based on the one or more changes in the electrical characteristic of the nanopore, a sequence for the target molecule.(h) A kit for producing a chip for performing nanopore-based sequencing comprising an LNP or a composition comprising an LNP as described herein.Exemplary embodiments of the disclosure are described in the following items:1. A lipid nanoparticle (LNP) comprising: a lipid, a saposin, and a nanopore.2. The LNP of item 1, wherein the nanopore is suitable for nanopore-based sequencing of a target molecule.3. The LNP of any one of the preceding items, wherein the nanopore comprises a nanopore-forming protein selected from a-hemolysin (aHL), Mycobacterium smegmatis porin A (MspA), OmpG, OmpF, OmpC, gramicidin A, maltoporin, PhoE, Tsx, F-pilus, SPP1, mitochondrial porin (VDAC)XX and Tom40.4. The LNP of any one of the preceding items, wherein the nanopore comprises a- hemolysin (aHL).5. The LNP of items 3 or 4. wherein the aHL comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1.6. The LNP of any one items 3-5, wherein the aHL comprises the amino acid sequence of SEQ ID NO: 1.7. The LNP of any one of the preceding items, wherein the lipid is chosen from POPG, POPE, DPhPC, DMPC and DPhPE8. The LNP of any one of the preceding items, wherein the lipid is POPG.9. The LNP of any one of the preceding items, wherein the saposin is Saposin A (SapA).10. The LNP of any one of the preceding items, wherein the saposin comprises the amino acid sequence of SEQ ID NO: 2.11. The LNP of any one of the preceding items, wherein the nanopore is a aHL nanopore, and wherein optionally the lipid is POPG and the saposin is SapA.12. The LNP of any of the preceding items, wherein the LNP is detergent-free.13. A composition comprising the LNP of any one of items 1-12.14. The composition of item 13, wherein the total concentration of detergents in the composition is below their critical micelle concentration.15. The composition of item 13 or 14, wherein the total concentration of detergents in the composition is below 0.01% (w / v).16. The composition of any one of items 13-15, wherein the composition is detergent-free.17. The composition of any one of items 13-16, wherein the molar ratio of saposin:lipid:nanopore in the composition is 1:50:0.2.18. A method for producing a lipid nanoparticle (LNP), comprising the steps:(1) mixing a first solution comprising a nanopore-forming protein solubilized by a detergent, a second solution comprising a lipid solubilized by a detergent, and a third solution comprising a saposin, and(2) reducing the total concentration of detergents comprised in the product of step (1).19. The method of item 18, wherein the step (1) comprises the substeps:(la) mixing the first solution comprising a nanopore-forming protein solubilized by a detergent and the second solution comprising a lipid solubilized by a detergent to produce a first mixture, and(lb) mixing the product of step (la) with the third solution comprising a saposin to produce a second mixture.20. The method of item 19, wherein the step (la) further comprises incubating the first mixture for a first time period, and step (lb) further comprises incubating the second mixture for a second time period.21. The method of any one of items 18-20, wherein step (2) is conducted by adding a detergent-free dilution solution to the product of step (1), by adsorbing detergents to beads and / or by dialysis.22. The method of any one of items 18-21, wherein step (2) is conducted by adding a detergent-free dilution solution to the product of step (1).23. The method of item 21 or 22, wherein the detergent-free dilution solution is a detergent-free buffer solution.24. The method of item 21, wherein the beads are polystyrene beads.25. The method of any one of items 18-24, further comprising the step:(3) purifying the LNP present in the product of step (2).26. The method of item 25, wherein step (3) comprises size exclusion chromatography (SEC).27. The method of any one of items 18-26. wherein the total concentration of detergents is reduced to below their critical micelle concentration in step (2).28. The method of any one of items 18-27, wherein detergents are completely removed.29. The method of any one of items 18-28, wherein the molar ratio of saposin:lipid:nanopore is 1:50:0.2.30. The method of any one of items 18-29, wherein the nanopore is suitable for sequencing a target molecule.31. The method of any one of items 18-30, wherein the nanopore comprises a nanoporeforming protein selected from a-hemolysin (aHL), Mycobacterium smegmatis porin A (MspA), OmpG, OmpF, OmpC, gramicidin A, maltoporin, PhoE, Tsx, F-pilus, SPP1, mitochondrial porin (VDAC)XX and Tom40.32. The method of any one of items 18-31, wherein the nanopore comprises a-hemolysin (aHL).33. The method of item 31 or 32, wherein the aHL comprises an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 .34. The method of any one of items 31-33, wherein the aHL comprises the amino acid sequence of SEQ ID NO: 1.35. The method of any one of items 18-34. wherein the lipid is chosen from POPG, POPE, DPhPC, DMPC and DPhPE.36. The method of any one of items 18-35, wherein the lipid is POPG.37. The method of any one of items 18-36, wherein the saposin is Saposin A (SapA).38. The method of any one of items 18-37, wherein the saposin comprises the amino acid sequence of SEQ ID NO: 2.39. The method of any one of items 18-38, wherein the nanopore comprises aHL, the lipid is POPG and the saposin is SapA.40. A lipid nanoparticle (LNP) produced by the method of any one of items 18-39.41. A chip for performing nanopore-based sequencing, comprising:(a) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(b) a first electrolyte solution on the cis side of the barrier;(c) a second electrolyte solution on the trans side of the barrier;(d) the LNP of any one of items 1-12 or 40 inserted into the barrier, wherein the nanopore comprised in the LNP has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and(e) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.42. The chip of item 41, wherein the barrier is a lipid bilayer membrane.43. A chip for performing nanopore-based sequencing, comprising:(a) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(b) a first detergent-free electrolyte solution on the cis side of the barrier;(c) a second detergent-free electrolyte solution on the trans side of the barrier;(d) a nanopore inserted into the barrier, wherein the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and(e) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule, wherein the barrier is a lipid bilayer membrane that is detergent-free.44. A method for producing a chip for performing nanopore-based sequencing, comprising the steps of:(a) providing a solid support comprising an aperture,(b) forming an electrochemically resistive barrier over the aperture, such that the barrier separates a cis side from a trans side;(c) providing a first electrolyte solution on the cis side of the barrier,(d) providing a second electrolyte solution on the trans side of the barrier,(e) contacting the barrier with the LNP of any one of items 1-12 or 40 or with the composition of any one of items 13-17, to insert the nanopore comprised in the LNP into the barrier, such that the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier permitting ion exchange between the first electrolyte solution and the second electrolyte solution, and(f) positioning a working electrode to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.45. The method of item 44, wherein the barrier is a lipid bilayer membrane.46. The method of item 44 or 45, wherein the solid support comprises a plurality of apertures, wherein a barrier is formed over each of the plurality of apertures, and wherein each of the barriers is contacted with the LNP or with the composition.47. A chip for performing nanopore-based sequencing produced by the method of any one of items 44 to 46.48. A system for performing nanopore-based sequencing, comprising the chip of any one of items 41-43 or 47 and a computing system adapted to record changes in one or more electrical characteristics of the nanopore, wherein the working electrode in the chip is in electronic communication with the computing system, and wherein the electrode is positioned to transmit any detected change(s) in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule to the computing system.49. A method for sequencing a target molecule, comprising:(a) providing a chip of any one of items 41-43 or 47;(b) contacting the chip with a target molecule;(c) applying a voltage across the barrier of the chip;(d) determining, by the working electrode, one or more changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by the target molecule; and(e) determining, based on the one or more changes in the electrical characteristic of the nanopore, a sequence for the target molecule.50. The method of item 49. wherein the target molecule is a nucleic acid, modified nucleic acid, or other organic polymer.51. The method of item 49 or 50, wherein the target molecule comprises an Xpandomer.52. The method of any one of items 49-51 , wherein the target molecule translocates through the nanopore.53. The method of any of items 49-52, wherein the determined sequence of the target molecule determines the sequence of a target nucleic acid sequence.54. The method of any of items 49-53, wherein the electrical characteristic of the nanopore is measured as a current change or a voltage change.55. The method of item 54, wherein the electrical characteristic of the nanopore is measured as a current decrease or a voltage increase.56. A kit for producing a chip for performing nanopore-based sequencing comprising the LNP of any one of items 1-12 or 40 or the composition of any one of items 13-17.57. The kit of item 56. further comprising a solid support comprising an aperture and an electrode.58. The kit of item 56 or 57, further comprising an electrolyte solution.59. The kit of any one of items 56-58, further comprising a lipid solution.60. The kit of any one of items 56-59, wherein the solid support comprises- an aperture configured to allow disposing an electrochemically resistive barrier over the aperture to separate a cis side from a trans side; and- an electrode on one side of the aperture.61. The kit of any one of items 56-60, wherein the solid support comprises- an aperture, wherein an electrochemically resistive barrier is disposed over the aperture, wherein the barrier separates a cis side from a trans side;- an electrode on the trans side of the barrier.62. The kit of item 60 or 61, wherein the solid support comprises a plurality of apertures, and a plurality of electrodes.63. The kit of item 61 or 62, wherein the solid support comprises a plurality of apertures, and wherein a barrier is disposed over each of the plurality of apertures.64. The kit of any one of items 60-63, wherein the barrier is a lipid bilayer membrane.BRIEF DESCRIPTION OF THE DRAWINGSFIG. 