Modified triblock copolymer compounds and their uses

Abstract

This application discloses triblock copolymer (TBC) molecules having modified chemical head groups. The head groups include azides, triazoles, and other chemical moieties, which make the TBC molecules useful as components of polymerosomes, vesicles, and membrane compositions (e.g., synthetic membranes used in nanopore sequencing devices). This application also discloses methods for preparing and using modified TBC molecules.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 464,053, filed May 4, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [Technical Field] This application relates to triblock copolymer compounds having modified chemical head - group moieties that exhibit properties useful as components in vesicles, such as membranes used in nanopore sequencing devices, polymersomes, and membrane compositions.

Background Art

[0003] Nanopore - based nucleic acid sequencing is an attractive approach that has been widely studied. In one approach, a single - stranded polynucleotide sequence is detected by changes in ionic current as the polynucleotide moves through a nanopore embedded in a lipid bilayer membrane that separates the two sides of an electrochemical cell. During the movement of the polynucleotide, partial occlusion of the nanopore aperture changes the ionic current over time, resulting in a change in the current that can be measured by the cell.

[0004] Sequencing by Expansion ("SBX") is a nanopore-based nucleic acid sequencing method that uses a biochemical process to transfer the DNA sequence to a measurable polymer molecule called "Xpandomer." See, for example, U.S. Patent No. 7,939,259 entitled "High Throughput Nucleic Acid Sequencing by Expansion" and PCT Public International Publication No. 2020236526 entitled "Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing." In the SBX process, the target nucleic acid sequence is encoded along the mainchain Xpandomer sequence using a reporter construct located approximately 10 nm apart, designed to provide well-distinguished response signals with a high signal-to-noise ratio during nanopore transfer. The enhanced signal-to-noise ratio provided by the distinct response signals significantly improves the sequence read efficiency and accuracy of the Xpandomer compared to the native nucleic acid molecule.

[0005] Nanopore-based sequencing-by-synthesis ("SBS") uses a polymerase (or other chain elongation enzyme) covalently bound to a nanopore to synthesize a DNA strand (i.e., a copy strand) complementary to a target sequence template. Nanopores embedded in the membrane of an electrochemical cell are used to simultaneously detect the identity of each nucleotide monomer as each monomer is added to its growth strand. See, for example, U.S. Patent Publications 2013 / 0244340(A1), 2013 / 0264207(A1), 2014 / 0134616(A1), 2015 / 0368710(A1), and 2018 / 0057870(A1), and International Publication 2019 / 166457. Each attached nucleotide monomer is detected by monitoring the signal resulting from the change in ion flow through the nanopore as the tag portion attached to each attached nucleotide monomer enters the nanopore and alters the ion flow. For optimal performance, the tag portion must be present in the nanopore for a sufficient time to provide a detectable, identifiable, and reproducible signal related to the change in ion flow through the nanopore (relative to the baseline "open current" flow), allowing the specific nucleotide associated with the tag to be clearly distinguished from other tagged nucleotides in the SBS solution.

[0006] However, nanopore-based sequencing faces the burden of having to distinguish small current signal differences buried in large background noise within a minute volume electrochemical cell. This measurement challenge is complicated by small changes in materials and parameters that affect the electrochemical cell, including but not limited to the performance of lipid bilayers with embedded nanopores.

[0007] There is a need for triblock copolymer polymer compounds with improved properties for use in polymersomes and membrane materials, such as membranes in nanopore-based nucleic acid sequencing devices. [Overview of the project]

[0008] This disclosure relates to triblock copolymer (TBC) compounds having modified chemical head group moieties, and methods of making and using these compounds, and methods of using them in membranes and polymerosome compositions. This summary is intended to introduce the subject matter of the disclosure but does not purport to cover all embodiments, combinations, or variations contemplated and described within the disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0009] In at least one embodiment, the disclosure also provides a compound of the formula: R 2 , , ,

[0012] ,

[0011] , , 1 , 2 , -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 wherein R 1 is a terminal head group containing an azide or a triazole; PMOXA is a poly(2-methyl-2-oxazoline) subunit; PDMS is a poly(dimethylsiloxane) subunit; R 2 is a terminal head group containing -OH, an azide, or a triazole; and the average values of n and m are such that m is from 4 to 20 and n is from 20 to 60.

[0010] In at least one embodiment of the novel TBC composition, R 1 is a terminal head group containing a triazole, R 2 is -OH, or R 1 and R 2 are terminal head groups containing a triazole.

[0011] In at least one embodiment, the terminal head group containing a triazole further comprises a group selected from alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

[0012] In at least one embodiment, the triazole-containing terminal head group may further include a fluorescent moiety such as a coumarin moiety or a coumarin derivative moiety.

[0013] In at least one embodiment, the terminal head group comprising triazole is selected from the following:

[0014] TIFF2026515985000001.tif213157 TIFF2026515985000002.tif241157 TIFF2026515985000003.tif44157

[0015] In at least one embodiment of the novel TBC composition, R 1 is Azid, R 2 Is -OH or R 1 and R 2 It is Azid. [Brief explanation of the drawing]

[0016] A better understanding of the novel features and advantages of this disclosure can be obtained by referring to the following detailed description illustrating exemplary embodiments in which the principles of this disclosure are utilized, and to the accompanying drawings (hereinafter also referred to as "Figures" and "FIG.").

[0017] [Figure 1] An embodiment of cell 100 in a nanopore-based sequencing chip is shown. [Figure 2] This document describes one embodiment of a cell 200 that performs nucleotide sequencing using Nano-SBS technology. [Figure 3] This shows one embodiment of a cell that is attempting to perform nucleotide sequencing using pre-loaded tags. [Figure 4] This document illustrates one embodiment of process 400 for nucleic acid sequencing using pre-loaded tags. [Figure 5] This shows one embodiment of a circuit 500 within a cell of a nanopore-based sequencing chip. [Figure 6] One embodiment of the circuit 600 within a cell of a nanopore-based sequencing chip is shown, in which the voltage applied to the nanopore can be configured to vary over a period of time during which the nanopore is in a specific detectable state. [Figure 7] This diagram outlines the generalized characteristics of XNTP and its functionality in Nano-SBX technology. [Figure 8] This diagram outlines the generalized characteristics of XNTP and its functionality in Nano-SBX technology. [Figure 9] This diagram outlines the generalized characteristics of XNTP and its functionality in Nano-SBX technology. [Figure 10] This diagram outlines the generalized characteristics of XNTP and its functionality in Nano-SBX technology. [Figure 11] This is a schematic diagram showing further details of one embodiment of XNTP. [Figure 12] This is a schematic diagram showing one embodiment of Xpandomer passing through biological nanopores. [Figure 13A] Heatmap images and plots are shown illustrating the effect of different amounts of TBC in a DPhPE membrane on the population of sequenceable cells in a nanopore. Figure 13A shows results using a DPhPE-only membrane. A bimodal distribution on the vertical axis in the left-hand inset of each image indicates the presence of a population of cells that cannot be sequenced. A single distribution indicates a single population of cells that can be sequenced. [Figure 13B] Heatmap images and plots are shown illustrating the effect of different amounts of TBC in a DPhPE membrane on the population of sequenceable cells in a nanopore. Figure 13B shows the results using a hybrid membrane with the following percentages of TBC Ch111 relative to DPhPE: 20% Ch111 (Figure 13B). A bimodal distribution on the vertical axis in the left-hand inset of each image indicates the presence of a population of cells that cannot be sequenced. A single distribution indicates a single population of cells that can be sequenced. [Figure 13C] Heatmap images and plots are shown illustrating the effect of different amounts of TBC in a DPhPE membrane on the population of sequenceable cells in a nanopore. Figure 13C shows the results using a hybrid membrane with the following percentages of TBC Ch111 relative to DPhPE: 30% Ch111 (Figure 13C). A bimodal distribution on the vertical axis in the left-hand inset of each image indicates the presence of a population of cells that cannot be sequenced. A single distribution indicates a single population of cells that can be sequenced. [Figure 13D] Heatmap images and plots are shown illustrating the effect of different amounts of TBC in a DPhPE membrane on the population of sequenceable cells in a nanopore. Figure 13D shows the results using a hybrid membrane with the following percentages of TBC Ch111 relative to DPhPE: 40% Ch111 (Figure 13D). A bimodal distribution on the vertical axis in the left-hand inset of each image indicates the presence of a population of cells that cannot be sequenced. A single distribution indicates a single population of cells that can be sequenced. [Figure 14A] The exemplary 1H-NMR spectrum of the "living" polymer TBC compound α,ω-(hydroxy-,2-methyl-2-oxazolinium triflate)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) in CD2Cl2 after evaporation of the reaction solvent is shown. This compound has the TBC formula PMOXA8-PDMS39-PMOXA8, with 68 wt% PDMS, Mn=4.2 kDa, f(2-methyl-2-oxazolinium triflate)-=70%, fhydroxy-=30%, and is normalized to 4H (m,0.55-0.45 ppm, -CH2- is adjacent to -Si(-CH3)2-O-). [Figure 14B]The exemplary 1H-NMR spectra in CDCl3 of the "Ch111 type" TBC compound in Scheme 2 after dialysis and fractionation are shown. This compound has the TBC formula PMOXA12-PDMS50-PMOXA12, with 64 wt% PDMS, MnCH111=5.7kDa, facetyloxy-=7%, fhydroxy-=93%, and is normalized to 4H (m, 0.55-0.45 ppm, -CH2- adjacent to Si(-CH3)2-O-). [Figure 14C] The following shows an exemplary 1H-NMR spectrum in CD2Cl2 of the purified α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) described in Scheme 3. This compound has the TBC formula PMOXA11-PDMS56-PMOXA11, is 69 wt% PDMS, Mn=6kDa, f-azido- about 90%, and f-HO- about 10%, normalized to 4H (m, 0.58-0.46 ppm, -CH2- adjacent to Si(-CH3)2-). [Figure 14D] The FT-IR spectra of α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline)TBC in Scheme 3 after solvent evaporation (gray), ultrafiltration (blue), and cosolvent fractionation (red) are superimposed. [Figure 14E] An example of the 1H-NMR spectrum in CD2Cl2 of the "Ch127 / 128 type" TBC polymer α,ω-(hydroxy-,5-(1-polymer-1H-1,2,3-triazole-4-yl)isophthalic acid)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) in Scheme 4 after dialysis and fractionation is shown. The compound has the TBC formula PMOXA9-PDMS30-PMOXA9, with 58 wt% PDMS, MnCH127=3.8kDa, fH5-(1-polymer-1H-1,2,3-triazole-4-yl)isophthalic acid=76%, fhydroxy-=24%, and is normalized to 4H (m,0.59-0.46 ppm, -CH2 is adjacent to -Si(-CH3)2-O-). [Modes for carrying out the invention]

[0018] This disclosure provides hybrid lipid bilayer compositions comprising phospholipids, triblock copolymers, and molecules having pores connecting both sides of the bilayer, and the use of these lipid bilayer compositions in electrochemical cells. Electrochemical cells are useful for performing nanopore-based methods for assaying nucleic acids, including nanopore-based sequencing. The features of the hybrid lipid bilayer compositions and the electrochemical cells containing them result in a remarkable advantage: a reduction in effects that induce adverse errors during the use of cells in nanopore-based assays. Adverse effects, sometimes called protopore formation, can cause undesirable orientations in sequencing, rapidly clogging ports and / or nanopores, thereby significantly reducing the accuracy and efficiency of nanopore-based assays. This disclosure also provides methods for using electrochemical cells and compositions in nanopore-based nucleic acid detection techniques such as nanopore-based sequencing-by-synthesis (Nano-SBS) and nanopore-based sequencing-by-expansion.

