Adapter for characterizing analytes, characterization method and application thereof

Abstract

A new method for blocking motor proteins in nanopore sequencing is provided. During the loading process of the motor protein, a blocking band chain that can simultaneously bind to the motor protein and non-covalently bind to the molecule to be characterized is used to block / stagnate the motor protein at a specified position, thereby controlling the movement of the motor protein without setting a spacer. During the process of the biopolymer molecule to be characterized entering the nanopore, the indirect binding between the motor protein and the molecule to be characterized through the blocking band chain is released, thereby achieving reversible release of the blockage / stagnation.

Description

Technical Field

[0001] The present invention relates to the field of next-generation sequencing, in particular to transmembrane nanopore sequencing technology, to an adapter for characterizing an analyte and a characterization method using the adapter. Background Art

[0002] As a new generation of sequencing, nanopore sequencing technology has the characteristics of single-molecule sequencing, long read length, fast sequencing speed, and real-time data monitoring. It can also directly read / detect modification information of analytes such as nucleic acids or polypeptides (for example, methylation and acetylation of nucleic acids, myristoylation of polypeptides, etc.), which has significant advantages over other sequencing technologies.

[0003] In the current common nanopore sequencing methods, polynucleotide binding proteins (e.g., helicases) or protein translocases that bind to the analyte generally act as motor proteins to control the speed of the sequence through the pore. However, the motor protein itself cannot accurately control the timing of the analyte entering the pore and moving. Therefore, it is necessary to be able to control the time point when the analyte, such as a polynucleotide or polypeptide, begins to enter the pore as the starting point of nanopore real-time sequencing to avoid missing sequences.

[0004] It has been reported that by setting a spacer sequence or spacer on a polynucleotide sequence, the helicase can be stagnant in a specified region of the target sequence, blocking the movement of the sequence to be tested caused by the helicase; and when it is decided to start sequencing, by contacting the helicase with the polynucleotide complex with the transmembrane pore and applying an electric potential, the stagnant helicase can be passed through the spacer, recovering the relative movement along the sequence to be tested, and finally completing sequencing (see CN105209634A). However, the formation of the spacer requires special modification of the nucleic acid adapter, which increases the synthetic complexity of the adapter, and the modification efficiency is low and costly. In addition, due to the lack of interaction between the spacer and the enzyme to stabilize the combination of the two, it is easy to cause the enzyme to fall off from the adapter in long-term sequencing, reducing sequencing efficiency. There is an urgent need in this area to be able to achieve the same technical means for effectively controlling the initiation of nanopore sequencing without using a spacer. Summary of the invention

[0005] The present application proposes a new strategy for blocking motor proteins in nanopore sequencing. During the loading process of the motor protein, a blocking belt chain that can simultaneously bind to the motor protein and non-covalently bind to the molecule to be characterized is used to achieve the blocking / stagnation of the motor protein at a specified position, so that the movement of the motor protein can be controlled without setting a spacer. In the process of the biopolymer molecule to be characterized entering the nanopore, the indirect binding effect between the motor protein and the molecule to be characterized through the blocking belt chain is released, thereby achieving reversible release of the blockage / stagnation. Through this blocking strategy, there is no need to perform complex spacer modifications on the biopolymer molecules used for nanopore sequencing, which reduces the synthesis difficulty and synthesis cost of nanopore sequencing, and the interaction between the blocking belt chain and the molecule to be characterized also increases the interaction between the motor protein and the molecule to be characterized, making the formed loading complex more stable. Optionally, the covalent binding mode of the blocking belt chain and the motor protein may also additionally achieve the effect of locking the enzyme.

[0006] Therefore, on the one hand, the present invention provides a linker for characterizing an analyte, which includes a main chain for connecting to the analyte and a blocking band chain for stalling a motor protein, wherein the main chain contains a main blocking element and a loading area for accommodating the motor protein in a stalled state, and the blocking band chain contains a blocking ligand and a linker, and the motor protein is stalled in the loading area of ​​the main chain by forming a non-covalent bond between the blocking ligand and the main blocking element, and by forming a bond between the linker and the motor protein.