1A is a graph showing the results of gel filtration chromatography of Saposin A, in accordance with certain example embodiments.FIG. IB is an image showing an SDS-PAGE of the gel filtration chromatography peak fractions from FIG. 1A, in accordance with certain example embodiments.FIG. 2A is a graph showing the gel filtration elution profile of Saposin A, in accordance with certain example embodiments.FIG. 2B is a graph showing the gel filtration elution profile of DPhPC Saposin A lipid particles, in accordance with certain example embodiments.FIG. 2C is a graph showing the gel filtration elution profile of DMPC Saposin A lipid particles, in accordance with certain example embodiments.FIG. 2D is a graph showing the gel filtration elution profile of POPE Saposin A lipid particles, in accordance with certain example embodiments.FIG. 2E is a graph showing the gel filtration elution profile of DPhPE Saposin A lipid particles, in accordance with certain example embodiments.FIG. 2F is a graph showing the gel filtration elution profile of POPG Saposin A lipid particles, in accordance with certain example embodiments.FIG. 3A is a graph showing a gel filtration elution profile of protein nanopore reconstituted into POPG SapA LNP, in accordance with certain example embodiments. As shown, two fractions were identified, corresponding to Peak 1 (Pl) and Peak 2 (P2).FIG. 3B is an image showing an SDS-PAGE of gel filtration chromatography peak fractions from FIG. 3A, in accordance with certain example embodiments.FIG. 4A is a histogram showing the number of aligned high quality XP captures per functional minute per cell (throughput), in accordance with certain example embodiments. More particularly, throughput was evaluated using sequencing chips into which four different concentrations of POPG SapA LNPs, along with reconstituted a-hemolysin nanopores, were electroporated. A chip into which detergent-solubilized a-hemolysin nanopores were electroporated was used as a control. As shown, throughput was comparable between the POPG SapA LNPs and controls.FIG. 4B is a histogram showing nanopore functional lifetime, in accordance with certain example embodiments. More particularly, nanopore functional lifetime was evaluated as the described above for FIG. 4A. As shown, nanopore functional lifetime was comparable between the POPG SapA LNPs and controls.FIG. 4C is a histogram showing XP accuracy, in accordance with certain example embodiments. More particularly, XP accuracy was evaluated using sequencing chips into which four different concentrations of POPG SapA LNPs, along with reconstituted a-hemolysin nanopores, were electroporated. A chip into which detergent-solubilized a-hemolysin nanopores were electroporated was used as a control. As shown, XP sequencing accuracy was improved relative to controls.FIG. 4D is a histogram showing XP read length, in accordance with certain example embodiments. More particularly, XP read length was evaluated as the described above for FIG. 4C. As shown, read length was similar to controls.FIG. 4E is a histogram showing XP insertion error, in accordance with certain example embodiments. More particularly, XP insertion error was evaluated using sequencing chips into which four different concentrations of POPG SapA LNPs, along with reconstituted a-hemolysin nanopores, were electroporated. A chip into which detergent-solubilized a-hemolysin nanopores were electroporated was used as a control. As show n, a decrease in insertions was observed versus control, indicating that the increase in sequencing accuracy is due to a decrease in insertions.FIG. 4F is a histogram showing XP deletion error, in accordance with certain example embodiments. More particularly, the XP deletion error was evaluated as the described above for FIG. 4E. As shown, there was no change in deletions versus control.DETAILED DESCRIPTION OF THE DISCLOSUREThe disclosure will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton (1994), Hale (1991) and Walker (1988) provide one of skill with a general dictionary' of many of the terms used in this disclosure. Practitioners are particularly directed to Sambrook (1989), and Ausubel (1993), for definitions and terms of the art. It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary.As used herein, the singular forms “a,’’ “an” and “the” include plural referents unless the context clearly dictates otherwise.Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.The headings provided herein are not limitations of the various aspects or embodiments of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.I. TermsAmino acid extension: An “amino acid extension” refers to a stretch of amino acids that is fused to the N-terminus or C-terminus of a given amino acid sequence. An amino acid extension is typically of limited length, such as a length of 1 to 120, such as 1 to 104, 1 to 50, 1 to 30. 1 to 20, 1 to 10 or 1 to 6 amino acids.Detergent-free: The term “detergent-free” means that a given composition, solution, etc. is devoid of detergents of any type.Lipid nanoparticle: a “lipid nanoparticle” generally refers to a particle in the low nanometer scale (such as a diameter at its widest segment of 50 nm or less, such as 20 nm or less) that comprises at least one lipid.Monomer subunit: A structural subunit of a multimeric protein complex. For example, a heptameric a-hemolysin pore comprises seven a-hemolysin monomer subunits. A monomer subunit that has not been oligomerized into a multimeric subunit is referred to herein as a “nonoligomerized monomer subunit.”Nanopore: The term “nanopore” generally refers to a pore in the nanometer scale.Nanopore-forming protein: The term “nanopore-forming proteins’" generally refers to proteins that are capable of forming a nanopore, either alone or in association of more than one nanopore-forming protein. An example is a-hemolysin that can form heptameric oligomers to form a nanopore.Organic polymer: An “organic polymer” generally refers to any type of polymeric molecule composed of several (e.g. at least three) or many organic subunits. In the context of sequencing, an organic polymer will be a type of polymer that is amenable to sequencing, e.g. by nanopore-based sequencing. For example, nucleic acids, e.g. DNA or RNA, Xpandomers, or polypeptides can be organic polymers.Percent identity: The term “% identity” in the context of nucleic acid or amino acid sequences refers to the level of sequence identity between a nucleic acid sequence and a reference nucleic acid sequence or between an amino acid sequence and a reference amino acid sequence, when aligned using a sequence alignment program. For example, as used herein, 80% identity indicates that a sequence has greater than 80% sequence identity over a length of the reference sequence. Exemplary levels of sequence identity include, but are not limited to, 80% or more, 85% or more, 90% or more, 95% or more, and 98% or more sequence identity to a reference sequence, e.g., the wildtype sequence for any one of the polypeptides described herein. Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet. See also, Altschul I and Altschul II. Sequence searches are typically carried out using the BLASTN program when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program may be used for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTP program may be used for searching amino acid sequence against amino acid sequences in the GenBank Protein Sequences and other public databases. All of BLASTN, BLASTX and BLASTP are run using 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, e.g., Altschul II). In certain example embodiments, an alignment of selected sequences in order to determine “% identity” between two or more sequences, is performed using for example, the CLUSTAL-W program in MacVector version 13.0.7, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0. 1. and a BLOSUM 30 similarity matrix.Solution: a ‘'solution” as used herein refers to an aqueous solution, unless specified otherwise.Expandable NTP: An "‘expandable NTP” or “XNTP” refers to 5' a-phosphate modified non-natural nucleoside triphosphate (NTP) molecules compatible with template dependent enzymatic polymerization. Each XNTP has two distinct functional regions, i.e., a selectively cleavable bond (e.g. a phosphoramidate bond) linking the 5’ a-phosphate to a sugar comprised in a nucleoside and a tether that is attached within the XNTP at positions that allow for controlled expansion by cleavage of the cleavable bond (e.g. a tether linking the 5' a-phosphate and the nucleobase). An XNTP can thus be present in a constrained configuration (when the cleavable bond is still intact) or in an expanded configuration (when the cleavable bond has been cleaved, e.g. via acid treatment).Xpandomer: An “Xpandomef ’ refers to a molecule comprising at least two XNTPs. An Xpandomer is obtainable, for example, by polymerase-mediated synthesis of a complementary strand to a template nucleic acid using XNTPs as polymerase substrates. An expanded configuration of the Xpandomer can be obtained by cleavage of the phosphoramidate bond in the XNTPs, e.g. via acid treatment.A recitation of “a”, “an”, or “the” is intended to mean “one or more” unless specifically indicated to the contrary. The use of “or” is intended to mean an “inclusive or,” and not an “exclusive or” unless specifically indicated to the contrary. Reference to a “first” component does not necessarily require that a second component be provided. Moreover, reference to a “first” or a “second” component does not limit the referenced component to a particular location unless expressly stated. The term “based on” is intended to mean “based at least in part on.”II. Lipid nanoparticles (LNPs) and method for producing LNPsThe disclosure provides a lipid nanoparticle (LNP) comprising a lipid, a saposin and a nanopore. In certain examples, the LNP comprises of lipids, saposin proteins and a nanopore.As explained in more detail below in the context of the method of producing an LNP, the LNPs spontaneously form upon withdrawal of detergents that are used to initially solubilize the nanopore and / or the lipids. Without wishing to be bound by any theory , it is expected that the LNPs (and compositions comprising the same) described herein inherently comprise detergents at low enough concentrations to allow the LNP to exist. The total concentration of detergents will thus generally be below their critical micelle concentration. In some embodiments, the LNP is detergent-free.The disclosure further provides a composition comprising a LNP as disclosed herein. In certain examples, the composition comprises of water and LNPs, and optionally a buffering agent and / or a salt. In other examples, the composition is a lyophilized composition comprising theLNP, and optionally a buffering agent and / or a salt.In some embodiments, the composition is detergent-free.In some embodiments, the composition comprises a detergent or mixture of detergents at a total concentration below the critical micelle concentration (CMC) of the detergent(s). For example, the total concentration of detergents in the composition can be below 0.01% (w / v). In some embodiments, the total concentrations of detergents in the composition is ImM or less, 200 pM or less, 100 pM or less, 10 pM or less, 1 pM or less, 100 nM or less, or 10 nM or less. For example, the total concentration of detergents in the compositions may be 50 pM or less. Examples of detergents include sodium cholate, n-Dodecyl-B-D-Maltoside (DDM), Triton-X 100, Triton-X 114, NP-40, Brij-35, Brij-58. Tween-20, Tween-80, octyl-beta-glucoside, octylthioglucoside, sodium dodecyl sulfate (SDS) and CHAPS. In some embodiments, the only detergent present in the composition is DDM. For example, DDM is the only detergent present in the composition and is present at a concentration below 0.01% (w / v). In another example, DDM is the only detergent present in the composition and is present at a concentration below 200 pM. Thus, in some embodiments, the composition is free of micelles. The skilled person is readily able to determine the presence of micelles in a composition, for instance by cryo transmission electron microscopy.The molar ratio between lipid, saposin and nanopore in the composition is not further limited, as long as the composition comprises an LNP as described herein. The skilled person is readily able to determine the presence of LNPs, e.g. by electron microscopy as described in Frauenfeld, by SDS-PAGE and / or by size exclusion chromatography (SEC).In some embodiments, the molar ratio of saposimlipid in the composition is between 1:5 and 1:500, such as between 1:10 and 1 :250, including between 1 :25 to 1 : 100. For example, the molar ratio of saposimlipid can be 1:50.In some embodiments, the molar ratio of saposimnanopore in the composition is between 1 :0.04 and 1 : 1, such as between 1 :0.08 and 1 :0.5, including between 1 :0. 