[0019] In this specification and the accompanying claims, the singular forms "a" and "an" include multiple references unless the context explicitly indicates otherwise. Thus, for example, a reference to "protein" includes multiple proteins, and a reference to "compound" refers to multiple compounds. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" is interchangeable and not intended to be limiting. Where the description of various embodiments uses the term "comprising," it should be further understood that in some particular examples the embodiment could be described alternatively using the terms "essentially consisting of" or "consisting of."

[0020] Where a range of values ​​is provided, unless the context clearly indicates otherwise, each intervening integer between the upper and lower limits of that range, one-tenth of each intervening integer of that range, and any other listed or intervening values ​​within the stated range are understood to be included in the invention, unless the context clearly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are also included in the invention, subject to any specifically excluded limitations within the stated range. Where a stated range includes one or both of the limit values, the range excluding (i) one or (ii) both of the included limit values ​​is also included in the invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0021] In general, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly used by those skilled in the art, such as Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition), Vols. 1-3, Cold Spring Harbor, NY, 2012 (hereinafter "Sambrook"); and Current Protocols in Molecular Biology, edited by FMAusubel et al. (first published in book form in 1987 by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., regularly supplemented until 2011, and now available in online journal format as Current Protocols in Molecular Biology, Vols. 00-130, (1987-2020) published by Wiley & Sons, Inc. in the Wiley Online Library) (hereinafter "Ausubel").

[0022] All publications, patents, patent applications, and other documents referenced in this disclosure are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure belongs. It should be understood that the terms used herein are intended to describe only specific embodiments and are not intended to limit them. For the purposes of interpreting this disclosure, the following definitions of terms apply, and where appropriate, singular terms are also included in their plural forms and vice versa.

[0024] Triblock copolymer having a modified head base portion This disclosure describes a novel class of (PMOXA) with modified terminal head groups. m -(PDMS) n -(PMOXA) m We provide triblock copolymer compounds of this class (PMOXA) m -(PDMS) n -(PMOXA) m Triblock copolymer compounds have one or two terminal head groups containing an azide group or a triazole group. As shown in the examples and elsewhere in this specification, these TBC compounds with novel terminal head group modifications can be used in membrane compositions, including vesicle systems, e.g., polymerosome compositions, and hybrid or fully synthetic membrane compositions. As shown in the examples, when incorporated into the membrane of a device, these membrane compositions can provide improved properties to nanopore-based devices used for sequencing.

[0025] A remarkable advantage of the TBC compounds of this disclosure is that the chemical structure of the head group can be easily modified with various groups, and further optimized to improve the functional properties of the TBC when used in polymerosomes and membrane compositions. As described elsewhere in this specification, including the following examples, the formula R of this disclosure 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 Various head groups have been prepared for the TBC compound.

[0026] Generally, TBC compounds with modified head groups are prepared using standard CuAAC click chemistry. As a result, terminal head group R 2 and / or R 1 The triazole moiety is included, and CuAAC synthesis enables a wide range of TBC compounds having various different head groups. For example, in at least one embodiment, the terminal head group R 2 and / or R 1 This may include fluorescent moieties such as coumarin moieties or coumarin derivative moieties. A wide range of fluorescent moieties are known in the art and can be incorporated into the TBC of this disclosure as HG using standard CuAAC click chemistry, as described in the examples and elsewhere in this specification.

[0027] In at least one embodiment, terminal head base R 2 and / or R 1 It may contain a group selected from hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

[0028] Table 1 below shows specific exemplary terminal head structures useful for TBCs in this disclosure.

[0029] [Table 1]

[0030] In at least one embodiment, the TBC compound may contain different terminal head groups R1 and R2, with only one of the head groups (e.g., R1) containing a triazole group. Thus, the other terminal head group may contain other more standard head group moieties. Exemplary head group moieties include, but are not limited to, hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine. Specific head group moiety structures are shown in the examples.

[0031] The preparation and use of TBCs containing the specific exemplary head groups shown in Table 1 are described in the Examples. Furthermore, it is further intended that additional head groups based on chemical derivatives of these exemplary head group structures may be prepared and tested for use in various TBC applications, such as hybrid membranes for nanopore sequencing. Such chemical derivatives can be prepared from the alkyne form of the desired compound using standard CuAAC click chemistry, as described in the Examples. The chemical derivatives may comprise any of the above specific structures further modified on an alkyl chain or aryl ring with a desired chemical group such as a halogen, hydroxyl, amine, or charged group (e.g., sulfate, carboxylate).

[0032] As described above, the TBC compounds having the modified head groups of this disclosure are synthesized using standard CuAAC click chemistry as described in the examples. In short, R1 and / or R2 have terminal azide groups, or one or both of them. 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2A TBC precursor is prepared and then contacted with an alkyne derivative of the desired modified head group under appropriate CuAAC click chemistry reaction conditions. Examples of alkyne derivatives useful for preparing modified head groups include, but are not limited to, propargyl alcohol, hexynyl-PE, 3-(dimethyl(propa-2-in-1-yl) ammonia), propane-1-sulfonate, 5-hexic acid, sodium 2-propyne-1-sulfonate, octa-7-in-1-amine, 5-ethynyl-1,3-benzenedicarboxylic acid, penta-4-in-1-yl dihydrogen phosphate, 2-(hepta-6-in-1-yl)propanedioic acid, (2R)-2-(penta-4-inamide)butanedioic acid, (S)-2-aminohepta-6-inoic acid, and 7-(diethylamino)-2-oxo-N-(propa-2-in-1-yl)-2H-chromen-3-carboxamide.

[0033] Use of Triblock Copolymer Membranes, as materials and components, are essential to the functionality of a wide variety of processes and devices. In at least one embodiment, it is conceivable that the TBC compounds having modified head groups of the present disclosure can be used in hybrid membrane compositions. These compositions comprise a lipid bilayer containing a mixture of phospholipids and TBC compounds. A remarkable property of the hybrid lipid bilayer compositions prepared using these TBC compounds is the improved accuracy and efficiency when used in certain nanopore-based electrochemical devices for nucleic acid detection and / or sequencing. As described in the examples and elsewhere in this specification, when incorporated into nanopore-based devices for nucleic acid sequencing, hybrid lipid bilayer compositions containing the TBC compounds having modified head groups of the present disclosure can increase the number of molecules sequenced per experiment by 1.1 to 5 times compared to the same device using a standard non-hybrid lipid bilayer without TBCs.

[0034] The hybrid membrane compositions of this disclosure exhibiting these improved characteristics are based on a lipid bilayer comprising a mixture of phospholipids and a triblock copolymer (or TBC). Typically, the amount of triblock copolymer is about 2% to about 40% of the mass of solids in the mixture with the phospholipids forming the bilayer. Methods for preparing hybrid membranes containing TBCs are known in the art and are described in the examples. Generally, the desired composition of phospholipids and TBCs is prepared as a mixture in a solution of silicone oil and hexadecane.

[0035] The triblock copolymers used in the mixture can have molecular weights ranging from approximately 3500 Daltons to approximately 6500 Daltons. Triblock copolymers typically also exhibit polydispersity of approximately 1.1 to 1.8. However, a broader range of polydispersity is considered potentially useful in various hybrid membrane applications.

[0036] As described in the examples and elsewhere in this specification, the extensive exemplary hybrid films of this disclosure are poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) triblock copolymer (PMOXA) m -(PDMS) n -(PMOXA) m Based on a lipid bilayer composition using a triblock copolymer (also known as a triblock copolymer). Structural formula (PMOXA) m -(PDMS) n -(PMOXA) m While triblock copolymers are known in the art, compositions incorporating such TBCs into nanopore-embedded lipid bilayers, and the use of these hybrid membrane structures in nanopore-based sequencing methods, are not known.

[0037] Examples of TBCs useful in the lipid bilayer composition of this disclosure include TBCs having the following formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m-R 2 (In the formula, R 1 R is a terminal head group containing a triazole moiety. 2 (where is a triazole moiety or a terminal head group containing a hydroxyl group). In this formula, "(PMOXA)" is a poly(2-methyl-2-oxazoline) subunit of the polymer structure. Each of two (PMOXA) polymers, consisting of m subunits, is adjacent and covalently bonded to a central poly(dimethylsiloxane) (or "PDMS") polymer, consisting of n subunits. 1 and optionally R 2 Each terminal head group (or "HG") is a chemical group covalently bonded to the terminal (PMOXA) subunit. In at least one embodiment, TBC is symmetric, and R 1 and R 2 Both are the same terminal HG. In at least one embodiment, the TBC is asymmetric, and R 1 and R 2 These are different terminal HGs. In at least one embodiment, the TBC is asymmetric, and R 2 The group is a terminal hydroxyl group (or "-OH"), R 1 The base is terminal HG.