[0007] In some embodiments, the adapter of the present invention further comprises a connecting strand, wherein the connecting strand comprises a complementary region that is complementary to a partial region in the main strand and a side chain region for binding to an external connector.

[0008] In some embodiments, the non-covalent binding of the main blocking element to the blocking ligand can be selected from one or more of ionic bonds, affinity interactions, salt bridges, hydrogen bonds, van der Waals interactions, polar interactions, Coulomb attraction, hydrophobic interactions, and close molecular packing. In a preferred embodiment, the non-covalent binding of the main blocking element to the blocking ligand is selected from hydrogen bonds or affinity interactions, for example, hydrogen bonds can be selected from base complementary pairing between DNA and DNA, DNA and RNA, DNA and PNA, and PNA and PNA, and affinity interactions can be selected from affinity interactions between ligands and ligands, affinity interactions between antigens and antibodies, and affinity interactions between nucleic acid aptamers and ligands.

[0009] In some embodiments, the bulk blocking element is selected from any one of nucleotides or analogs thereof, amino acids or analogs thereof, polypeptides or analogs thereof, or any combination thereof.

[0010] In some embodiments, the blocking ligand is selected from any one of nucleic acid, peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), closed loop nucleic acid (LNA), bridge nucleic acid (BNA), or any combination thereof.

[0011] In some embodiments, the main blocking element and the blocking ligand are selected from any of the following combinations in pairs: (1) alpha-fetoprotein (AFP) / antibody; (2) human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), luteinizing hormone (hLH) / antibody; (3) oxidized low-density lipoprotein (oxLDL) / CD36 receptor; (4) vascular endothelial growth factor receptor (sVEGFR-1) / VEGF; (5) uric acid / uric acid enzyme; (6) folate receptor / folate; (7) SNAP-25 peptide / botulinum neurotoxin serotype A light chain (BoNT-LcA); (8) prostate-specific antigen (PSA), interleukin-6 (IL-6) / antibody; (9) HIV envelope protein gp120 / CD4 / antibody; (10) apurinic / apyrimidinic endonuclease 1 (APE-1) / antibody; (11) cardiac troponin I (cTnI) / antibody; (12) 8-hydroxy-2 ′-deoxyguanosine (8-OHdG) / antibody; (13) carbohydrate antigen 15-3 (CA15-3) / antibody; (14) vascular endothelial growth factor (VEGF) / antibody; (15) thyroid hormone receptor (TRβ) mutant / agonist; (16) ferritin / antibody; (17) folate receptor / folate; (18) bacterial lipopolysaccharide (LPS) / anti-LPS peptide; (19) human epidermal growth factor receptor (HER-3) / antibody; (20) epidermal Growth factor receptor (EGFR) / GE11 peptide; (21) dengue virus / C-type lectin domain family 5 receptor; (22) IL-6, IL-8, VEGF, VEGF-C / antibody; (23) immunoglobulin A (IgA) / antibody; (24) glucose / glucose oxidase; (25) cholera toxin B (CTB) / ganglioside GM1; (26) streptavidin / biotin; or (27) at least one pair of nucleotides that form complementary base pairs.

[0012] In some embodiments, the main body chain comprises a main body arrest element and a loading region in sequence along the direction in which the motor protein moves when characterizing an analyte.

[0013] In some embodiments, the linker can be selected from an irreversible covalent linker, a reversible covalent linker, a click chemistry reactive group; preferably, the irreversible covalent linker, the reversible covalent linker, the click chemistry reactive group comprises an azide group, an alkenyl group, an alkynyl group, a tetrazine group, a maleimide group, and / or a carboxyl group; more preferably, the irreversible covalent linker, the reversible covalent linker, the click chemistry reactive group comprises a cyclooctenyl group (such as trans-cyclooctenyl (TCO)), a cyclooctyne group (such as dibenzocyclooctyne group (DBCO)), a difluorinated cyclooctyne group (DIFO), a bicyclononynyl group (BCN), a 1,2,3,4-tetrazine group, a 1,2,3,5-tetrazine group, a 1,2,4,5-tetrazine group, a maleimide group, and / or a carboxyl group; more preferably, it is a DBCO or a maleimide group; most preferably, it is DBCO;

[0014] In some embodiments, the linker is bound to an amino acid side chain of the motor protein.