1 to 1 :0.4. For example, the molar ratio of saposimlipid can be 1:0.2.In some embodiments, the molar ratio of saposimlipid in the composition is between 1:5 and 1:500, such as between 1 : 10 and 1:250, including between 1 :25 to 1 : 100, and the molar ratio of saposimnanopore in the composition is between 1:0.04 and 1 : 1, such as between 1:0.08 and 1 :0.5, including between 1 :0.1 to 1:0.4.In some embodiments, the molar ratio of saposimlipid in the composition is 1:50, and the molar ratio of saposimnanopore in the composition is between 1:0.04 and 1: 1, such as between 1 :0.08 and 1:0.5. including between 1:0.1 to 1 :0.4.In some embodiments, the molar ratio of saposimlipid in the composition is between 1:5 and 1:500, such as between 1 : 10 and 1:250, including between 1 :25 to 1 :100, and the molar ratio of saposimnanopore in the composition is 1 :0.2.In some embodiments, the molar ratio of saposin:lipid:nanopore in the composition is 1 :50:0.2.The disclosure further provides a method for producing a LNP as disclosed herein. Generally, the LNPs disclosed herein can be produced by forming a mixture comprising a lipid, a saposin and a nanopore, in which the lipid and the nanopore have been solubilized by a detergent. Upon withdrawal of the detergent from the mixture, LNPs spontaneously form in which a nanopore is embedded in lipids and stabilized by the saposin. Conversely, if the total concentration of detergents in the mixture is too high, no stable and homogenous LNPs will form. The LNPs produced can be optionally purified by any suitable method. The production of LNPs comprising saposin and membrane proteins as well as the structure of such LNPs has been described by Frauenfeld (2016) and Flayhan (2018), for example, the contents of which are incorporated by reference herewith.In some embodiments, a method for producing a LNP is provided, comprising the steps:(1) mixing a first solution comprising a nanopore-forming protein solubilized by a detergent, a second solution comprising a lipid solubilized by a detergent, and a third solution comprising a saposin, and(2) reducing the total concentration of detergents comprised in the product of step (1).Typically, the LNP thus produced comprises a lipid, a saposin and a nanopore.Thus, in some embodiments, a method is provided for producing a LNP comprising a lipid, a saposin and a nanopore, the method comprising the steps:(1) mixing a first solution comprising a nanopore-forming protein solubilized by a detergent, a second solution comprising a lipid solubilized by a detergent, and a third solution comprising a saposin, and(2) reducing the total concentration of detergents comprised in the product of step (1).In some embodiments, step (1) comprises the substeps:(la) mixing the first solution comprising a nanopore-forming protein solubilized by a detergent and the second solution comprising a lipid solubilized by a detergent to produce a first mixture, and(lb) mixing the product of step (la) with the third solution comprising a saposin to produce a second mixture.Thus, in some embodiments, a method is provided for producing a LNP (comprising a lipid, a saposin and a nanopore), comprising the steps:(la) mixing the first solution comprising a nanopore-forming protein solubilized by a detergent and the second solution comprising a lipid solubilized by a detergent to produce a first mixture,(1 b) mixing the product of step (1 a) with the third solution comprising a saposin to produce a second mixture, and(2) reducing the total concentration of detergents comprised in the product of step (1).In some embodiments, the step (la) further comprises incubating the first mixture for a first time period, and / or step (lb) further comprises incubating the second mixture for a second time period.Typically, the solutions in the method will be aqueous solutions. The components of the solutions are not particularly limited, as long as they do not interfere with the formation of the LNPs.The type of detergent used in the first and / or second solution is not particularly limited, as long as it allow solubilization of the nanopore and / or the lipid. Examples of suitable detergents include sodium cholate, n-Dodecyl-B-D-Maltoside (DDM), Triton-X 100, Triton-X 114, NP-40, Brij-35, Brij-58, Tween-20, Tween-80, Octyl-beta-glucoside, octylthioglucoside, sodium dodecyl sulfate (SDS) and CHAPS.In some embodiments, the detergent in the first solution is DDM. In some embodiments, DDM is the only detergent present in the first solution.In some embodiments, the detergent in the second solution is DDM. In some embodiments, DDM is the only detergent present in the second solution.In some embodiments, the detergent in the first and the second solution is DDM. In some embodiments, DDM is the only detergent present in the first and second solution.In some embodiments, DDM is the only detergent present in the product of step (1).The total concentrations of detergent in the first and / or second solutions are not particularly limited as long as they suffice to solubilize the nanopore or the lipid used. Solubilization of a given nanopore or lipid can be readily determine using known methods, e g. by SDS-PAGE and Westemblot on solubilized fractions, or by determining turbidity upon titration of the detergent.In some embodiments, the total concentration of detergent in the first solution is 0.02% (wt / v) or more, such as between 0.02% (w / v) and 4% (w / v), between 0.04% (w / v) and 2% (w / v), between 0.08% (w / v) and 1% (w / v) or between 0.1% (w / v) and 0.5% (w / v). For example, the total concentration of detergent in the first solution can be 0.2% (w / v).In some embodiments, the total concentration of detergent in the second solution is 0.02% (wt / v) or more, such as between 0.02% (w / v) and 4% (w / v), between 0.04% (w / v) and 2% (w / v), between 0.08% (w / v) and 1% (w / v) or between 0.1% (w / v) and 0.5% (w / v). For example, the total concentration of detergent in the second solution can be 0.2% (w / v).In some embodiments, the total concentration of detergent in each of the first and second solution is 0.02% (wt / v) or more, such as between 0.02% (w / v) and 4% (w / v), between 0.04%(w / v) and 2% (w / v), between 0.08% (w / v) and 1% (w / v) or between 0. 1% (w / v) and 0.5% (w / v). For example, the total concentration of detergent in each of the first and second solution can be 0.2% (w / v).In some embodiments, the total concentration of detergent in the first solution is 400 pM or more, such as between 400 pM and 80 mM. between 800 pM and 40 mM, between 1.5 mM and 20 mM or between 2 mM and 10 mM. For example, the total concentration of detergent in the first solution can be 4 mM.In some embodiments, the total concentration of detergent in the second solution is 400 pM or more, such as between 400 pM and 80 mM. between 800 pM and 40 mM, between 1.5 mM and 20 mM or between 2 mM and 10 mM. For example, the total concentration of detergent in the second solution can be 4 mM.In some embodiments, the total concentration of detergent in each of the first and second solution is 400 pM or more, such as between 400 pM and 80 mM, between 800 pM and 40 mM, between 1.5 mM and 20 mM or between 2 mM and 10 mM. For example, the total concentration in each of detergent in the first and second solution can be 4 mM.Typically, the third solution is detergent-free. However, given that the concentration of detergent is reduced in step (2), the presence of a detergent in the third solution is of no general harm to the method.In some embodiments, the first solution further comprises a buffering agent.In some embodiments, the second solution further comprises a buffering agent.In some embodiments, the third solution further comprises a buffering agent.In some embodiments, the first and second solution further comprises a buffering agent.In some embodiments, the first, second and third solution further comprises a buffering agent.The type of buffering agent is not particularly limited, as long as it does not interfere with formation of LNPs. Examples of buffering agents include HEPES, Tris buffer, phosphate buffer and MES buffer. In some embodiments, the buffering agent in the first and / or second solution is HEPES. In some embodiments, the buffering agent in the third solution is a Tris buffer, such as Tris-HCl.The concentration of buffering agent is not particularly limited, as long as it does not interfere with formation of LNPs. The concentration of buffering agent can be, for example, between ImM and 300mM, such as between 5mM to 200mM, between lOmM and lOOmM, such as 20mM or 50mM. In some embodiments, the concentration of the buffering agent in the first and / or second solution is 50mM. In some embodiments, the concentration of the buffering agent in the third solution is 20mM.In some embodiments, the first and second solution comprises 50 mM HEPES and the third solution comprises 20mM Tris.The type of the solutions is not particularly limited, as long as it does not interfere with formation of LNPs. The pH of the first solution can be, for example, between 5 and 10, such as between 6 and 9, between 7 and 8. such as 7.5.The pH of the second solution can be, for example, between 5 and 10, such as between 6 and 9, between 7 and 8, such as 7.5.The pH of the third solution can be, for example, betw een 5 and 11, such as between 6 and 10, between 7 and 9, such as 8.0.In some embodiments, the first and second solution has a pH of 7.5 and the third solution has a pH of 8.0.In some embodiments, the first and second solution comprises 50 mM HEPES at pH 7.5 and the third solution comprises 20mM Tris at pH 8.0.In some embodiments, the first solution further comprises a salt.In some embodiments, the second solution further comprises a salt.In some embodiments, the third solution further comprises a salt.In some embodiments, the first and second solution further comprises a salt.In some embodiments, the first, second and third solution further comprises a salt.The type of salt is not particularly limited, as long as it does not interfere with formation of LNPs. The salt can be, for example, NaCl or KC1.The concentration of salt is not particularly limited, as long as it does not interfere with formation of LNPs. The salt can be present, for example, at a concentration of between lOmM and IM, such as between 20mM and 500mM, between 50mM and 250mM or between lOOmM and 200mM, such as 150mM or 200mM. In some embodiments, the salt concentration in the first and / or second solution is 150mM. In some embodiments, the salt concentration in the third solution is 200mM. In some embodiments, the salt concentration in the first and second solution is 150mM and the salt concentration in the third solution is 200mM.In some embodiments, the first and / or second solution comprises 50mM HEPES, pH7.5, 150mM NaCl and 0.2%DDM.In some embodiments, the third solution comprises 20mM Tris-Cl, pH8.0, and 200mM NaCl.In some embodiments, the first solution comprises water, 50mM HEPES. pH7.5. 