[0038] formula R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 The number of PMOXA and PDMS subunits in the polymer structure is defined by integer values ​​"m" and "n". In the exemplary TBCs of this disclosure, m is between 4 and 20, and n is between 20 and 60. As shown in the table illustrating the exemplary TBCs in the examples, the structure of a TBC can be represented by two m values ​​and one n value. For example, "12-50-12" is a TBC of the formula R 1 -(PMOXA) 12 -(PDMS) 50 -(PMOXA) 12 -R 2The TBC is shown. The m and n values ​​shown in the formulas of the exemplary TBCs of this disclosure are understood to be the average of the ranges of m and n values ​​that occur in the population of polymer molecules that occur during the synthesis of the TBCs of this disclosure. That is, the TBC shown as "12-50-12" is actually a distribution of TBC molecules where the average values ​​of m and n are 12 and 50, respectively. As disclosed elsewhere in this specification, including the following examples, the specific m and n values ​​that determine the ratio of PMOX and PDMS polymer units in the TBC can be varied to provide a TBC with optimized functional properties when used in hybrid membranes.

[0039] Nanopore-based devices for detecting nucleic acids Nanopore-based devices for detecting nucleic acids have been developed for rapid sequencing, and various designs and uses are known in the art. See, for example, U.S. Patents 9,494,554 (B2), 9,567,630 (B2), 9,557,294 (B2), and 9,605,309 (B2), respectively, which are incorporated herein by reference. These devices generally include an electrochemical cell having a chamber containing nanopores embedded in a membrane. The membrane serves to separate the cell chamber into two subchambers, which are called the cis and trans sides of the cell, and each contains an electrode.

[0040] Generally, the membranes of such devices may be organic membranes such as lipid bilayers, or synthetic membranes made from polymer materials that do not exist naturally. This disclosure envisions nanopore-based devices comprising hybrid membranes comprising a bilayer mixture of phospholipids and triblock copolymers. As shown in the examples herein, such hybrid membranes having TBCs can embed nanopores and, when used in nanopore-based devices, can provide improvements in nucleic acid detection and sequencing. Therefore, the following general disclosures of nanopore-based devices, their manufacture and use should be considered to include the hybrid membranes of this disclosure.

[0041] The pores of the device are provided by nanopores embedded in the membrane, which function as channels (or passages) between the cis and trans sides of the cell. Depending on the nanopores used, the pores may have a width or diameter ranging from about 1 angstrom to about 10,000 angstroms. The nanopores may be naturally occurring pore-forming proteins such as α-hemolysin from Staphylococcus aureus (S. aureus), or non-naturally occurring variants or variants of wild-type pore-forming proteins. A variety of naturally occurring and non-naturally occurring nanopores with various pore sizes and properties are known in the art. See, for example, U.S. Patent Nos. 10351908(B2), 10934582(B2), and 10227645(B2).

[0042] Within an electrochemical cell, nanopores embedded in a membrane are positioned in close proximity to electrodes coupled to a sensing circuit, such as a complementary metal-oxide-semiconductor (CMOS) or field-effect transistor (FET) circuit. When a potential is applied across the nanopores (through the electrodes) while they are immersed in a conductive fluid, a small current can be observed due to the ion flow through the nanopores. This ion flow is sensitive to the pore size, and therefore, molecules entering the pores affect the ion flow and voltage measured through this sensor circuit.

[0043] Electrochemical cells for nanopore-based sequencing of nucleic acids are typically used in a large-scale parallel configuration, where thousands of such cells are configured as an array within a single device, often referred to as a chip (or biochip). Typically, a nanopore-based sequencing chip device may incorporate an array of more than one million electrochemical cells, containing 1,000 rows x 1,000 columns of such cells (see, for example, a chip manufactured by Roche Sequencing Solutions in Santa Clara, California, USA). Methods for manufacturing and using such nanopore array microchips can also be found in U.S. Patent Applications Publications 2013 / 0244340(A1), 2013 / 0264207(A1), 2014 / 0134616(A1), 2015 / 0368710(A1), and 2018 / 0057870(A1), as well as International Publication 2019 / 166457(A1), which are incorporated herein by reference, respectively. Each well in the array is manufactured using a standard CMOS process with surface modifications that allow for sustained contact between the bioreagent and the conductive salt. Each well can support a phospholipid bilayer membrane embedded with a nanopore polymerase conjugate. The electrodes in each well are individually addressable by a computer interface. All reagents used are introduced into a simple flow cell above the array microchip using a computer-controlled syringe pump. The chip supports analog-to-digital conversion and reports electrical measurements independently from all electrodes at speeds exceeding 1000 points / second. Nanopore measurements are performed asynchronously at least once every 1 millisecond (msec) on each of the 8M addressable nanopore-containing films in the array and can be recorded to an interfaced computer. Further description of exemplary electrochemical cells useful for nanopore-based nucleic acid assays such as sequencing is provided below, including chambers and electrode materials, buffers, sensing circuits, array devices, and their use in various applications.

[0044] Figure 1 shows an exemplary embodiment of an electrochemical cell 100 found in an array of such cells useful for nanopore-based sequencing. A membrane 102 is formed on the surface of the cell. In some embodiments, the membrane 102 is a lipid bilayer. A bulk electrolyte solution 114 containing nanopores or "protein nanopore transmembrane complexes" (PNTMCs) 104 is placed directly on this surface within the cell, and a single PNTMC 104 is inserted into the membrane 102 using electroporation. The individual nanopore-embedded membranes within each cell of the array are not chemically or electrically connected to one another. Thus, each cell is an independent sequencing machine, generating data specific to a single polymer molecule associated with the PNTMC.

[0045] Continuing to refer to Figure 1, the thin film of electrolyte solution 108 is isolated from the bulk electrolyte solution 114 by the ion-impermeable membrane 102. Thus, the nanopore-embedded membrane separates the electrochemical cell 100 into two chambers: a cis chamber containing the bulk electrolyte solution 114 and a trans chamber containing the thin film of electrolyte solution 108. The thin film of electrolyte solution 108 in the trans chamber is in contact with the metallic working electrode 110 connected to the analog measurement circuit 112. The bulk electrolyte solution in the cis chamber is in contact with the counter electrode 116 and the reference electrode 117.

[0046] The pores in PNTMC 104 provide channels through membrane 102 that allow analytes (e.g., nucleic acids) and ions to flow, resulting in the regulation of ionic current across the impermeable bilayer. Thus, the pores in PNTMC 104 provide the sole pathway for ionic current to flow from the metallic working electrode 110 to the bulk electrolyte solution 114 in contact with the counter electrode 116. The electrochemical cell also includes a reference electrode 117 to further enhance its sensitivity as an electrochemical potential sensor.

[0047] A thin film of electrolyte constitutes a femtoliter volume of solution in a transformer chamber that is in direct contact with the working electrode. This small amount of electrolyte in contact with the electrode of the electrochemical cell must receive repeated voltage pulses to sense the ion flow through the nanopore. The need to maintain the ion flow through the nanopore using these repeated electrochemical measurements with electrodes in a small amount of electrolyte, and repeated measurements corresponding to individual molecular parts moving through the pore, requires a highly sensitive sensor system. This electrochemical measurement system is highly sensitive to any precipitate or aggregated molecular entities that could clog the pore or otherwise interfere with the ion flow between the working and counter electrodes. Therefore, the electrolyte salt, electrochemical cell material, and electrochemical measurement conditions should be carefully controlled to prevent or reduce harmful precipitate formation or electrode wear in the system.

[0048] The electrolyte solution 108 may contain one of the following: lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, ammonium chloride, ammonium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2). In some embodiments, the film of the electrolyte solution has a thickness of about 3 microns (μm). The thickness of the film of the electrolyte solution may be in the range of 0 to 5 microns.

[0049] In addition to the salts described above, the electrolyte membrane may contain redox-active ions such as ferrocyanide ions and ferricyanide ions. These redox ions are used in certain nanopore-based sequencing methods performed in electrochemical cells. For example, Nano-SBX measurements performed in the cells of this disclosure may contain ferricyanide and ferrocyanide ions. Such redox-active ions may undergo irreversible reactions depending on their concentration and the specific electrochemical conditions at the working and counter electrodes. Indeed, as described elsewhere in this specification, the irreversible formation of Prussian blue, a precipitate in electrochemical cells, is highly detrimental to any nanopore-based measurements in the cell. Therefore, the concentrations of redox-active ions such as ferricyanide and ferrocyanide should be carefully controlled. In at least one embodiment of this disclosure, the total concentration of ferricyanide and ferrocyanide ions in the solution within the electrochemical cell should be maintained at approximately 25 mM to 250 mM.

[0050] It has also been found that the salt used in the electrolyte solution of an electrochemical cell can affect the ability of the cell to be used for highly sensitive nanopore-based nucleic acid measurements. For example, many nanopore-based sequencing protocols use ammonium chloride as a buffer salt in the electrolyte solution provided in the cell chamber. It has been found that controlling the concentration of ammonium chloride by substituting it with ammonium acetate as the salt in the electrolyte solution during nanopore-based nucleic acid sequencing methods such as Nano-SBX can help reduce or prevent the formation of redox precipitates such as Prussian blue, which are highly detrimental to the accuracy and sensitivity of the electrochemical cell as described above. Therefore, in some embodiments of the electrochemical cell of this disclosure, the buffer composition used in the cell contains ammonium acetate at a concentration of about 0 mM to about 1500 mM and / or ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition contains ammonium acetate at a concentration of about 0 mM to 1500 mM and ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM.

[0051] Dielectric materials are typically used to form an oxide layer 106 that defines the transformer chamber of the cell. Useful dielectric materials include glass, oxides, and silicon mononitride (SiN). In some embodiments, the upper surface of the dielectric oxide layer 106 in contact with the bulk electrolyte 114 may be silanized. Silanization forms a hydrophobic layer on top of the dielectric layer. In some embodiments, this hydrophobic layer has a thickness of about 1.5 nanometers (nm). Alternatively, a hydrophobic dielectric material such as hafnium oxide can be used to form the upper part of the dielectric layer.

[0052] As shown in Figure 1, the membrane is a lipid bilayer at least partially formed on the dielectric oxide layer 106 and spans a well containing a thin film of electrolyte 108. For example, the membrane can be formed on a hydrophobic layer on the dielectric oxide layer, and when the membrane reaches the opening of the well, the lipid monolayer migrates into a lipid bilayer that extends across the opening of the well. The hydrophobic layer can facilitate the formation of a lipid monolayer on the dielectric layer and the migration from the lipid monolayer to a lipid bilayer.