[0015] In some embodiments, the linker is covalently linked to the side chain of Cys, Tyr, or Lys of the motor protein. In a preferred embodiment, the linker is linked to the motor protein by covalent crosslinking between DBCO and Tyr or its derivatives, by covalent crosslinking between DBCO and covalently crosslinked Tyr-Tyr or its derivatives, by covalent crosslinking between maleimide group and Cys, Tyr, or Lys, by covalent linking between ester bond or thioester bond and Tyr or Cys, or by covalent linking to Lys through amide bond.

[0016] In some embodiments, the blocking band chain further comprises a water-affinity group for increasing the solubility of the blocking band chain. In some embodiments, the water-affinity group is a hydrophilic amino acid, or a dipeptide, oligopeptide or polypeptide composed thereof. In some embodiments, the hydrophilic amino acid is selected from the group consisting of arginine, alanine, glycine, proline, serine, cysteine, lysine and threonine. In a preferred embodiment, the hydrophilic amino acid is selected from the group consisting of arginine and proline. In a more preferred embodiment, the hydrophilic amino acid is a dipeptide consisting of arginine and proline.

[0017] In some embodiments, the motor protein is a polynucleotide binding protein or a protein translocase. In some embodiments, the polynucleotide binding protein is selected from at least one of a DNA polymerase, an RNA polymerase, a helicase, an endonuclease, an exonuclease, a DNA protease, a topoisomerase, a nucleic acid translocase, a nucleic acid nicking enzyme, or any fusion protein thereof. In some embodiments, the protein translocase is an unfolding enzyme, preferably an unfolding enzyme driven by NTP, more preferably an AAA+ enzyme, most preferably a ClpX unfolding enzyme, a VAT unfolding enzyme, a PAN unfolding enzyme, an AMA unfolding enzyme, an MBA unfolding enzyme, a SAMP unfolding enzyme, a ClpA unfolding enzyme, a ClpC unfolding enzyme, or a ClpE unfolding enzyme, or a functional mutant thereof.

[0018] In some embodiments, the main body chain further comprises a linking region for binding the analyte to be characterized, and the linking region is preferably located at either end of the main body chain.

[0019] In some embodiments, the main chain does not include a spacer region for blocking the relative movement of the motor protein thereon.

[0020] In some embodiments, the main chain further comprises a spacer region for blocking the relative movement of the motor protein thereon.

[0021] In some embodiments, the backbone strand further comprises a leader sequence.

[0022] In another aspect, the present invention also provides a construct comprising an analyte to be characterized and an adaptor of the present invention covalently bound thereto.

[0023] On the other hand, the present invention also provides an adaptor protein complex, which comprises the adaptor of the present invention and a motor protein covalently bound to the linker of the arresting band chain in the adaptor. In some embodiments, the motor protein in the complex is arrested in the loading zone in the main chain.

[0024] On the other hand, the present invention also provides a construct protein complex, which comprises the construct of the present invention and a motor protein, wherein the motor protein is covalently bound to the linker of the arresting band chain in the adapter. In some embodiments, the motor protein in the complex is arrested in the loading zone in the main chain.

[0025] On the other hand, the present invention also provides a modified motor protein, which is obtained by covalently linking the motor protein and the arresting belt chain of the present invention.

[0026] On the other hand, the present invention provides a method for characterizing an analyte, comprising the following steps (a) and (b): (a) using the adapter, construct, adapter protein complex or construct protein complex of the present invention to prepare a characterization mixture comprising a complex formed by the adapter, analyte and motor protein of the present invention; (b) contacting the characterization mixture of step (a) with a hole, allowing the analyte to pass through and move relative to the hole, and obtaining one or more measurement values, which represent one or more characteristics of the analyte.