150mM NaCl, 0.2%DDM and solubilized nanopore protein, the second solution comprises water, 50mM HEPES, pH7.5, 150mM NaCl, 0.2%DDM and solubilized lipids and the third solution comprises water, 20mM Tris-CIn some embodiments, the first time period is between 1 min and 3h. such as between 2min and 1.5h, between 4min and 0.75h, between 8min and 30min or between lOmin and 20min.In some embodiments, the second time period is between 1 min and 4h, such as between 2min and 2h, between 5min and Ih or between 15min and 45min. such as 30min.In some embodiments, step (2) is conducted by adding a detergent-free dilution solution to the product of step (1), by adsorbing detergents to beads and / or by dialysis.In some embodiments, step (2) is conducted by adding a detergent-free dilution solution to the product of step (1). In some embodiment, the detergent-free dilution solution is a detergent- free buffer solution. In some embodiments, step (2) can be conducted simultaneously with step (lb). In such embodiments, the third solution acts as a detergent-free dilution solution.Typically, the dilution solution will be an aqueous solution. In some embodiments, the dilution solution is the same as the first solution but without nanopore and without detergent. In such embodiments, concerning the components of the dilution solution, their concentration and the pH of the dilution solution, the same applies as described above for the first solution.In some embodiments, the addition of the dilution solution is followed by incubating for a third time period. For example, the third time period can be between Imin and 60min, such as between 2min and 30min or between 5min and 20min, such as lOmin.The incubation temperature can be, for example, between 0°C and 40°C, such as between 10°C and 30°C or between 15°C and 25°C.In some embodiments, step (2) is conducted by adsorbing detergents to beads. In some embodiments, the beads are polystyrene beads.In some embodiments, step (2) is conducted by dialysis. Dialysis can be done by known methods and the dialysis solutions used are not particularly limited, as long as they do not disrupt the LNP.In some embodiments, the method further comprises the step:(3) purifying the LNP present in the product of step (2).For example, step (3) can comprise a centrifugation step, a size exclusion chromatography (SEC) step, etc.. In some embodiments, step (3) comprises a centrifugation step followed by subjecting the supernatant to a SEC step.The rotation speed during the centrifugation step can be, for example, lOOOOrpm to 16000rpm, such as 13000rpm. The length of the centrifugation step can be, for example, between Imin and lOmin. such as 5min.The buffer used for the SEC is not particularly limited as long as it does not disrupt the LNP. Typically, the buffer used for SEC will be detergent-free. For example, the buffer used for the SEC can be 20 mM Tris, pH8.0, 200Generally, it is sufficient to reduce the total concentration of detergents in step (2) to such an extent that LNPs will spontaneously form. The presence of LNPs can be readily tested by, e.g., by electron microscopy (described for example in Frauenfeld), SDS-PAGE and / or SEC. Generally, LNPs will spontaneously form when the total concentration of detergents is reduced to such an extent that the solution is (substantially) free from micelles. Thus, in some embodiments, the total concentration of detergents is reduced to below their critical micelle concentration (CMC) in step (2). For example, the total concentration of detergents is reduced to below 0.01% (w / v) in step (2). In some embodiments, the total concentrations of detergents is reduced to below ImM, below 100 pM, below 10 pM, below 1 pM, below 100 nM, or below 10 nM. For example, the total concentration of detergents is reduced to below 50 pM. The CMC can be readily determined for a given detergent or mixture of detergent using known methods, e.g. using fluorescence spectroscopy (see e.g. Wu). Of note, the CMC decreases with increasing salt concentrations. When determining the CMC of a given detergent or mixture of detergents, the applied salt concentration is to be taken into account.In some embodiments, the only detergent present is DDM. For example, DDM is the only detergent present and is present at a concentration below 0.01% (w / v). In another example, DDM is the only detergent present and is present at a concentration below 200 pM.In some embodiments, the detergents are completely removed after step (1). Complete removal can be done, for example, in step (2) and / or (3). For example, the total concentration of detergents can be reduced in step (2) to such an extent that LNPs will spontaneously form, followed by a removal of residual detergents in step (3). In an alternative example, the detergents can be completely removed already in step (2).The molar ratio between lipid, saposin and nanopore in the product of step (1 ) is not further limited, as long as it allows formation of LNPs as described herein upon reduction of the total concentration of detergents in step (2). The skilled person will be readily able to determine molar ratios suitable for formation of LNPs.In some embodiments, the molar ratio of saposimlipid in the product of step (1) is between 1 :5 and 1 :500, such as between L 10 and 1 :250, including between L25 to 1 : 100. For example, the molar ratio of saposimlipid can be 1:50.In some embodiments, the molar ratio of saposimnanopore in the product of step (1) is between 1:0.04 and 1 : 1, such as between 1:0.08 and 1:0.5, including between 1:0.1 to 1:0.4. For example, the molar ratio of saposimlipid can be 1 :0.2.In some embodiments, the molar ratio of saposimlipid in the product of step (1) is between 1 :5 and 1:500, such as between 1: 10 and 1 :250, including between 1:25 to 1: 100, and the molarratio of saposin:nanopore in the product of step (1) is between 1 :0.04 and 1 : 1, such as between 1 :0.08 and 1:0.5, including between 1 :0.1 to 1 :0.4.In some embodiments, the molar ratio of saposinlipid in the product of step (1) is 1 :50, and the molar ratio of saposinmanopore in the composition is between 1:0.04 and 1:1. such as between 1:0.08 and 1 :0.5, including between 1:0.1 to 1 :0.4.In some embodiments, the molar ratio of saposin:lipid in the product of step (1) is between 1 :5 and 1:500, such as between 1: 10 and 1 :250, including between 1:25 to 1: 100, and the molar ratio of saposinmanopore in the composition is 1 :0.2.In some embodiments, the molar ratio of saposimlipidmanopore in the product of step (1) is 1 :50:0.2.The disclosure further provides a LNP produced by the method disclosed herein.In any of the embodiments disclosed herein, the nanopore can comprise aHL, the lipid can be POPG or POPE and the saposin can be Sap A, for example. For instance, the nanopore can be aHL nanopore, the lipid can be POPG or POPE and the saposin can be SapA.In any of the embodiments disclosed herein, the nanopore can comprise aHL, the lipid can be POPG and the saposin can be SapA, for example. For instance, the nanopore can be aHL nanopore, the lipid can be POPG and the saposin can be SapA.Further details on the nanopore, lipid and saposin are as follows.A. NanoporeA “nanopore” generally refers to a pore in the nanometer scale. For example, the nanopore can be suitable for nanopore-based sequencing of a target molecule, such as a single stranded nucleic acid. For instance, a nanopore that is suitable for nanopore-based sequencing may generally have an inner diameter (at its narrowest segment) of 0.5 nm to 4 nm. such as 1 nm to 3 nm. For example, the nanopore may have an inner diameter (at its narrowest segment) of 1 nm to 2 nm, such as 1.4 nm.In the context of nanopore-based sequencing, the nanopore is formed or otherwise provided in an electrically-resistive barrier (such as a lipid bilayer membrane, a silicon layer, a polymeric layer, or a graphene layer) such that an ionic current may pass through the nanopore (in open state).In the context of this disclosure, the nanopore is typically a biological nanopore. The ty pe of biological nanopore is not particularly limited, and can be, for example, a biological nanopore comprising a nanopore-forming protein selected from a-hemolysin (aHL), Mycobacterium smegmatis porin A (MspA), outer membrane porin G (OmpG), outer membrane porin F (OmpF), outer membrane porin C (OmpC), gramicidin A, maltoporin, PhoE, Tsx, F-pilus, SPP1 , mitochondrial porin (VDAC)XX, Tom40, leukocidin, cytolysin A (ClyA), outer membranephospholipase A, Neisseria autotransporter lipoprotein (NalP). WZA, Nocardia farcinica NfpA / NfpB cationic selective channel, lysenin, aerolysin, and Curlin sigma S-dependent growth subunit G (CsgG). For example, the nanopore may comprise odTL.In some embodiments, the nanopore is “aHL nanopore'’, meaning a nanopore formed by seven aHL monomer subunits. A cross-section of an exemplary aHL nanopore is illustrated at e.g. Figure 3 of WO 2022 / 263496 AL Wildtype aHL is found under SEQ ID NO: 1.The aHL monomer subunits of the nanopore may comprise modifications that confer specific characteristics on the pore. One example includes substitutions that control the ability of non-oligomerized monomer subunits to self-oligomerize. For example, aHL monomer subunits having substitutions at H35 (e.g., H35G / L / D / E substitutions) are substantially non-oligomerized as long as they are kept at room temperature or below (e.g. 25 °C or lower), but will stably oligomerize when the temperature is raised to a higher temperature (e.g. 35 °C). Other examples of substitution strategies for controlling self-oligomerization and / or directing specific patterns of oligomerization are disclosed in, for example, WO 2017 / 050718. Another example includes substitutions that improve the expression level of the aHL monomer subunit(s) in a recombinant cell used to express the monomer subunit(s). Other examples include substitutions that reduce coefficient of variation of the arrival rate of the pore (CV), such as D227N.In some embodiment, the aHL comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 1 (and that is capable of forming a nanopore). The aHL optionally further comprises an N-terminal and / or C-terminal amino acid extension of 1 to 30, such as 1 to 20, 1 to 10 or 1 to 6 amino acids. An amino acid extension can be a 6xHis tag, for example, that is typically N-terminally fused. The aHL can also comprise a methionine at the N-terminus. For example, the aHL can comprise SEQ ID NO: 1 with an N-terminal and / or C-terminal amino acid extension of 1 to 30, such as 1 to 20, 1 to 10 