[0053] The bulk electrolyte 114 may further comprise one of lithium chloride (LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithium acetate, sodium acetate, potassium acetate, calcium chloride (CaCl2), strontium chloride (SrCl2), manganese chloride (MnCl2), and magnesium chloride (MgCl2). Therefore, in some embodiments of the electrochemical cells of this disclosure, the buffer composition of the bulk electrolyte used in the cell comprises ammonium acetate at a concentration of about 0 mM to about 1500 mM and / or ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the buffer composition comprises ammonium acetate at a concentration of about 0 mM to 1500 mM and ammonium chloride at a concentration of less than 2000 mM. In at least one embodiment, the concentration of ammonium acetate is 0 mM and the concentration of ammonium chloride is about 600 mM. Similarly, in some embodiments, the total concentration of ferricyanide and ferrocyanide ions in the bulk electrolyte solution of the electrochemical cell should be maintained at approximately 25 mM to 250 mM.

[0054] As mentioned above regarding electrolyte thin films, the composition of the salts used in the bulk electrolyte (e.g., ammonium chloride versus ammonium acetate) and the redox-active ions can affect the accuracy and sensitivity of nanopore-based measurements performed using electrochemical cells. Depending on the type of electrochemical measurement being performed, particularly the potential applied to the electrode in contact with the solution, it is possible to adjust the composition of the solution to reduce or prevent the formation of harmful precipitates.

[0055] To perform the necessary insertion and removal of solutions, the electrochemical cells of this disclosure include inlet and outlet ports operably connected to the cell's chamber or reservoir. Although the schematic diagram of the electrochemical cell in Figure 1 does not show the inlet and outlet ports, the incorporation of such ports into electrochemical cells is known in the art. The inlet and outlet ports allow for the insertion of reagents necessary for nanopore-based nucleic acid detection, including materials for fabricating membranes with embedded nanopores, nucleotide synthesis reagents, and electrolytes and buffers. Typically, the inlet and outlet ports include metal (e.g., stainless steel) tubing attached to the cell to provide a fluid connection from the outside to the cell's chamber, which can be used to insert or remove solutions. Thus, at least a portion of the inlet and outlet port tubing is in contact with the electrochemical solution in the cell chamber. Optimally, the composition of the ports should be electrochemically inert at a voltage used to induce ion flow through the nanopores and to perform perceptual measurements of the change in ion flow as molecular portions enter and / or move through the nanopores.

[0056] The electrochemical cell 100 includes a counter electrode (CE) 116 and a reference electrode 117 that functions as an electrochemical potential sensor. In some embodiments, the counter electrode 116 is shared among multiple cells and is therefore also called a common electrode. The common electrode can be configured to apply a common potential to the bulk liquid in contact with nanopores in the multiple cells. The common potential and common electrode are common to all measurement cells.

[0057] In at least one embodiment, a non-Faraday electrochemical cell having a titanium nitride (TiN) working electrode has been found to be advantageous for nanopore-based sequencing of nucleic acids. This cell has a general structure similar to the cell in Figure 1, but includes a TiN working electrode that exhibits increased electrochemical capacity. The electrochemical cell further includes a conductive or metallic layer connecting the working electrode of the cell to the circuit of the array in which the cell is part.

[0058] Therefore, in at least one embodiment, the metallic working electrode 110 is a titanium nitride (TiN) metal electrode with increased electrochemical capacitance. The electrochemical capacitance associated with the metallic working electrode 110 can be increased by maximizing the specific surface area of ​​the electrode. The specific surface area of ​​the metallic working electrode 110 is per unit mass (e.g., m²). 2 / kg) or per unit volume (e.g., m 2 / m 3 or m -1 ) or per unit base area (for example, m 2 / m 2 This is the total surface area of ​​the electrodes. A larger surface area increases the electrochemical capacitance of the metal working electrode, allowing more ions to move at the same applied potential before the capacitor is charged. The surface area of ​​the working electrode 110 can be increased by making the TiN electrode "sponge-like" or porous. A TiN sponge absorbs the electrolyte and creates a large effective surface area that comes into contact with the electrolyte.

[0059] Capacitance related to film (C membrane ) and capacitance (C) related to the working electrode electrochemical The ratio to ) can be adjusted to achieve optimal overall system performance. Improvement in system performance is C electrochemical Maximizing C membrane This can be achieved by reducing C membrane It is tuned to generate the required RC time constant without requiring additional on-chip capacitance, thereby enabling a significant reduction in cell and chip size. The base surface area of ​​the metallic working electrode 110 is greater than or equal to the surface area of ​​the opening of the transside well containing the electrolyte 108, which has walls defined by the oxide layer 106. Thus, the two base surface areas are C membrane and C electrochemicalIt can be independently optimized to provide a desired ratio between and . By using a spongy, porous TiN working electrode, the electrolyte can diffuse vertically down the uncoated portion of the working electrode through the spaces between the columnar TiN structures, and then horizontally into the coated portion of the working electrode 1102 beneath the dielectric layer 1104. As a result, the effective surface area of ​​the TiN in contact with the electrolyte is maximized, and C electrochemical This is the maximum value.

[0060] Further descriptions of non-Faraday electrochemical cells, TiN working electrodes, and other nanopore-based cell designs useful for the devices, compositions, and methods of this disclosure can be found, for example, in U.S. Patent No. 10174371(B2), which is incorporated herein by reference.

[0061] As described elsewhere in this specification, the electrochemical cells of this disclosure can be used as devices for nanopore-based nucleic acid detection and measurement assays, including nucleic acid sequencing. Generally, nanopore-based sequencing of target nucleic acids can be performed using the electrochemical cell of the above design and associated solution compositions comprising electrolytes and buffers that reduce the precipitate formation described above. The present method provides an electrochemical cell comprising (a) an electrochemical cell having (i) a nanopore embedded in a membrane, the membrane separating the cell into cis and trans chambers operably connected by the nanopore, the cis and trans chambers each comprising an electrode, a solution containing ferrocyanide ions, ferricyanide ions, and a buffer composition, and (ii) an inlet port and an outlet port containing a metal operably connected to the cell; (b) adding a molecule derived from a target nucleic acid to the cis chamber; (c) applying a voltage to the cell that causes at least a portion of the molecule to move through the nanopore; and (d) detecting a change in the voltage flow within the cell as the molecule moves through the nanopore, wherein the change in the voltage flow indicates the sequence of the target nucleic acid.

[0062] As described elsewhere in this specification, the specific potentials applied, their timing, and duration can significantly affect the highly sensitive changes in ion flow conductance through the nanopore in the presence of nucleic acid molecular portions to be detected when performing the sequencing method. Exemplary methods and parameters for applying potentials to an entire nanopore-based electrochemical cell array for sequencing are known in the art and are described, for example, in U.S. Patent No. 11150216 and U.S. Patent No. 11029306, each of which is incorporated herein by reference. The applied potentials, their pulse timing, and duration also affect the redox chemistry within the cell, which can lead to the formation of harmful precipitates such as Prussian blue. A remarkable advantage of the electrochemical cells of this disclosure and the associated solutions used therein is that, in some embodiments, the nanopore sequencing method using the electrochemical cells of this disclosure can be performed using alternating current (AC) applied to the cell for 10 hours in a voltage range of 450 mV to 1200 mV, without the visible formation of Prussian blue within the cell.

[0063] In some embodiments of the sequencing method of this disclosure, the applied voltage includes applying a baseline potential to the cell. The typical baseline voltage applied may be in the range of about 55 mV to about 95 mV.

[0064] In some embodiments of the sequencing method, the applied voltage can include pulsed voltages. Useful pulsed voltages for this method may range from approximately 320 mV to approximately 550 mV. The duration of the pulsed voltage is typically approximately 5 μs to approximately 15 μs. In at least one embodiment, the time between pulsed voltages is approximately 0.2 ms to 5 ms. In at least one embodiment, the applied voltage supplies alternating current to an electrochemical cell. For example, the resulting alternating current may have a periodicity of approximately 0.4 seconds to approximately 6 seconds.

[0065] In at least one embodiment, it is conceivable that a parallel sequencing method can be performed using an array of electrochemical cells of this disclosure. Such parallel nanopore-based sequencing of nucleic acids using a parallel array of electrochemical cells has been used in conjunction with nanopore-based sequencing-by-synthesis (Nano-SBS) technology. System compositions and methods for Nano-SBS are known in the art. See, for example, U.S. Patent Application Publications 2013 / 0244340(A1), 2013 / 0264207(A1), 2014 / 0134616(A1), 2015 / 0368710(A1), and 2018 / 0057870(A1), and International Publication 2019 / 166457(A1). These known systems, compositions, and methods are conceivable to be used or adapted for use with the electrochemical cells of this disclosure. The following provides a description of the use of Nano-SBS in the context of the electrochemical cells and compositions of this disclosure.

[0066] Figure 2 shows an embodiment of an electrochemical cell 200 used to perform nanopore-based nucleic acid sequencing using Nano-SBS technology. In Nano-SBS technology, a template 202 and primers to be sequenced are introduced into the electrochemical cell 200. Four different tagged nucleotides 208 are added to the bulk aqueous phase of this template-primer complex. Once the correctly tagged nucleotides form a complex with polymerase 204, the tag tails are positioned within the barrel of the nanopore 206. The tags held within the barrel of the nanopore 206 generate a unique ion-barrier signal 210, thereby electronically identifying the attached bases based on the tag-specific chemical structure differences.

[0067] Figure 3 shows one embodiment of a cell in which nucleotide sequencing is to be performed using a pre-loaded tag. A nanopore 301 is formed within the membrane 302. An enzyme 303 (e.g., a polymerase such as DNA polymerase) associates with the nanopore. In some cases, the enzyme 303 is covalently bonded to the nanopore 301. The polymerase 303 associates with the nucleic acid molecule 304 to be sequenced. The associated nucleic acid molecule 304 may be linear or cyclic. In some embodiments, a nucleic acid primer 305 hybridizes to a portion of the nucleic acid molecule 304. The polymerase 303 uses the single-stranded nucleic acid molecule 304 as a template to catalyze the incorporation of nucleotide 306 into the primer 305. As described above, nucleotide 306 contains a tag species ("tag") 307 that allows it to be distinguished from the other three nucleotides.

[0068] Figure 4 shows one embodiment of process 400 for nucleic acid sequencing using pre-loaded tags. In step A, the tagged nucleotides (one of four different types: A, T, G, or C) are not associated with the polymerase. In step B, the tagged nucleotides are associated with the polymerase. In step C, the polymerase is close to the nanopore. The tags are drawn into the nanopore by the electric field generated by a voltage applied to the membrane and / or the nanopore. Some of the associated tagged nucleotides do not form base pairs with the nucleic acid molecule. These unpaired nucleotides are typically rejected by the polymerase within a timescale shorter than the timescale in which properly paired nucleotides remain associated with the polymerase. Since the unpaired nucleotides only transiently associate with the polymerase, process 400 shown in Figure 4 typically does not proceed beyond step B.