[0027] In some embodiments, the pores of the present invention are selected from nanopores, transmembrane pores, biological pores, solid-state pores or biological and solid-state hybrid pores. In preferred embodiments, the biological pores of the present invention are derived from hemolysin, leukocidin, CsGG, Mycobacterium smegmatis porin A (MspA), porin B, porin C, porin D, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria autotransporter, and WZA; the solid-state pores of the present invention are preferably derived from graphene nanopores, MoS2 nanopores, BN nanopores or PA63 nanopores.

[0028] In some embodiments, the analyte of the present invention is selected from polynucleotides, polypeptides, polysaccharides, or lipids.

[0029] In some embodiments, in step (a) of the characterization method of the present invention, the motor protein is arrested at the loading region of the main strand via the arresting belt strand or via the arresting belt strand and the spacer.

[0030] In some embodiments, in step (b) of the characterization method of the present invention, the motor protein is released from the loading zone by eliminating the non-covalent binding between the main chain and the arresting band chain, thereby allowing the analyte to pass through and move relative to the transmembrane nanopore.

[0031] In another aspect, the present invention also provides a kit for characterizing an analyte, which comprises the adaptor, construct, adaptor-protein complex, construct-protein complex, or modified motor protein of the present invention.

[0032] On the other hand, the present invention also provides the use of the adapter, construct, adapter protein complex, construct protein complex, modified motor protein or kit of the present invention in characterizing biopolymers, controlling the movement of biopolymers, or preparing products for characterizing biopolymers or controlling the movement of biopolymers. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is an exemplary structure of a stalled complex comprising an adaptor, a motor protein, and an analyte molecule of the present invention. Wherein, 0 represents a leader sequence, 1 represents a main blocking element, 2 represents a loading region, 3 represents an analyte connected to the main chain, 4 represents a blocking ligand, 5 represents a linker bound to the motor protein, 6 represents a motor protein, which is stalled in the loading region on the main chain, 7 represents a partial region in the main chain, 8 represents a complementary region in the connecting chain, and 9 represents a side chain region in the connecting chain, which can be further connected to an external linker.

[0034] Figures 2A to 2C Some exemplary structures of the host chain of the present invention are shown.

[0035] Figure 3 shows the electrophoresis results of exemplary stalling complexes of the present invention;

[0036] Figure 4 The results of purification of an exemplary stalled complex of the present invention through a DNAPac PA200 oligonucleotide HPLC column are shown;

[0037] Figure 5 The figure shows the electrophoresis results of each purified component after the exemplary stalled complex of the present invention is purified by DNAPac PA200 oligonucleotide HPLC column;

[0038] Figure 6 The stability analysis results of the exemplary stalled complex of the present invention under different ATP conditions are shown. The X-axis shows the ATP concentration of the complex, and the Y-axis shows the proportion of the helicase in the complex that falls off from the nucleic acid chain to which it is bound;

[0039] Figure 7 Results of nanopore sequencing using exemplary adapters of the invention are shown. DETAILED DESCRIPTION

[0040] definition

[0041] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure will have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

[0042] As used herein, the term "comprising" or "including" means that the sequence, composition and method include the recited components or steps, but do not exclude other components or steps. "Mainly composed of...", when used to define compositions and methods, should mean excluding any other components or other steps that are obviously important for the technical effect it should achieve. "Consisting of..." should mean excluding other components and steps not mentioned.

[0043] Unless otherwise specified herein, the nouns or terms used herein should be understood according to the conventional knowledge and usage of ordinary technicians in the art. Unless otherwise specified, the specific operating methods (including: preparation process, experimental steps, detection means, etc.) used in this application adopt conventional biochemical experiments, cell biology experiments, molecular biology experiments, gene editing (e.g., recombinant DNA technology), and conventional techniques in related fields in the field of this technology. These technologies have been fully described in the existing literature, and can be specifically referred to, for example, by Sam brook et al. Molecular Cloning: a Laboratory Manual 4th Edition, Cold Spring Harbor Laboratory Press, 2012; Ausubel et al., Current Protocols in Molecular Biology, Wiley Online Publishing House, updated from time to time.