or 1 to 6 amino acids, and optionally a methionine at the N-terminus (and that is capable of forming a nanopore). For example, the aHL can comprise SEQ ID NO: 1 with an N-terminally fused 6xHis tag, and optionally a methionine at the N-terminus.Each monomer subunit of the aHL nanopore may have the same primary amino acid sequence (termed a “homoheptamer”), or at least one monomer subunit of the heptamer may have an amino acid sequence that is different from the amino acid sequence of the other monomer subunits (termed a “heteroheptamer”). In some embodiments, each monomer subunit of the aHL nanopore is provided as a polypeptide that does not contain additional monomer subunits (termed herein a “non-oligomerized monomer subunit”). Exemplary methods of making homoheptamers and heteroheptamers from non-oligomerized monomer subunits are disclosed at US 2017-0088890 ALAs mentioned, in some embodiments, the nanopore can be a biological nanopore formed by nanopore-forming proteins other than oHL. Exemplary non-oHL nanopore-forming proteins include Mycobacterium smegmatis porin A (MspA; canonical full-length unprocessed sequence disclosed at Uniprot Accession No. A0QR29-1), outer membrane porin G from Escherichia coli (OmpG; canonical full-length unprocessed sequence disclosed at Uniprot Accession No. P76045- 1), outer membrane porin F (OmpF), outer membrane porin C (OmpC), gramicidin A, maltoporin, PhoE, Tsx, F-pilus, DNA packaging motor of bacteriophage SPP1 (SPP1), mitochondrial porin (VDAC)XX, Tom40, leukocidin, cytolysin A (ClyA), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP), WZA, Nocardia farcinica NfpA / NfpB cationic selective channel, lysenin. aerolysin. and Curlin sigma S-dependent growth subunit G (CsgG), dodecameric connector channel from bacteriophage phi29 DNA packaging motor (Phi29), Bacillus anthracis protective antigen, PAes (PA63), and ferric hydroxamate uptake component A (FhuA) from E. coli. Reviews of the use of various nanopore proteins can be found at, for example, Gari (OmpG), Haque etal. (MspA and Phi29), and Wang II (Phi29, MspA, CsgG, PA63, ClyA, FhuA, SPP1).In some embodiments, at least one nanopore-forming protein is attached to an enzyme, such as a helicase or a polymerase, for example by covalent linkage. Embodiments in which a polymerase is attached are especially useful for performing SBT methods. In some embodiments, a single polymerase is attached to a nanopore-forming protein. Exemplary polymerases include those derived from DNA polymerase Clostridium phage phiCPV4 (described by GenBank Accession No. YP_00648862, referred to herein as “Pol6”), phi29 DNA polymerase, T7 DNA pol, T4 DNA pol, E. coli DNA pol 1, KI enow fragment, T7 RNA polymerase, and E. coli RNA polymerase, as well as associated subunits and cofactors. In some embodiments, the polymerase is a DNA polymerase derived from Pol6. Exemplary Pol6 derivatives useful in nanopore-based sequencing are disclosed at, for example, US 2016 / 0222363, US 2016 / 0333327, US 2017 / 0267983, US 2018 / 0094249, and US 2018 / 0245147. Exemplary methods of attaching a polymerase to an aHL nanopore include SpyTag / SpyCatcher peptide system (Zakeri), native chemical ligation system (Thapa), sortase system (Wu; Heck), transglutaminase systems (Dennler), formylglycine linkage systems (Rashidian), click chemistry attachment systems, or other chemical ligation techniques known in the art. In yet other embodiments, one of the monomer subunits of the nanopore-forming proteins is expressed as a fusion protein with the polymerase. In some embodiments, the polymerase is attached to an amino acid side chain of one of the monomer subunits.In other embodiments, the nanopore-forming protein(s) is / are not attached to an enzyme, such as a helicase or polymerase. Nanopores formed with such nanopore-forming proteins are especially useful for performing sequencing by expansion methods, for example.B. LipidThe type of lipid is not particularly limited, as long as it can form an LNP in association with a given saposin and a given nanopore. As mentioned, the formation of LNPs can be readily tested, e.g. by electron microscopy.The lipid can be, for example, a phosphatidylcholine-based lipid, phosphoethanolamine- based lipid, or a synthetic derivative thereof. In some embodiments, the lipid is chosen from POPG, POPE, DPhPC, DMPC and DPhPE. More particularly, the lipid can be chosen from POPG and POPE, and most particularly can be POPG.The examples show that POPG and POPE are particularly suitable for forming LNPs in conjunction with SapA. Moreover, the examples show that POPG is particularly suitable for forming LNPs in conjunction with SapA and aHL.C. SaposinThe type of saposin is not particularly limited, as long as it can form an LNP in association with a given lipid and a given nanopore. As mentioned, the formation of LNPs can be readily tested, e.g. by electron microscopy. The specificity of different types of saposins to different types of lipids has been described in e.g. Flayhan.Human saposins A-D are cleavage products from the same prosaposin precursor protein (Uniprot Accession No. P07602). The saposin A cleavage product corresponds to residues 60 to 140 (SEQ ID NO: 2), saposin B cleavage product corresponds to residues 195 to 273 (SEQ ID NO: 3). the saposin C cleavage product corresponds to residues 311 to 390 (SEQ ID NO: 4), and the saposin D cleavage product corresponds to residues 407 to 484 (SEQ ID NO: 5) of prosaposin.In some embodiment, the saposin is (human) SapA. In some embodiments, SapA comprises the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 2 (and that has at least the same capacity of forming LNPs with POPG compared to the amino acid sequence of SEQ ID NO: 2). More particularly, SapA can comprise the amino acid sequence of SEQ ID NO: 2, optionally with aN- and / or C-tenninal amino acid extension, and optionally a methionine at the C-terminus.In some embodiments, the saposin is (human) SapB. In some embodiments, SapB comprises the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 3 (and that has at least thesame capacity of forming LNPs with POPG compared to the amino acid sequence of SEQ ID NO:3). More particularly, SapB can comprise the amino acid sequence of SEQ ID NO: 3, optionally with aN- and / or C-terminal amino acid extension, and optionally a methionine at the C-terminus.In some embodiments, the saposin is (human) SapC. In some embodiments, SapC comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 4 (and that has at least the same capacity of forming LNPs with POPG compared to the amino acid sequence of SEQ ID NO:4). More particularly, SapC can comprise the amino acid sequence of SEQ ID NO: 4, optionally with aN- and / or C-terminal amino acid extension, and optionally a methionine at the C-terminus.In some embodiments, the saposin is (human) SapD. In some embodiments. SapD comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 5 and that has at least the same capacity of forming LNPs with POPG compared to the amino acid sequence of SEQ ID NO: 5. More particularly, SapD can comprise the amino acid sequence of SEQ ID NO: 5, optionally with a N- and / or C-terminal amino acid extension, and optionally a methionine at the C-terminus.Saposins from species other than Homo sapiens can also be used, such as Equus caballus, Bos taurus, Mus musculus, Oryctolagus cuniculus, Rattus norvegicus, Xenopus laevis.Any of the saposins described herein can comprise an N- and / or C-terminal amino acid extension, e.g. for purification, such as a SUMO tag (e.g. the amino acid sequence of SEQ ID NO: 6) and / or 6xHis tag and the like. For example, a SUMO tag (e.g. the amino acid sequence of SEQ ID NO: 6) can be N-terminally fused to a saposin. For example, a 6xHis tag can be C-terminally fused to a saposin. Such amino acid extensions typically have a length of 1 to 120, such as 1 to 104, 1 to 50, 1 to 30. 1 to 20, 1 to 10 or 1 to 6 amino acids. Any of the saposins described herein can further comprise a methionine atthe N-terminus. Thus, in some embodiments, the saposin can comprise any one of SEQ ID NOs 2-5 (e.g. SEQ ID NO: 2) with an N-terminal and / or C-terminal amino acid extension of 1 to 120, such as 1 to 104, 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 6 amino acids, and optionally a methionine at the N-terminus (and that has at least the same capacity of forming LNPs with POPG compared to the amino acid sequence of the SEQ ID NOs 2-5, respectively; i.e. when the saposin comprises SEQ ID NO: 2, the reference sequence is SEQ ID NO: 2, etc.). More specifically, an N-terminal amino acid extension may have a length of 1 to 120, such as 1 to 104 amino acids, for example. A C-terminal amino acid extension may have a length of 1 to 30, 1 to 10 or 1 to 6 amino acids, for example. Thus, in some embodiments, the saposin can comprise any one of SEQ ID NOs 2-5 (e.g. SEQ ID NO: 2) with an N-terminal amino acid extension of 1 to 120 or 1 to 104 amino acids and / or a C-terminal amino acid extension of,l to 30, 1 to 20, 1 to 10 or 1 to 6 amino acids, and optionally a methionine at the N-terminus (and that hasat least the same capacity of forming LNPs with POPG compared to the amino acid sequence of the SEQ ID NOs 2-5, respectively). In more specific embodiments, the saposin can comprise any one of SEQ ID NOs 2-5 (e.g. SEQ ID NO: 2) fused N-terminally to a SUMO tag (e.g. the amino acid sequence of SEQ ID NO: 6) and fused C-terminally to a 6xHis tag, and optionally a methionine at the N-terminus (and that has at least the same capacity of forming LNPs with POPG compared to the amino acid sequence of the SEQ ID NOs 2-5, respectively).III. Nucleic acid sequencing systems and methodsThe disclosure also provides chips, systems and methods for performing sequencing using the disclosed LNPs. Systems for nanopore-based sequencing (e.g. of nucleic acids or Xpandomers) generally comprise a chip with a plurality of nanopores and a computing system adapted to record changes in one or more electrical characteristics of the nanopores.In some embodiments, the disclosure provides a chip for performing nanopore-based sequencing, produced using the LNP disclosed herein.As those skilled in the art will appreciate, chips for nanopore-based sequencing and systems comprising the same have been developed for rapid sequencing of polymers and various designs and methods of use are known in the art. See, e.g., US9494554B2, US9567630B2, US9557294B2, US9605309B2, each of which is hereby incorporated by reference herein in their entirety. These chips generally comprise an electrochemical cell with a chamber containing a nanopore within a barrier. The barrier acts to separate the cell chamber into two sub-chambers, referred to as the cis and trans sides, each of which normally contain an electrode. The barrier can be, for example, an organic membrane, such as a lipid bilayer, or a synthetic membrane made of a non-naturally occurring polymeric material. For sequencing operation, the cis side and the trans side are filled with electrolyte solutions, such that the pore of the nanopore acts as a channel in the membrane permitting ion exchange between the electrolyte solutions. A non-limiting example of an electrochemical cell within a chip for performing nanopore-based sequencing is illustrated at, e.g., Figure 2 of WO 2018 / 069302 Al and the description thereof in