[0069] Before polymerase docks with the nanopore, the conductance of the nanopore is approximately 300 picosiemens (300 pS). In step C, the conductance of the nanopore is approximately 60 pS, 80 pS, 100 pS, or 120 pS, depending on which of the four types of tagged nucleotides is used. Polymerase incorporates the nucleotide into the growing nucleic acid molecule via isomerization and transphosphorylation reactions, releasing the tag molecule. In particular, once the tag is retained within the nanopore, a unique conductance signal (see signal 210 in Figure 2, e.g.) is generated due to the different chemical structures of the tag, thereby electronically identifying the added base. By repeating the cycle (i.e., steps A-E or A-F), sequencing of the nucleic acid molecule becomes possible. In step D, the released tag passes through the nanopore.

[0070] In some cases, as seen in step F of Figure 4, tagged nucleotides that are not integrated into the growing nucleic acid molecule also pass through the nanopore. While unintegrated nucleotides may be detectable by the nanopore in some cases, this method provides a means for distinguishing between integrated and unintegrated nucleotides based at least partially on the time it takes for the nucleotide to be detected in the nanopore. Tags bound to unintegrated nucleotides pass through the nanopore rapidly and are detected for only a short period (e.g., less than 10 ms), while tags bound to integrated nucleotides are loaded into the nanopore and are detected for a longer period (e.g., at least 10 ms).

[0071] Figure 5 shows one embodiment of circuit 500 within an electrochemical cell of a nanopore-based sequencing chip. As described above, when a molecular part (e.g., a tag) enters the nanopore 502, a unique conductance signal (see signal 210 in Figure 2, e.g.) is generated due to its different chemical structure and how it fits into the nanopore, thereby electronically identifying the added base. The circuit in Figure 5 maintains a constant voltage across the nanopore 502 while the ionic conductance is being measured. In particular, this circuit maintains a constant voltage across the nanopore 502. a or V b Includes an operational amplifier 504 and a pass device 506 that maintain a constant voltage equal to the capacitor n. The ion flow through the nanopore 502 is transmitted to capacitor n. cap The signal is integrated in 508 and measured by the analog-to-digital (ADC) converter 510. However, circuit 500 measures only a unidirectional current flow. Furthermore, temperature drift within the operational amplifier 504 can cause the actual voltage applied across the nanopore 502 to vary across different electrochemical cells in the array. The actual voltage applied to the nanopore 502 may drift by tens of millivolts above or below the desired value, thereby causing significant measurement inaccuracies. Reducing the size of the operational amplifier in a large array may introduce other performance issues.

[0072] Figure 6 shows one embodiment of circuit 600 for use in an electrochemical cell of a nanopore-based sequencing chip, where the voltage applied to the nanopore can be configured to vary over a period of time during which the nanopore is in a particular detectable state. One possible state of the nanopore is the open-channel state when no molecular parts (e.g., tags) are present within the barrel of the nanopore. Other possible states of the nanopore correspond to the states when four different types of molecular parts are present within the barrel of the nanopore. Yet another possible state of the nanopore is when the membrane has ruptured. Figure 6 shows a nanopore 602 inserted into a membrane 612, where the nanopore 602 and membrane 612 are positioned between the cell working electrode 614 and the counter electrode 616 so that a voltage is applied across both ends of the nanopore 602. The nanopore 602 is also in contact with the bulk liquid / electrolyte 618. Note that the nanopore 602 and membrane 612 are drawn upside down compared to the nanopore and membrane in Figure 1. Hereinafter, an electrochemical cell means that it includes at least a membrane, a nanopore, a working electrode, and associated circuitry. In some embodiments, the counter electrode is shared among multiple cells (e.g., in an array) and is therefore also called a common electrode. The common electrode may be configured to apply a common potential to the bulk liquid in contact with the nanopore in the measurement cell. The common potential and common electrode are common to all measurement cells. Within each measurement cell there is a metallic working cell electrode, and in contrast to the common electrode, the metallic working cell electrode 614 may be configured to apply a separate potential independent of the working cell electrodes of other measurement cells.

[0073] In addition to the Nano-SBS sequencing described above, in some embodiments, parallel sequencing of nucleic acids can be performed using nanopore-based sequencing-by-expansion (Nano-SBX) technology with the electrochemical cells of this disclosure. See, for example, U.S. Patent No. 7,939,259. The SBX technology is based on the polymerization of highly modified non-natural nucleotide analogs called "XNTPs". Typically, SBX uses biochemical polymerization to transfer the DNA template sequence onto a measurable polymer called "Xpandomer". The transferred sequence is encoded along the Xpandomer backbone in a high-signal-to-noise reporter about 10 nm away, designed for high-signal-to-noise, high-differentiation response. These differences significantly improve the sequence read efficiency and accuracy of Xpandomer compared to natural DNA. A general description of the SBX process is shown in Figures 7, 8, 9, and 10.

[0074] XNTPs are expandable 5'-triphosphate modified unnatural nucleotide analogs that are compatible with template-dependent enzymatic polymerization. A highly simplified XNTP is shown in Figure 7, which highlights the intrinsic characteristics of these unnatural substrates. XNTP100 has two distinct functional regions: a selectively cleavable phosphoramide bond 110 that links the 5'α-phosphate 115 to the nucleic acid base 105, and a symmetrically synthesized reporter tether (SSRT) 120 bound within the nucleoside triphosphoramide at a position that allows for controlled expansion by cleavage of the phosphoramide bond. The SSRT contains linkers 125A and 125B separated by the selectively cleavable phosphoramide bond. Each linker is bound to one end of the reporter code 130. XNTP100 is shown in a "constrained configuration" characteristic of XNTP substrates and daughter chain products of template-dependent polymerization. The constrained configuration of polymerized XNTPs is a precursor to the expanded configuration, as seen in the Xpandomer product. The transition from a constrained configuration to an extended configuration occurs when the PN bond of the phosphoramidate within the primary backbone of the daughter chain is cleaved.

[0075] The synthesis of the Xpandomer polymer is summarized in Figures 8 and 9. As shown in Figure 8, during assembly, the monomer XNTP substrates 145 (XATP, XCTP, XGTP, and XTTP) are polymerized at the elongable ends of the nascent daughter strands 150 by a template-directed polymerization process using a single-strand template 140 as a guide. Generally, this process is initiated from the primer and proceeds in the 5' to 3' direction. Generally, DNA polymerase or other polymerase is used to form the daughter strands, and conditions are selected to obtain a complementary copy of the template strand. After the daughter strands are synthesized, the coupled SSRTs form constrained Xpandomer, which further forms the daughter strands. The SSRTs in the daughter strands have a "constrained configuration" of the XNTP substrates. The constrained configuration of the SSRTs is a precursor to the extended configuration, as seen in the Xpandomer product.

[0076] As shown in Figure 9, the transition from the constrained configuration 160 to the extended configuration 165 is due to the cleavage of selectively cleavable phosphoramide bonds (shown by unshaded ellipses for simplification) within the primary backbone of the daughter strand. In this embodiment, the SSRT includes one or more reporters or reporter codes 130A, 130C, 130G, or 130T that are specific to the nucleic acid bases to which they are linked, thereby encoding the sequence information of the template. In this way, the SSRT provides a means to extend the length of the Xpandomer and reduce the linear density of the sequence information of the parent strand.

[0077] Figure 10 shows the Xpandomer 165 moving from the cis chamber 175 to the trans chamber 185 through the nanopore 180. Upon passing through the nanopore, each reporter code of the linearized Xpandomer generates a distinct and reproducible electronic signal specific to the nucleic acid base to which it is linked. This signal is shown by the superimposed trace 190.

[0078] Figure 11 shows in more detail a generalized structure of one embodiment of XNTP. XNTP 200 comprises a nucleoside triphosphoramide 210 having linker arm portions 220A and 220B separated by selectively cleavable phosphoramide bonds 230. SSRT 275 is linked to the nucleoside triphosphoramide by linking groups 250A and 250B, with the first SSRT terminus linked to a heterocycle 260 (represented here by cytosine, although the heterocycle may be any one of the four standard nucleic acid bases, A, C, G, or T), and the second SSRT terminus linked to an α-phosphate 270 of the nucleic acid base backbone. Those skilled in the art will understand that the final XNTP substrate product can be formed using many suitable coupling chemistry known in the art, for example, that SSRT conjugation can be achieved by the formation of a triazole linking group.

[0079] In this embodiment, the SSRT275 includes several functional elements, or “features,” such as polymerase-enhancing regions 280A and 280B, reporter codes 285A and 285B, and translation control elements (TCEs) 290A and 290B. In other embodiments, the SSRT may include a single TCE. Each of these features plays a unique function during the migration of the Xpandomer through the nanopore, generating a series of distinctive and reproducible electronic signals. The SSRT275 is designed to control the rate of Xpandomer migration by the TCE via a combination of steric hindrance and / or electrorepulsion, as discussed elsewhere in this specification. Different reporter codes are sized to block ion flow through the nanopore at different measurable levels.

[0080] Specific SSRT polymer sequences can be efficiently synthesized using phosphoramidite chemistry, which is typically used in oligonucleotide synthesis. Reporter codes and other features can be designed by selecting specific phosphoramidite sequences from commercially available and / or proprietary libraries. Such libraries include, but are not limited to, polyethylene glycols having a length of 1 to 12 or more ethylene glycol units, and aliphatic polymers having a length of 1 to 12 or more carbon units. In certain embodiments, the SSRT includes a feature called a "polymerase-enhancing region" at the terminal of the SSRT proximal to the nucleotide triphosphoamide diester. The polymerase-enhancing region may include a positively charged polyamine spacer (e.g., primary, secondary, tertiary, or quaternary amine) or a triamine spacer (three secondary amines separated by three carbons each) that facilitates the uptake of the XNTP structure by nucleic acid polymerase. In certain embodiments, the polymerase-enhancing region comprises two repeating units of spermine, the spermine portion of which is provided by a phosphoramidite monomer having the following structure (as those skilled in the art will recognize, the trifluoroacetamide protecting group is removed at the end of the SSRT synthesis to expose the amine group on spermine). [ka]

[0081] Where used throughout this disclosure, the term “reporter construct” refers to an element of an SSRT comprising reporter codes, a symmetric chemical branched chain, and a translocation control element. In certain embodiments, the reporter construct is a polymer comprising a first reporter code, a symmetric chemical branched chain supporting a translocation control element, and a second reporter code, continuous from a first end to a second end. The term “supports” refers to a covalent bond between the symmetric branched chain and the translocation control element, which results in a favorable orientation of the translocation control element relative to the two reporter codes. The symmetric chemical branched chain may be represented by the letter “Y”, with the two reporter codes linked to the arms of the Y and the translocation control element linked to the stem of the Y. Thus, the two reporter codes are linked inline by the branched chain, which supports the translocation control element in a linear, inline, and perpendicular orientation to the SSRT.

[0082] As used throughout this disclosure, the terms “linker A” and “linker B” refer to a polymerase-enhancing region and one or more migration-decelerating features or regions, respectively, as well as a region of the SSRT that includes a spacer region containing, for example, a polymer of PEG6, which can be customized to adjust the length of the SSRT traversing within the nanopore.

[0083] In certain embodiments, XNTP may be a compound having the following generalized structure. [ka]

[0084] In one embodiment, for example, when sequencing a DNA template using a compound, R may be H. In another embodiment, for example, when sequencing an RNA template using a compound, R may be OH. In certain embodiments, the nucleic acid base is adenine, cytosine, guanine, thymine, uracil, or a nucleic acid base analog. As those skilled in the art will understand, adenine, cytosine, guanine, thymine, and uracil are naturally occurring nucleic acid bases. As used herein, the term “nucleic acid base analog” refers to a non-naturally occurring nucleic acid base that can form a Watson-Crick base pair with a complementary nucleic acid base on an adjacent single-stranded nucleic acid template. Exemplary nucleic acid base analogs include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- This list includes, but is not limited to, mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid(v), wibutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid(v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, 3-nitropyrrole, 8-aza-7-deazaguanine, 8-aza-7-deazynosine, and 8-aza-7-deazaadenine.

[0085] As discussed elsewhere in this specification, the reporter construct is a polymer having a first end and a second end, comprising, continuously from the first end to the second end, a first reporter code, a symmetric chemical branched chain carrying a movement control element, and a second reporter code. This set of features reflects the symmetric structure of the reporter construct (as well as the entire SSRT including the symmetric linkers, linker A and linker B), where the sequences of the two reporter codes are identical and linked in line in opposite directions by the symmetric chemical branched chain. Briefly, synthesis proceeds in the 3' to 5' direction, starting at the 3' end of the TCE. By adding the symmetric branched chain to the 5' end of the TCE, simultaneous polymerization of the first and second reporter codes from each arm of the branched chain, followed by the simultaneous synthesis of linkers A and B, is enabled, terminating at the 5' ends of the first and second ends of the SSRT. The inline redundancy provided by two identical reporter codes separated by a symmetric branching chain supporting a movement control element has been found to offer several advantages during nanopore sequencing. For example, if the Xpandomer moves in either direction, it can potentially be read by the nanopore; that is, the Xpandomer can be read either "forward" or "backward." This flexibility enables other methods of sequencing, such as the "ratchet" method and "flooring" based on the AC pattern of voltage application, which will be discussed further herein.

[0086] Figure 12 shows one embodiment of a cleaved Xpandomer in the process of passing through a nanopore. The nanopore is embedded in a lipid bilayer membrane that separates and electrically isolates two electrolyte chambers. A typical electrolyte contains 1M ammonium chloride buffered to pH 6.0–8.0. When a small voltage of typically 85mV is applied to the bilayer, the nanopore provides the only channel for potential-induced ion flow and is the primary source of resistance in the circuit. The Xpandomer reporter code is designed to give a specific ion flow cutoff level, and since pulses of potential result in an array of reporter code moving through the nanopore, the array information can be read by measuring the array of ion flow levels. In the case of an α-hemolysin nanopore, the nanopore is typically embedded in the membrane so that movement occurs by entering on the cis-antibiotic side and exiting on the trans-basal side. As shown in Figure 12, the nanopore is oriented to initially capture the Xpandomer from the basal side. As the Xpandomer passes through the nanopore, the reporter enters the base until its movement control element stops at the base entrance. The reporter is held at the base until the TCE enters and can pass through the base, after which movement proceeds to the next reporter. In this embodiment, the passage of the TCE into the base is made possible by disengaging the movement control portion from the TCE. [Examples]

[0087] Various features and embodiments of this disclosure are shown in the following representative examples, which are illustrative and not limiting. Those skilled in the art will readily understand that certain embodiments are merely illustrative of the invention, which are more fully described in the subsequent claims. All embodiments and features described herein should be understood to be interchangeable and combinable with all embodiments contained herein.

[0088] Example 1: Synthesis of a triblock copolymer having a modified head group This example illustrates the synthesis of various triblock copolymer compounds having modified head groups for use in lipid bilayers embedded with the nanopores of the present disclosure.

[0089] A. Synthesis of α,ω-(hydroxy-,2-methyl-2-oxazolinium triflate)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline).

[0090] This member of the PMOXA-PDMS-PMOXA family of "living" triblock copolymer (TBC) compounds has the structural formula shown in Scheme 1 and is a precursor for the synthesis of other α,ω-(hydroxyl- / methylester-)- and α,ω-(hydroxyl- / azido-)-terminated TBC compounds described below. [ka]

[0091] The synthesis of the TBC compound in Scheme 1 was carried out as follows, following the method generally described in Lorcher, S.; Meier, W. “Cosolvent fractionation of PMOXA-b-PDMS-b-PMOXA: Bulk separation of triblocks from multiblocks”. European Polymer Journal 2017, 88, 575-585: 6.42 g (2.29 mmol, Mn 1H-NMRAnhydrous α,ω-bis(carbinol)-terminated polydimethylsiloxane (2800 g / mol) and 0.67 mL (4.84 mmol) of anhydrous triethylamine were dissolved in 100 mL of anhydrous hexane and cooled to -25°C. A solution of 0.83 mL (4.91 mmol) of trifluoromethanesulfonic acid anhydride in 15 mL of anhydrous hexane was added dropwise to the cooled polydimethylsiloxane solution. The reaction mixture was stirred under an inert atmosphere at -25°C for 3 hours. The formed precipitate of triethylammonium trifluoromethanesulfonate was filtered off under an inert atmosphere at -20°C, and the hexane was evaporated at room temperature and 0.3 mbar. The obtained α,ω-bis(triflate)-terminated polydimethylsiloxane was further dried at 30°C and 0.3 mbar for 1 hour, after which 45 mL of anhydrous chloroform, 19 mL of anhydrous acetonitrile, and 3.3 mL (38.9 mmol) of anhydrous 2-methyl-2-oxazoline were added. The reaction mixture was held at 42°C for 65 hours under an inert atmosphere, and then passively cooled to room temperature. Polymerization was stopped before the monomers were completely converted using various termination agents (nucleophiles) under established reaction conditions.

[0092] The "living" polymer of Scheme 1 obtained from polymerization 1 The 1H-NMR (400MHz, CD2Cl2, δ) parameters are as follows: 4.97(t, 2.51H, -CH2- is the -N head group of 2-methyl-2-oxazolinium triflate). + = adjacent to), 4.40 (t, 2.82H, -CH2- is adjacent to the -O- of the 2-methyl-2-oxazolinium triflate head group), 3.96 (br, 2.73H, -CH2- is adjacent to the -N of the 2-methyl-2-oxazolinium triflate head group) += adjacent), 3.85-3.70 (br, 4H, the -CH2- adjacent to -OH overlaps with the -CH2- adjacent to -CH2-OH), 3.7-3.2 (br, 72.55H, the -CH2-CH2- adjacent to -NC(O)CH3 of the PMOXA skeleton overlaps with the -CH2-CH2-O-CH2-CH2-CH2-linker), 2.48 (br, 4.11H, 2-methyl-2-oxazolinium triflate) The values ​​for the head group (-CH3), 2.26-1.98 (br, 49.57H, -CH3 of the 2-methyl-2-oxazoline side chain), 1.63-1.5 (br, 3.98H, -CH2- is adjacent to -CH2-Si(-CH3)2-O-), 0.59-0.47 (m, 4H (ref.), -CH2- is adjacent to -Si(-CH3)2-O-), and 0.08 (br, 235.46H, CH3- of the PDMS skeleton), all in ppm units, are shown in Figure 14A.

[0093] B. Synthesis of α,ω-(acetyloxy-,hydroxy-)-terminal poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline).

[0094] This member of the PMOXA-PDMS-PMOXA family of TBC compounds has the structural formula shown in Scheme 2 and is referred to herein as the "Ch111 type" TBC. [ka]

[0095] The polymerization of 2-methyl-2-oxazoline was terminated by adding triethylamine: water (1:4 v / v) (triethylamine was 4.3 molar equivalents of the polydimethylsiloxane macroinitiator) and mixing at room temperature for 18 h. Volatiles were evaporated and the polymer was dried at 50 °C and 0.3 mbar for 3 h. The polymer was dissolved in water: ethanol (1:1 v / v, 7 mg / mL) and low molecular weight ethanol / water-soluble impurities were removed by ultrafiltration (e.g., dialysis with ethanol: water (1:1 v / v) using a centrifugal filter with a regenerated cellulose membrane having a MWCO of 2 kDa). After ultrafiltration, water and ethanol were evaporated and the polymer was dried at 50 °C and 0.3 mbar for 3 h. The dialyzed polymer was dissolved in methanol: hexane (1:1, 6 mg / mL) and the solution was co-solvent fractionated by mixing at room temperature for 18 h and at -60 °C for 3 h. The cooled solution was warmed to room temperature in a separatory funnel until clear phase separation occurred. The bottom fraction was collected, the solvent was evaporated and the polymer was dried at 50 °C and 0.3 mbar for 3 h.

[0096] Determined for the Ch111 TBC compound prepared above 1 The 1H-NMR (400 MHz, CDCl3, δ) parameters are as follows. 4.38 (br, 0.71H, -CH2- adjacent to -COOCH3), 3.77 (br, 3.92H, -CH2- adjacent to -OH), 3.68 - 3.25 (br, 98.24H, -CH2-CH2- adjacent to -NC(O)CH3 of the PMOXA backbone overlaps with -CH2- adjacent to -CH2-OH, -CH2- adjacent to -CH2-COOCH3, and -CH2- adjacent to -CH22-CH2-Si(-CH3)22-O-), 2.2 - 2.04 (br, 67.93H, -NC(O)CH3), 1.65 - 1.5 (br, 4.51H, -CH2- adjacent to -CH2-Si(-CH3)2-O-), 0.55 - 0.45 (m, 4H (ref.), -CH2- adjacent to -Si(-CH3)2-O-) and 0.07 (br, 309.71H, CH3- of the PDMS backbone), all in ppm units and shown in Figure 14B.