[0051] -

[0060] of that document, incorporated herewith by reference.In some embodiments, the disclosure provides a chip for performing nanopore-based sequencing, comprising:(a) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(b) a first electrolyte solution on the cis side of the barrier;(c) a second electrolyte solution on the trans side of the barrier;(d) the LNP disclosed herein inserted into the barrier, wherein the nanopore comprised in the LNP has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and(e) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.In some embodiments, the working electrode is positioned on the trans side. In some embodiments, the chip comprises a working electrode positioned on the trans side and a counterelectrode positioned on the cis side.The working electrode can be a metal electrode, for example. For non-faradaic conduction, the working electrode can be made of metals or other materials that are resistant to corrosion and oxidation, such as, for example, platinum, gold, titanium nitride, and graphite. For example, the working electrode can be a platinum electrode with electroplated platinum. In another example, the working electrode can be a titanium nitride (TiN) working electrode.In some embodiments, the chip comprises a plurality of apertures, a barrier disposed over each aperture, a first electrolyte solution on the cis side and a second electrolyte solution on the trans side of each barrier, an LNP as disclosed herein inserted into each barrier, and a plurality of working electrodes positioned to detect changes in an electrical characteristic of each nanopore associated with occupation of a given nanopore by a molecule.The nature of the barrier is not particularly limited, but the barrier will typically be a lipid bilayer membrane. In some embodiments, the barrier is detergent free. For example, the barrier is a lipid bilayer membrane that is detergent free.The disclosure also provides a chip for performing nanopore-based sequencing, comprising:(a) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(b) a first detergent-free electrolyte solution on the cis side of the barrier;(c) a second detergent-free electrolyte solution on the trans side of the barrier;(d) a nanopore as disclosed herein inserted into the barrier, wherein the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and(e) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule, wherein the barrier is a lipid bilayer membrane that is detergent-free.In some embodiments, the chip further comprises a saposin inserted into the barrier.In some embodiments, the nanopore is a oHL nanopore, wherein the entrance side is formed by the barrel side of the nanopore and the exit side is formed by the vestibule side of nanopore. This can be achieved, for example, by including the nanopore into the second electrolyte solution, followed by electroporation. aHL nanopores have the tendency to insert barrel first into a lipid bilayer membrane upon electroporation.The disclosure also provides a kit for producing a chip for performing nanopore-based sequencing as disclosed herein, comprising a LNP or a composition comprising a LNP as disclosed herein. The kit can further comprise a solid support comprising an aperture and an electrode, a lipid solution for forming a lipid bilayer membrane as a barrier, and / or an electrolyte solution.In some embodiments, the kit further comprises a solid support comprising an aperture and an electrode.In some embodiments, the kit further comprises a lipid solution.In some embodiments, the kit further comprises an electrolyte solution. In some embodiments, the kit further comprises a first and a second electrolyte solution. The first and second electrolyte solutions can be the same. The first and second electrolyte solutions can thus be provided as a single electrolyte solution with the kit. Suitable types of electrolyte solutions are as described for the chip for performing nanopore-based sequencing.In some embodiments, the kit comprises a LNP or a composition comprising a LNP as disclosed herein, a lipid solution and an electrolyte solution.Suitable lipids provided with the kit are the same as described for forming a barrier on the chip for performing nanopore-based sequencing.In some embodiments, the solid support comprises a plurality of apertures and a plurality of electrodes. The solid support provided with the kit is generally similar to a chip for performing nanopore-based sequencing except that it does not comprise a barrier (such as a lipid bilayer membrane), a nanopore, and / or first and second electrolyte solutions. The kit thereby allows producing a chip for nanopore-based sequencing by the methods as described herein.In some embodiments, the solid support comprises- an aperture configured to allow disposing an electrochemically resistive barrier over the aperture to separate a cis side from a trans side; and- an electrode on the trans side of the aperture.In some embodiments, the solid support comprises- an aperture, w herein an electrochemically resistive barrier is disposed over the aperture, wherein the barrier separates a cis side from a trans side;- an electrode on the trans side and an electrode on the cis side of the barrier.In some embodiments, the solid support comprises a plurality of apertures, and a barrier is disposed over each of the plurality of apertures.The barrier will typically be a lipid bilayer membrane.The disclosure further provides a method for producing a chip for performing nanoporebased sequencing using the LNP disclosed herein.In some embodiments, a method is provided for producing a chip for performing nanoporebased sequencing, comprising the steps of:(a) providing a solid support comprising an aperture,(b) forming an electrochemically resistive barrier over the aperture, such that the barrier separates a cis side from a trans side;(c) providing a first electrolyte solution on the cis side of the barrier,(d) providing a second electrolyte solution on the trans side of the barrier,(e) contacting the barrier with the LNP as disclosed herein or with the composition comprising an LNP as disclosed herein, to insert the nanopore comprised in the LNP into the barrier, such that the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier permitting ion exchange between the first electrolyte solution and the second electrolyte solution, and(I) positioning a working electrode to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.The barrier will typically be a lipid bilayer membrane.In some embodiments, the first and / or second electrolyte solution is detergent free.In some embodiments, the barrier is a lipid bilayer membrane and the nanopore is inserted into the barrier by electroporation. This can be achieved, for example, by including the nanopore on the trans side of the barrier, followed by electroporation. Alternatively, the nanopore can also be on the cis side of the barrier followed by electroporation. Without wishing to be bound by any theory, it is expected that all components of the LNP can be inserted into a lipid bilayer membrane via electroporation. It is thus expected that the lipid bilayer membrane comprises, after electroporation, a nanopore and a saposin, or even a nanopore, a saposin and a lipid originating from the LNP.In some embodiments, the solid support comprises a plurality of apertures, wherein a barrier is formed over each of the plurality of apertures, and wherein each of the barriers is contacted with the LNP or with the composition.The disclosure also provides a chip produced by the methods for producing a chip disclosed herein.The lipid to form a lipid bilayer membrane in any of the embodiments described herein can be a single type of lipid or a mixture of lipids. The type of molecule to form the lipid bilayer membrane is not particularly limited as long as it can form an electrochemically resistive barrier. Examples of suitable molecules to form the lipid bilayer membrane include amphiphilic lipids and synthetic polymers. Amphiphilic lipids can be naturally occurring lipids, such as phospholipids, or synthetic lipids. Exemplary naturally occurring lipids include various phospholipids such as 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), palmitoyl-oleoyl-phosphatidyl- choline (POPC), dioleoyl-phosphatidyl-methylester (DOPME), l,2-diphytanoyl-sn-glycero-3- phosphatidylcholine (DPhPC) dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin.Exemplary synthetic polymers to form a lipid bilayer membrane include molecules such as poly(n-butyl methacrylate-phosphorylcholine), poly(ester amide)-phosphorylcholine, polylactide-phosphorylcholine, polyethylene glycol-poly(caprolactone)-di- or tri-blocks, polyethylene glycol-polylactide di- or tri-blocks and polyethylene glycol-poly(lactide-glycolide) di- or tri-blocks.A lipid bilayer membrane can comprise further components in addition to the lipid or mixture of lipids. For example, such further components can be bilayer additives and / or cosolvents.The first electrolyte solution and second electrolyte solution are generally aqueous solutions buffered to an optimum ion concentration and maintained at an optimum pH to keep the nanopore open and the barrier intact as long as possible. In any of the embodiments described herein, the first and second electrolyte solutions can be the same or different. The components of the first and second electrolyte solutions are not particularly limited, but in general the first and / or second electrolyte solution will be detergent-free. The types of electrolytes are not particularly limited as long as they enable an ionic current through the nanopore during nanopore-based sequencing. Examples of suitable electrolytes include one or more of ammonium chloride (NH4Q), lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (K.C1), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCk), strontium chloride (SrCh), manganese chloride (MnCh), and magnesium chloride (MgCh).The electrolyte solutions can independently further comprise a buffering agent. The buffering agent is not particularly limited as long as it does not interfere with nanopore-based sequencing. Examples of suitable buffering agents include HEPES, Tris buffer, phosphate buffer and MES buffer. When the sequencing is conducted using non-faradaic current, a redox pairpresent in the electrolyte solutions can slow down the voltage drop that is inherent to non-faradaic operation. Thus, in some embodiments, the first and second electrolyte solutions can further comprise a redox pair. The redox pair is not particularly limited as long as it does not interfere with nanopore-based sequencing. Examples of suitable redox pairs include ferrocyanide / ferri cyanide.The first electrolyte solution can further comprise a target molecule to be sequenced.Apart from the above, the electrolyte solutions can further comprise additional components, such as chaotropic denaturants or crowding agents that increase the localized concentrations of a target molecule near the nanopore, as long as these components do not interfere with nanopore-based sequencing.The disclosure further provides a system for performing nanopore-based sequencing, comprising the chip as disclosed herein.In some embodiments, the system comprises the chip as disclosed herein and a computing system adapted to record changes in one or more electrical characteristics of the nanopore, wherein the working electrode in the chip is in electronic communication with the computing system, and wherein the working electrode is positioned to transmit any detected change(s) in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule to the computing system.The disclosure also provides a method for sequencing a target molecule using the chip as disclosed herein.It has been found that using the LNPs disclosed herein to produce a chip for nanoporebased sequencing can increase the sequencing accuracy, mainly due to the decrease of insertions. Without wishing to be bound by any theory, it is assumed that the usage of less detergent or even no detergent at all during nanopore delivery' leads to a higher stability of the membrane surrounding the nanopore and to more stable voltage signals used for detection of changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.In some embodiments, the method for sequencing a target molecule comprises:(a) providing a chip as disclosed herein;(b) contacting the chip with a target molecule;(c) applying a voltage across the membrane of the chip;(d) determining, by the working electrode, one or more changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by the target molecule; and(e) determining, based on the one or more changes in the electrical characteristic of the nanopore, a sequence for the target molecule.In some embodiments, the target molecule is a nucleic acid, modified nucleic acid, or other organic polymer. For example, the target molecule may be a DNA sequence or RNA sequence, or in some instances a DNA sequence or RNA sequence that has been modified for improved sequencing or other applications. For example, US 10,851,405 describes a modified target molecule that includes a hydrophobic capture element. The hydrophobic capture element, for example, increases the affinity of the target molecule to the lipid membrane and hence improves nanopore capture of the target molecule. See US 10,851,405, which is hereby incorporated herein in its entirety. As another example, the other organic polymer can comprise an Xpandomer. Thus, in some embodiments, the target molecule comprises an Xpandomer. In more specific embodiments, the target molecule comprises an Xpandomer. In general terms, sequencing by expansion uses biochemical polymerization to transcribe the sequence of a DNA template onto a measurable polymer called an “Xpandomer”.More particularly, the sequencing by expansion technique is based on the polymerization of “expandable NTPs”, or in short “XNTPs” (see, e.g.. Figure 1 of WO 2020 / 236526 Al for an illustration of an exemplary' structure of an XNTP and the generation of Xpandomers). The XNTPs are expandable, 5' triphosphate modified non-natural molecules compatible with template dependent enzy matic polymerization. Each