[0097] Synthesis of C.α,ω-(azido-,hydroxy-)terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline)

[0098] This member of the PMOXA-PDMS-PMOXA family of TBC compounds has the structural formula shown in Scheme 3 and is a precursor used for the CuAAC click chemistry preparation of TBC compounds having the modified head groups described below. [ka]

[0099] The reaction solvents (e.g., chloroform and acetonitrile) were evaporated under Schlenk conditions, and the resulting α,ω-bis(2-methyl-2-oxazolinium triflate)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) "living" polymer was dried at 30°C and 0.03 mbar for approximately 30 minutes. The dried solid "living" polymer was redissolved in acetonitrile (approximately 90 mg / mL) and further mixed with sodium azide (70 equivalents of sodium azide per 1 equivalent of PDMS) under an inert atmosphere at 80°C for 45 hours. The reaction mixture was cooled to room temperature, and then the unreacted sodium azide was filtered off, and the solvent was evaporated. The resulting polymer was redissolved in ethanol, and the precipitated sodium azide was removed by centrifugation. Finally, the ethanol was evaporated, and the resulting polymer was dried at 30°C and 1 mbar for 1 hour, and then stored at -20°C until use.

[0100] The obtained α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) was determined. 1The H-NMR (400 MHz, CD2Cl2, δ) parameters were as follows: 3.7 (br, the -CH2- adjacent to -OH overlaps with the -CH2-CH2- derived from the PMOXA skeleton), 3.76-3.15 (br, 101, 14H, the -CH2-CH2- adjacent to -NC(O)CH3 of the PMOXA skeleton overlaps with the -CH2- adjacent to -CH2-OH, the -CH2- adjacent to -OH, the -CH2- adjacent to -N3, and the -CH2-CH2-Si(-CH3)2-O- (overlapping with the adjacent -CH2-), 2.22-1.96 (br, 66.61H, -NC(O)-CH3), 1.64-1.50 (br, 5.12H, -CH2- is adjacent to -CH2-Si(-CH3)2-O-), 0.58-0.46 (m, 4H (ref.), -CH2- is adjacent to -Si(-CH3)2-O-) and 0.08 (br, 334.64H, CH3- of the PDMS framework). An example NMR spectrum is shown in Figure 14C. FT-IR spectra (shown in Figure 14D) were obtained for α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-blocked polydimethylsiloxane-blocked poly(2-methyl-2-oxazoline) after solvent evaporation (gray), ultrafiltration (blue), and cosolvent fractionation (red). The azido head group was approximately 2100 cm⁻¹ in all samples. -1 It can be shown.

[0101] Synthesis of α,ω-(hydroxy-,5-(1-polymer-1H-1,2,3-triazole-4-yl)isophthalic acid)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) via D.CuAAC.

[0102] This member of the PMOXA-PDMS-PMOXA family of TBC compounds has the structural formula shown in Scheme 4 and is referred to herein as the "Ch127 / 128 type" TBC. [ka]

[0103] Reaction conditions for CuAAC (use of ethanol and copper nanoparticles, reaction time, and temperature) have been reported for PMOXA-PDMS-PMOXA synthesis (use of α,ω-bis(azide)-terminated PDMS and alkyne-terminated PPMeOxa) (see, for example, Isaacman, MJ; Barron, KA; Theogarajan, LS. Clickable amphiphilic triblock copolymers. Journal of Polymer Science Part A: Polymer Chemistry 2012, 50(12), 2319-2329), but the use of CuAAC for modification of TBC head groups has not been reported to date.

[0104] The synthesis of Ch127 / 128 type polymers based on CuAAC was carried out as follows: 0.45 g (0.1 mmol) of α,ω-(azido-,hydroxy-)-terminated poly(2-methyl-2-oxazoline)-block-polydimethylsiloxane-block-poly(2-methyl-2-oxazoline) and 0.54 g (2.85 mmol) of alkyne, in this example 5-ethynyl-1,3-benzenedicarboxylic acid, were dissolved in 4 mL of ethanol, and the resulting solution was purged with an inert gas for 30 minutes. After purging, 60-100 mg of copper nanoparticles (25-60 nm size) were added to the polymer solution under an inert atmosphere, and the reaction mixture was stirred at 60°C for 2-3 hours under an inert atmosphere. After cooling to room temperature, the reaction mixture was diluted with ethanol, and the copper nanoparticles were removed by centrifugation. Trace amounts of copper were further removed by passing the polymer solution through a copper capture column several times. Unreacted alkynes and ethanol / water-soluble impurities were removed by ultrafiltration (e.g., dialysis of ethanol:water (0.8:0.2-1:1 v / v) using a centrifugal filter with a regenerated cellulose membrane containing 2 kDa MWCO). After ultrafiltration, water and ethanol were evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours. The dialyzed polymer was dissolved in methanol:hexane (1:1; 6 mg / mL), and the solution was cosolvent-fractionated by mixing at room temperature for 18 hours and at -60°C for 3-5 hours. The cooled solution was warmed at room temperature in a separatory funnel until clear phase separation occurred. The bottom fraction was collected, the solvent was evaporated, and the polymer was dried at 50°C and 0.3 mbar for 3 hours.

[0105] The above CH127 / 128 type polymer 1¹H-NMR (400MHz, CD2Cl3, δ): 8.7-8.2 (br, 6.1H, aromatic protons from triazole and aromatic ring), 4.7 (br, 2.96H, -CH2- is adjacent to triazole), 3.93 (br, 3.31H, -CH2- is adjacent to -CH2-triazole), 3.79-2.95 (br, 82.44H, -CH2-CH2- adjacent to -NC(O)CH3 of PMOXA skeleton is -CH2-CH2-O-CH2 -CH2-CH2- (overlapping with the -CH2- of the linker), 2.32-1.86 (br, 55.31H, -NC(O)-CH3), 1.68-1.48 (br, 4.46H, -CH2- is adjacent to -CH2-Si(-CH3)2-O-), 0.59-0.46 (m, 4H (ref.), -CH2- is adjacent to -Si(-CH3)2-O-) and 0.08 (br, 177.35H, CH3- of the PDMS skeleton), all in ppm units. An example NMR spectrum is shown in Figure 14E.

[0106] Table 2 (below) provides alkynes that can be used for further synthesis of PMOXA-PDMS-PMOXA TBCs with different MW and PDMS content and different head groups.

[0107] [Table 2] TIFF2026515985000012.tif127157

[0108] The various alkynes shown in Table 1 can be used in CuAAC-based synthesis using α,ω-(azido-,hydroxy-) terminal PMOXA-PDMS-PMOXA to provide various TBCs having different terminal head groups containing triazole groups as shown in Schemes 5 to 17 below. The reaction conditions used are the same as those described above for CuAAC using 5-ethynyl-1,3-benzenedicarboxylic acid.

[0109] Scheme 5. Chemical structure of Ch118 / 122 [ka]

[0110] Scheme 6. Chemical structure of Ch112 / 113 / 114 [ka]

[0111] Scheme 7. Chemical structure of Ch119 / 123s [ka]

[0112] Scheme 8. Chemical structure of Ch115 / 116 [ka]

[0113] Scheme 9. Chemical structure of Ch120 / 124 [ka]

[0114] Scheme 10. Chemical structure of Ch125 / 126 [ka]

[0115] Scheme 11. Chemical structure of TBC-Ch129 / Ch130 having a phosphate head group [ka]

[0116] Scheme 12. Chemical structure of TBC-Ch131 / Ch132 having a propanediic acid head group [ka]

[0117] Scheme 13. Chemical structure of TBC-Ch133 / Ch134 having a butanediic acid head group [ka]

[0118] Scheme 14. Chemical structure of TBC-Ch135 / Ch136 having an aminoheptic acid head group [ka]

[0119] Scheme 15. Chemical structure of TBC with octic acid head group - next synthesis [ka]

[0120] Scheme 16. Chemical structure of Ch108 type TBC [ka]

[0121] Scheme 17. Chemical Structures of Ch137 and Ch138 Type TBCs [ka]

[0122] Example 2: Use of Hybrid Triblock Copolymer (TBC) Lipid Bilayer in Nanopore-Based Nucleic Acid Detection This example illustrates the study of the use of hybrid triblock copolymer (TBC) lipid bilayers in electrochemical cells during SBX nanopore sequencing. A series of experiments were conducted to demonstrate the effects of triblock copolymer size and concentration on sequencing results.

[0123] material and method: A. Preparation of DPhPE / TBC solution: In a vial, dissolve 30 mg by mass of DPhPE in 10 mL of a 9:1 mixture of PDM20 silicone oil and hexadecane. Stir the solution at ambient temperature and sonicate until completely dissolved to obtain a DPhPE solution of 3 mg / mL in 9:1 silicone oil and hexadecane. In another vial, add 9 mg of PMOXA-PDMS-PMOXA TBC. To the solid, add 2.7 mL of PDM20 silicone oil, followed by 0.3 mL of hexadecane. Then, sonicate the resulting suspension at ambient temperature for 1 hour or until completely dissolved to obtain a 3 mg / mL copolymer solution in 9:1 silicone oil and hexadecane. To prepare 15% TBC and 85% DPhPE solutions, add 150 μL of 3 mg / mL polymer solution to 850 μL of 3 mg / mL lipid solution.

[0124] B. Preparation of electrochemical cells having nanopore-embedded hybrid lipid bilayers: Preparation of nanopore-embedded lipid bilayers placed on an array of electrochemical cells for nanopore-based sequencing is described, for example, in U.S. Patent Application Publication 2013 / 0244340(A1) and U.S. Patent Application Publication 2014 / 0134616(A1). Standard nanopore-embedded bilayer preparations utilize DPhPE lipids as the sole component for membrane formation and subsequent sequencing. This example uses a hybrid bilayer-forming solution containing standard DPhPE lipids and various amounts of the synthetic triblock copolymer compounds described above. The resulting hybrid lipid / TBC solution is mixed with α-hemolysin nanopores and used to form the hybrid lipid bilayer on an electrochemical cell array according to a standard method (see, for example, U.S. Patent Application Publication 2013 / 0244340(A1)).