XNTP has two distinct functional regions, e.g., a selectively cleavable phosphoramidate bond linking the 5’ a-phosphate to a sugar comprised in a nucleoside and a symmetrically synthesized reporter tether (SSRT) that is attached within the nucleoside triphosphorami date at positions that allow for controlled expansion by cleavage of the phosphoramidate bond. In this embodiment, the SSRTs include one or more reporters or reporter codes specific for the nucleobase to which they are linked, e.g., A, C, G, or T. thereby encoding the sequence information of the template. The SSRT includes linkers separated by the selectively cleavable phosphoramidate bond. Each linker attaches to one end of a reporter code. While the XNTP substrates are bound to the daughter strand via template-dependent polymerization, they are present in a “constrained configuration” due to their size. The constrained configuration of polymerized XNTPs is the precursor to the expanded configuration, as found in Xpandomers. The transition from the constrained configuration to the expanded configuration occurs upon scission of the P-N bond of the phosphoramidate within the primary' backbone of the daughter strand. The transcribed nucleic acid sequence is thereby encoded along the daughter strand backbone in reporter moieties that are separated by ~10 nm and are designed for high-signal-to-noise, well- differentiated responses. These differences provide significant performance enhancements in sequence read efficiency and accuracy of Xpandomers relative to natural nucleic acids, such as DNA.During Xpandomer assembly, the monomeric XNTP substrates (XATP, XCTP, XGTP and XTTP) are polymerized on the extendable terminus of a nascent daughter strand by a process of template-directed polymerization using single-stranded template as a guide. This process is initiated, for example, from a primer and it proceeds in the 5’ to 3’ direction. Generally, a DNA polymerase or other polymerase is used to form the daughter strand, and conditions are selected so that a complimentary copy of the template strand is obtained. Immediately after synthesis, the daughter strand is constrained, in which the XNTPs have the “constrained configuration” of the XNTP substrates. The constrained configuration of the daughter strand is the precursor to the expanded configuration, as found in the Xpandomers.The transition from the constrained configuration to the expanded configuration results from cleavage of the selectively cleavable phosphoramidate bonds within the primary backbone of the daughter strand. In this manner, the SSRTs provide a means to expand the length of the Xpandomer and lower the linear density of the sequence information of the parent strand. More details on how to generate Xpandomers and sequencing the same using nanopore-based sequencing can be found in e.g. WO 2020 / 236526 Al .In some embodiments, the determined sequence of the target molecule determines the sequence of a target nucleic acid sequence. For example, the determined sequence of an Xpandomer can determine the sequence of a nucleic acid sequence used as template for Xpandomer synthesis, such as a DNA sequence.In any of the embodiments described herein, the electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule can be a change in the electrical resistance of the nanopore, for example. Generally, the electrical characteristic of the nanopore can be measured as a cunent change or a voltage change. For example, the electrical characteristic of the nanopore can be measured as a current decrease or a voltage increase.The following examples describe some ways of performing the disclosure, but are not meant as limiting. The scope of the disclosure is limited only by the claims, and the disclosure encompasses numerous alternatives, modifications and equivalents. The specific details of particular embodiments may be combined in any suitable manner without departing from the spirit and scope of embodiments of the invention.IV. ExamplesExample 1: Expression and purification of Saposin AThe gene encoding Saposin A was cloned into the vector pET 26b with SUMO tag and 6xHis tag at N- terminus and C-terminus respectively. The protein was expressed as describedpreviously (Frauenfeld) with minor modification. Briefly, the expression vector was transformed into E. coll Rosetta gami-21M(DE3). Cells were grown at 37 °C in Terrific broth supplemented with kanamycin and chloramphenicol and induced with ImM IPTG at OD= 0.5 for 3 hr. The cells were harvested by centrifugation at 4000 rpm for 30 min. The cell pellet was resuspended in the lysis buffer (20 mM Tris-Cl. pH 8.0, 300 mM NaCl, 10 mM imidazole) and disrupted by sonication. Lysates were subjected to centrifugation at 15,000rmp for 30 min and the supernatant was loaded onto a cobalt resin column followed by washing with 20 CV lysis buffer. The protein was eluted with 3 CV elution buffer( 20 mM Tris-Cl, pH 8.0, 300 mM NaCl, 150 mM imidazole). The elute was concentrated and loaded onto the Superdex 200 increase 10 / 300 column with running buffer 20mM Tris-Cl pH8.0 200mM NaCl. Peak fractions were pooled and concentrated. The protein sample was flash-frozen in liquid nitrogen and stored at -80 °C for further use. The gel filtration elution profile of SapA is shown in FIG. 1A. Further, fractions C1-C4 in FIG. IB represent the fractions collected from the main (single) peak of saposin A purification shown in the size exclusion column chromatogram in Fig. 1 A. The fractions are different in overall protein concentration and the components and purity is visualized in the SDS page gel in FIG. IB.Example 2: Generation of Saposin A lipid particlesSaposin A lipid particles were generated as described previously (Flayhan) with minor modification. In brief, the lipid was dissolved in the buffer 50mM HEPES pH7.5, 150mM NaCl, 0.2%DDM and further incubated for 1 hour at 37 C under shaking. The lipid and Saposin A were mixed at a molar ratio of 50: 1 and incubated at 37 C for 10 min. Dilute the mixture with detergent- free buffer 50mM HEPES pH7.5, 150mM NaCl to make the concentration of DDM below 0.01% and incubate the sample at 37 C for 10 min. The samples were centrifuged at 13,000 rpm for 5 min and transferred to a 30kD cut-off concentrator to concentrate dow n to around lOOuL Another 2ml detergent-free buffer 50mM HEPES pH7.5, 150mM NaCl was added to the sample and the mixture was concentrated down to around lOOuL. The sample was run on the SEC column Superose™ 6 Increase 5 / 150 GL in the buffer 20mM Tris pH8.0 200mM NaCl.To assess the efficiency of the lipids for the formation of SapA LNP, several lipids were screened, including DPhPC, DMPC, DPhPE, POPE and POPG. It w as determined that POPG and POPE can form SapA LNP with SapA more efficiently than the others (FIGS. 2A-2F).Example 3: Incorporation of nanopore into SapA LNPThe reconstitution was performed as described previously with minor modification (Flayhan). To reconstitute the nanopore into the Saposin A lipid particles, purified protein nanopore was mixed with the lipids solution solubilized in the buffer 50mM HEPES pH7.5,150mM NaCl, 0.2%DDM and incubated for 10-20 min at room temperature. Then SapA was added to the sample and the mixture was incubated for 30 min at room temperature. The ratio of SapA: Lipid: Pore is 1 : 50: 0.2. The samples were diluted with a detergent-free buffer to bring the concentration of detergent below the CMC and incubated for 10 min at room temperature. Finally, the samples were centrifuged at 13,000 rpm for 5 min followed by running on SEC column Superdex 200 increase 10 / 300 in the buffer 20mM Tris pH8.0 200mM NaCl.In this example, an a-hemolysin nanopore was reconstituted into POPG SapA LNP and POPE SapA LNP and it was determined that the nanopore can be incorporated into POPG SapA LNP more efficiently than into POPE SapA LNP (see FIGS. 3A-3B). Therefore. POPG SapA LNP was chosen for further nanopore reconstitution.Example 4: SequencingIn this example, a-hemolysin nanopores were prepared either incorporated into POPG SapA LNPs according to Example 3 or solubilized by detergent Polysorbate 20 / n-octyl-P-d- glucoside. The template for generating the Xpandomers for sequencing was a distributed Human Genomic library (HG001 -maternal -vl) with a median read length of 370 bases. Sequencing of Xpandomers was carried out according to WO 2020 / 236526. Briefly, for sequencing, Xpandomers were added to a sample buffer of 1.25M NH4CI, 187.5mM K3Fe(CN)6, 187.5mM K4Fe(CN)e, 125mM MES pH 6.2, 1000 mM Urea, 6.25% PEG8k. Protein nanopores were prepared by inserting WT a-hemolysin (either detergent-solublized or reconstituted in POPG SapA LNPs) into a DPhPE / hexadecane bilayer membrane in a buffer containing 2M NH4Q, 800mM Urea. 150mM K3lFe(CN)6], 150mM K4tFe(CN)6], lOOmM HEPES pH 7.4. This experiment used buffers of lOOmM MES pH 6.2, 800 mM Urea, 5% PEG8k, 1000 mM NH4Cl, 150mM IGFe(CN)6, 150mM K4Fe(CN)6in the cis well andlOOmM MES pH 6.2, 800mM Urea, 2MNH4C1, 150mM KsFe(CN)6, 150mM K4Fe(CN)6, 0.001% Tween 20 in the trans well. After diluting the Xpandomer, it flowed into the consumable device using the following flow schedule: 21 pL at 10 pL / sec followed by 45 pL at 0.013 pL / sec. The voltage parameters run were as follows: 70mV / 650mV / 6ps / l ,5ms (read voltage / pulse voltage / pulse voltage duration / pulse frequency). Primary' Analysis was performed using the in-house pipeline to look at the sequencing signal, and aggregation of the data was done with python scripts.Sequencing experiments were performed with both a-hemolysin nanopore in the buffer with detergent and separately with nanopore and in POPG SapA LNP. The results showed that throughput (FIG. 4A), functional lifetime (Fig. 4B), and read length (FIG. 4D) were comparable between them (i.e.. comparable versus control). However, the nanopore in the POPG SapA LNP condition showed higher sequencing accuracy (Fig. 4C), which was mainly due to the decrease ofinsertions (Fig. 4E and FIG. 4F). Therefore, using nanopores reconstituted into saposin LNPs provides a new and improved way for nanopore delivery onto a chip for sequencing.SEQUENCE LISTING FREE TEXTSEQ ID NO: 1 (WT «HL)ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNSEQ ID NO: 2 (SapA cleavage product)SLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESSEQ ID NO: 3 (SapB cleavage product)GDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQPKEICALVGFCDESEQ ID NO: 4 (SapC cleavage product)SDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGSEQ ID NO: 5 (SapD cleavage product)GFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVL1EILVEVMDPSFVCLKIGACPSSEQ ID NO: 6 (SUMO tag)GSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGGGSAll patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for all purposes. None is admitted to be prior art.NON-PATENT REFERENCESThe following non-patent references are referred to in the text by the name of the first author:1. Altschul et al., Basic local alignment search tool, 1990, J Mol Biol Vol. 215. Issue 3, pp. 403-10. ("‘Altschul I ")2. Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, 1997, Nucleic Acids Res. Vol. 25, Issue 17, pp. 3389- 402. (“Altschul II”)3. Ausubel et al., Current protocols in molecular biology, 1993, John Wiley & Sons, Inc.4. Chen & Liu, Fabrication and Applications of Solid-State Nanopores, 2019, Sensors, Vol. 19, Issue 8, E1886.5. Dennler et al., Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates, 2014, Bioconjug Chem Vol. 25, Issue 3, pp. 569-5786. Feng et al.. Nanopore-based Fourth-generation DNA Sequencing Technology, 2015, Genomics, Proteomics & Bioinformatics, Vol. 13, Issue 1, pp. 4-16.7. Flayhan et al., Saposin Lipid Nanoparticles: A Highly Versatile and Modular Tool for Membrane Protein Research, 2018, Structure, Volume 26, Issue 2, pp. 345- 355. e58. Frauenfeld et al., A saposin-lipoprotein nanoparticle system for membrane proteins, 2016, Nature Methods, Volume 13. pp. 345-3519. Gari et al., Quiet Outer Membrane Protein G (OmpG) Nanopore for Biosensing, ACS Sensors, April 14, 2019, Vol. 4, pp. 1230-35.10. Hale and Marham, The Harper Collins dictionary of biology, 1991, Harper Perennial, NY11. Haque et al. , Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA, Nano Today (Feb. 2013), Vol. 8, Issue 1, pp. 56-74.12. Heck et al., Enzyme-catalyzed protein crosslinking, 2013, Appl Microbiol Biotechnol, Volume 97, pp. 461-47513. Kumar et al., PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis, 2012, Scientific Reports, Vol. 2, Art. 684.14. Lee et al., Recent progress in solid-state nanopores, 2018, Advanced Materials, Vol. 30. Issue 42.Manrao et al. , Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase, 2012, Nature Biotechnology, Vol. 30, pp. 349-53. Rashidian et al. , Enzymatic labeling of proteins: techniques and approaches, 2013, Bio conjug Chem, Volume 24, pp. 1277-94 Sambrook et al., Molecular cloning: A laboratory manual, 1989, Cold Spring Harbor Laboratory Press Singleton et al.. Dictionary of microbiology and molecular biology, 2nded., 1994, John Wiley and Sons. New York (1994) Thapa et al.. Native chemical ligation: a boon to peptide chemistry, 2014, Molecules, Volume 19, pp. 14461-483 Walker and Cox, The Language of Biotechnology: A Dictionary of Terms. 1988, American Chemical Society, Washington, D C. ISBN-0-8412-1499-1 Wang et al., The evolution of nanopore sequencing, 2015, Frontiers in Genetics, Vol. 5, Art. 449 (“Wang I”). Wang et al., Engineering of Protein Nanopores for Sequencing, Chemical or Protein Sensing and Disease Diagnosis, 2018, Current Opinions in Biotechnology, Vol. 51. pp. 80-89 (“Wang II”). Wash et al.. Graphene-based nanopore approaches for DNA sequencing: A literature review, 2018, Biosensors and Bioelectronics, Vol. 1 19, pp. 191-203. Wu and Guo, Sortase-mediated transpeptidation for site-specific modification of peptides, glycopeptides, and proteins, 2012. J Carbohydr Chem, Volume 31, pp. 48-66 Wu et al., Determination of the critical micelle concentration of surfactants using fluorescence strategies, 2022, Soft Matter, Vol. 18, pp. 8920-8930 Zakeri et al., Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin, 2012, PNAS, Volume 109, pp. E690-E697

Claims

PATENT CLAIMS1. A lipid nanoparticle (LNP) comprising: a lipid, a saposin, and a nanopore.

2. The LNP of claim 1, wherein the nanopore comprises a nanopore-forming protein selected from a-hemolysin (aHL), Mycobacterium smegmatis porin A (MspA), OmpG, OmpF, OmpC, gramicidin A, maltoporin. PhoE, Tsx, F-pilus. SPP1, mitochondrial porin (VDAC)XX and Tom40.

3. The LNP of any one of the preceding claims, wherein the nanopore comprises a-hemolysin (aHL).

4. The LNP of any one of the preceding claims, wherein the lipid is chosen from POPG, POPE, DPhPC, DMPC and DPhPE.

5. The LNP of any one of the preceding claims, wherein the lipid is POPG.

6. The LNP of any one of the preceding claims, wherein the saposin is Saposin A (SapA).

7. The LNP of any of the preceding claims, wherein the LNP is detergent-free.

8. A composition comprising the LNP of any one of claims 1-7.

9. The composition of claim 8. wherein the total concentration of detergents in the composition is below their critical micelle concentration.

10. The composition of claim 8 or 9, wherein the composition is detergent-free.

11. A method for producing a lipid nanoparticle (LNP), comprising the steps:(1) mixing a first solution comprising a nanopore-forming protein solubilized by a detergent, a second solution comprising a lipid solubilized by a detergent, and a third solution comprising a saposin, and(2) reducing the total concentration of detergents comprised in the product of step (1).

12. The method of claim 11, wherein the step (1) comprises the substeps:(la) mixing the first solution comprising a nanopore-forming protein solubilized by a detergent and the second solution comprising a lipid solubilized by a detergent to produce a first mixture, and(lb) mixing the product of step (la) with the third solution comprising a saposin to produce a second mixture.

13. The method of claim 11 or 12, further comprising the step:(3) purifying the LNP present in the product of step (2).

14. The method of any one of claims 11-13. wherein the nanopore is suitable for sequencing a target molecule.

15. The method of any one of claims 11-14, wherein the nanopore comprises a nanoporeforming protein selected from a-hemolysin (aHL), Mycobacterium smegmatis porin A (MspA), OmpG, OmpF, OmpC, gramicidin A, maltoporin, PhoE, Tsx, F-pilus, SPP1 , mitochondrial porin (VDAC)XX and Tom40.

16. The method of any one of claims 11-15, wherein the nanopore comprises a-hemolysin (aHL).

17. The method of any one of claims 11-16, wherein the lipid is chosen from POPG, POPE, DPhPC, DMPC and DPhPE.

18. The method of any one of claims 11-17, wherein the lipid is POPG.

19. The method of any one of claims 11-18, wherein the saposin is Saposin A (SapA).

20. A lipid nanoparticle (LNP) produced by the method of any one of claims 11-19.

21. A chip for performing nanopore-based sequencing, comprising:(a) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(b) a first electrolyte solution on the cis side of the barrier;(c) a second electrolyte solution on the trans side of the barrier;(d) the LNP of any one of claims 1-7 or 20 inserted into the barrier, wherein the nanopore comprised in the LNP has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and(e) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.

22. The chip of claim 21, wherein the barrier is a lipid bilayer membrane.

23. A chip for performing nanopore-based sequencing, comprising:(a) an electrochemically resistive barrier disposed over an aperture on a surface of the chip, wherein the barrier separates a cis side from a trans side;(b) a first detergent-free electrolyte solution on the cis side of the barrier;(c) a second detergent-free electrolyte solution on the trans side of the barrier;(d) a nanopore inserted into the barrier, wherein the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier, such that the nanopore permits ion exchange between the first electrolyte solution and the second electrolyte solution, and(e) a working electrode positioned to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule, wherein the barrier is a lipid bilayer membrane that is detergent-free.

24. A method for producing a chip for performing nanopore-based sequencing, comprising the steps of:(a) providing a solid support comprising an aperture,(b) forming an electrochemically resistive barrier over the aperture, such that the barrier separates a cis side from a trans side;(c) providing a first electrolyte solution on the cis side of the barrier,(d) providing a second electrolyte solution on the trans side of the barrier,(e) contacting the barrier with the LNP of any one of claims 1-7 or 20 or with the composition of any one of claims 8-10, to insert the nanopore comprised in the LNP into the barrier, such that the nanopore has an entrance side on the cis side of the barrier and an exit side on the trans side of the barrier permitting ion exchange between the first electrolyte solution and the second electrolyte solution, and(1) positioning a working electrode to detect changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule.

25. The method of claim 24, wherein the barrier is a lipid bilayer membrane.

26. A chip for performing nanopore-based sequencing produced by the method of claim 24 or 25.

27. A system for performing nanopore-based sequencing, comprising the chip of any one of claims 21-23 or 25 and a computing system adapted to record changes in one or more electrical characteristics of the nanopore, wherein the working electrode in the chip is in electronic communication with the computing system, and wherein the electrode is positioned to transmit any detected change(s) in an electrical characteristic of the nanopore associated with occupation of the nanopore by a molecule to the computing system.

28. A method for sequencing a target molecule, comprising:(a) providing a chip of any one of claims 21-23 or 25;(b) contacting the chip with a target molecule;(c) applying a voltage across the barrier of the chip;(d) determining, by the working electrode, one or more changes in an electrical characteristic of the nanopore associated with occupation of the nanopore by the target molecule; and(e) determining, based on the one or more changes in the electrical characteristic of the nanopore, a sequence for the target molecule.

29. The method of claim 28, wherein the target molecule is a nucleic acid, modified nucleic acid, or other organic polymer.

30. A kit for producing a chip for performing nanopore-based sequencing comprising the LNP of any one of claims 1-7 or 20 or the composition of any one of claims 8-10.

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