[0125] C. Nanopore-based SBX sequencing using hybrid lipid bilayers: Nanopore-based SBX sequencing experiments are performed using an array of electrochemical cells containing buffer solutions with ferricyanide and ferrocyanide ions, as described, for example, in U.S. Patent No. 7,939,259, International Publication No. 2020, 236,526,A1 and U.S. Patent No. 63 / 337,413 filed on May 2, 2022. The main modification of the protocol is the composition of the membrane solution used to insert the biological nanopore and perform the sequencing experiment. Varying the properties of the triblock copolymer and the amount added to DPhPE can result in different sequencing characteristics, as detailed below.

[0126] A series of nanopore-based SBX sequencing experiments were performed over several months using an array of electrochemical cells in which nanopores were embedded in standard phospholipid bilayers, and compared with an array in which nanopores were embedded in hybrid triblock copolymer lipid bilayers (Table 3, Experiments 1-8).

[0127] [Table 3] TIFF2026515985000027.tif26157

[0128] Results: Cell failure due to non-sequencing "protopore" formation was found to be reduced by at least twofold when nanopores were embedded in a hybrid triblock copolymer lipid bilayer. In some cases, the use of certain polymer additives can completely eliminate the non-sequencing "protopore" population, which may account for up to 50% of the potential pores when using lipid-only membranes.

[0129] Initial tests in Experiment 1, Table 3, showed that adding polymer Ch111 in various amounts to DPhPE lipids could improve sequencing rates, measured as the number of sequenced molecules per cell per minute. With a standard DPhPE membrane alone, a rate of approximately 10 molecules / minute was achieved. With a 10% composition (or 0.3 mg / mL Ch111 + 2.7 mg / mL DPhPE), the rate improved, exceeding 19 sequenced molecules per cell per minute. As a final result, the total number of sequenced molecules increased despite an increase in the number of cells being sequenced with DPhPE alone. Tests with alternative polymers (e.g., Experiments 2 and 3) demonstrated that this was consistent across other polymer sizes, improving sequencing rates (measured as the total number of sequenced molecules or the number of sequenced molecules per cell per minute), and notably, these advantages could be achieved with a 10% polymer composition relative to the DPhPE lipids. In higher concentration tests (Experiment 4), it was shown that a 15% polymer composition was optimal for improving the processing rate when using polymer Ch111, compared to other conditions. Experiments 5-8 demonstrated the properties of other polymer species with varying monomer amounts, and the effects on sequencing were observed, particularly regarding processing rate and the cells used for sequencing. Notable results included (PMOXA)7-(PDMS) 21 -When using the (PMOXA)7 scaffold, Ch15 showed improved processing rates and a higher cell level for sequencing compared to other polymers. Using smaller scaffolds (e.g., Ch72 and Ch74 in Experiment 8) resulted in fewer cells for sequencing and fewer molecules sequenced per cell per minute compared to Ch15. Experiment 6 shows that polymers of similar size also yield similar results in terms of processing rates.

[0130] In the following series of experiments conducted by the inventors, the modification of polymer head groups was investigated. Using a novel synthesis method for PMOXA-PDMS-PMOXA polymers, azide-containing polymers were produced that could be modified via reaction with alkyne species, enabling modification of the head groups. A series of sequencing experiments were performed using these polymers as hybrid compositions, and the sequencing properties, as referenced in Table 4, were determined.

[0131] [Table 4] TIFF2026515985000029.tif242157 TIFF2026515985000030.tif237157 TIFF2026515985000031.tif187157

[0132] The experimental results in Table 4 confirm that various head group modifications can also modulate sequencing cell yield and the number of molecules sequenced per cell per minute. Compared to unmodified lipids, head groups modified as hybrid membranes for sequencing can improve the number of molecules sequenced per cell per minute (Table 4, Experiment 1), or improve the number of cells being sequenced while similarly achieving the number of molecules sequenced per cell per minute (Table 4, Experiment 2). Polymers with modified head groups may have a wider range of compositional tolerance than unmodified polymers, and the properties of the head groups can have a significant impact on yield and processing capacity. Comparing Experiment 1 with Experiments 2-4, it is shown that modification with the phosphatidylethanolamine head group is necessary to achieve good cell yields compared to the phosphatidylcholine head group, which exhibits significantly lower cell yields. Head groups containing carboxylic acids or phosphoric acids (Experiments 5, 6, 15, 17, 18, 19) can achieve higher throughput and similar cell yields than unmodified or standard polymers. Triazole-alcohol and triazole-octylamine head groups may be beneficial for cell yield, but may result in lower yields compared to carboxylate head groups. Sulfonic acid and sulfobetaine head groups may result in lower cell yields compared to other polymers.

[0133] As shown in Table 5 below, a third series of experiments was conducted to understand the resulting properties of membranes containing one or more added synthetic triblock copolymers.

[0134] [Table 5] TIFF2026515985000033.tif51157

[0135] Table 5, Experiment 1 again demonstrates the ability of these mixtures, containing DPhPE lipids and two or more additional triblock copolymers, to improve the number of molecules sequenced per cell per minute compared to standard DPhPE without the addition of triblock copolymers. In this case, more complex mixtures can also result in a considerable increase in the number of cells being sequenced. The polymer composition in the membrane, as a result of mixing unmodified polymers with head-modified polymers, can regulate the cell yield and processing rate for sequencing molecules within each cell.

[0136] A further advantage in all experiments utilizing synthetic triblock copolymers is the elimination of undesirable nanopore populations that cannot provide sequencing data. Sequencing experiments using only DPhPE lipids generate a significant amount of non-sequencing pores. As shown in the image in Figure 13A, using a lipid bilayer of only DPhPE yields a bimodal distribution in each left-hand inset on the vertical axis, indicating the presence of a considerable number of array cells with embedded nanopores that cannot be sequenced. As shown in the images in Figures 13B, 13C, and 13D, adding 20%, 30%, or 40% TBC Ch111 to DPhPE in the lipid bilayer eliminates non-functional populations and increases the probability of successful sequencing data.

[0137] All publications, patents, patent applications, and other documents cited herein are incorporated herein by reference in whole for all purposes to the same extent that each individual publication, patent, patent application, or other document is individually indicated as being incorporated herein by reference for all purposes.

[0138] Although various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 This is a terminal head group containing azide or triazole, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, R 2 This is a terminal head group containing -OH, azide, or triazole. (m is between 4 and 20, and n is between 20 and 60) A triblock copolymer compound.

2. R 1 is a terminal head group containing triazole, and R 2 is -OH, or R 1 and R 2 is a terminal head group containing triazole, the compound according to claim 1.

3. The compound according to any one of claims 1 to 2, wherein the terminal head group containing the triazole further comprises a group selected from hydroxyl, alcohol, azide, ester, amine, amide, alkyl, heteroalkyl, aryl, heteroaryl, carboxylate, terephthalate, phosphate, phosphatidylethanolamine, sulfonate, and sulfobetaine.

4. The terminal head group containing the aforementioned triazole is as follows: A compound selected from any one of claims 1 to 3.

5. The compound according to any one of claims 1 to 4, wherein the terminal head group containing the triazole further comprises a fluorescent moiety, and optionally the fluorescent moiety comprises a coumarin moiety or a coumarin derivative moiety.

6. R 1 is Azid and R 2 is -OH, or R 1 and R 2 The compound according to claim 1, wherein the compound is an azide.

7. The average of the values ​​of m and n is m=12, n=21; (Ch03) m=8, n=34; (Ch05) m=7, n=21; (Ch15) m=9, n=30; (Ch20 / Ch127) m=11, n=40; ​​(Ch42) m=9, n=37; (Ch67) m=5, n=18; (Ch71) m=5, n=17; (Ch72) m=4, n=18; (Ch74) m=11, n=53; (Ch93) m=9, n=37; (Ch103) m=12, n=53; (Ch104) m=9, n=37; (Ch106 / Ch118) m=13, n=59; (Ch108) m=13, n=52; (Ch109) m=11, n=49; (Ch110) m=12, n=50; (Ch111) m=9, n=36; (Ch112) m=9, n=42; (Ch113) m=12, n=55; (Ch114) m=9, n=39; (Ch115 / Ch117) m=11, n=49; (Ch116) m=10, n=38; (Ch119) m=10, n=40; ​​(Ch120) m=12, n=44; (Ch121) m=12, n=41; (Ch122) m=12, n=37; (Ch123) m=12, n=45; (Ch124) m=10, n=31; (Ch125) m=11, n=37; (Ch126) m=12, n=24; (Ch128) m=13, n=31; (Ch129) m=12, n=40; ​​(Ch130) m=9, n=33; (Ch131) m=11, n=34; (Ch132) m=9, n=32; (Ch133) m=11, n=33; (Ch134) m=9, n=39; (Ch135) m=11, n=36; (Ch136) and mixtures thereof A compound selected from any one of claims 1 to 6.

8. formula: R 1 -(PMOXA) m -(PDMS) n -(PMOXA) m -R 2 (In the formula, R 1 It is Azid, R 2 is -OH, azid, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, (m is between 4 and 20, and n is between 20 and 60) A method for synthesizing a triblock copolymer compound, The aforementioned method, (a) Formula: R A -(PMOXA) m -(PDMS) n -(PMOXA) m -R B (In the formula, R A and R B The process involves providing a compound (which is a 2-methyl-2-oxazolinium triflate group), (b) Dissolve the compound of (a) in acetonitrile, and mix it with sodium azide in a ratio of 1 equivalent of compound of (a) to 70 equivalents of sodium azide under an inert atmosphere at 80°C for 45 hours, (c) Cool to room temperature, filter off unreacted sodium azide, and evaporate the solvent. A method for synthesizing a triblock copolymer compound, including [the specified compound].

9. A method for synthesizing a triblock copolymer compound according to any one of claims 1 to 7, wherein the method is: (a) 2.5 to 3.5 equivalents of formula R in ethanol 1 - Alkynes, and / or R 2 - Alkyne compounds, formula for one equivalent: R A -(PMOXA) m -(PDMS) n -(PMOXA) m -R B (In the formula, R A It is Azid, R B is -OH, azid, PMOXA is a poly(2-methyl-2-oxazoline) subunit, PDMS is a poly(dimethylsiloxane) subunit, (m is between 4 and 20, and n is between 20 and 60) The process of mixing with the compound, (b) A step of purging the mixture with an inert gas, (c) Adding a catalytic amount of copper nanoparticles and stirring at 60°C for 2 to 3 hours in an inert atmosphere. Methods that include...

10. A membrane composition comprising the TBC compound according to any one of claims 1 to 7.

11. A polymerosome composition comprising the TBC compound according to any one of claims 1 to 7.

Images