[0044] It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. As used in this specification and the appended claims, unless the content clearly indicates otherwise, the singular forms "a / an" and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of two or more cells, or an entire culture of cells. Unless expressly provided or obvious from the context, as used herein, the term "or" is understood to be inclusive. Unless otherwise defined herein or and in the remainder of the specification below, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention belongs.

[0045] Unless expressly provided or obvious from the context, as used herein, the term "about" should be understood to be within the normal tolerance range of the field, such as within 2 standard deviations of the mean value. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% of the value. Unless otherwise obvious from the context, all numerical values ​​provided herein are modified by the term "about".

[0046] As used herein, the terms "A binds to B", "A (covalently / non-covalently) binds to B", or similar expressions refer to the indicated element A being molecularly coupled to the indicated element B in a controlled manner, or being physically close to, affinitized, adsorbed, or attracted to another indicated element B, or to the control element A being placed at a suitable position relative to element B in space, or the control element A being adjacent to element B in space, indicating the position / functional relationship of the two elements, and such binding does not occur from the outset, but is a binding that occurs under certain conditions or time (i.e., in a controlled manner). Exemplarily and not restrictively, in the context of "helicase binds to a nucleic acid chain", "binding" may mean that under certain conditions (e.g., providing a suitable buffer to provide a suitable ionic environment), the helicase is loaded into the DNA, for example, in the case where the helicase is a ring of two hexamers, the nucleic acid chain passes through the hexamer ring, and such loading is binding. Exemplarily but not limitingly, in the context of "capable of binding to the main chain", "binding" may mean that under certain conditions (e.g., providing a suitable buffer to provide a suitable ionic environment), at least a portion of the connecting chain can be non-covalently bound to the main chain (e.g., base complementary pairing, etc.). Exemplarily but not limitingly, in the context of "the blocking band chain is bound to the helicase", "binding" may mean that under certain conditions (e.g., the reaction conditions of the click chemistry reaction group), the click chemistry reaction group (e.g., DBCO group) on the blocking band chain undergoes a click chemistry reaction with a specific group (e.g., Tyr-Tyr) on the helicase.

[0047] As used herein, the term "stall" or similar expressions may refer to the fact that the relative spatial position of a motor protein and a characterized biological molecule (e.g., a polynucleotide binding protein and a nucleic acid chain, a protein translocase and a polypeptide chain) does not change to a certain extent or range, and such stalling is relatively stable and reversible under certain conditions (e.g., such stalling can be broken, released or overcome by changing ionic conditions or potential conditions).

[0048] As used herein, the term "motor protein" refers to any protein that can bind to a polynucleotide (such as but not limited to dsDNA, RNA-DNA hybrid duplex) or a polypeptide waiting to be characterized, and control its movement through a nanopore, and can also be referred to as a rate-controlling protein. Those skilled in the art can easily determine whether a protein is bound to a polynucleotide or a polypeptide. For example, a protein can be contacted with a polynucleotide or a polypeptide to measure the ability of the protein to bind to a polynucleotide or a polypeptide and move along the polynucleotide or polypeptide. The motor protein may include modifications that, for example, help the molecule to be characterized to bind, prevent it from detaching, and / or help it to be active at high salt concentrations and / or room temperature. As a preferred example, the "motor protein" of the present invention may list any known or unknown polynucleotide binding protein, including but not limited to DNA / RNA polymerase, helicase, endonuclease, exonuclease, DNA protease, topoisomerase, nucleic acid translocase, nucleic acid cutting enzyme, etc., and protein translocase includes but is not limited to unfolding enzymes, etc., but is not limited thereto. For examples of polynucleotide binding proteins, see Chinese Patent Application Publication No. WO2021253410A. As used herein, the term "protein translocase" refers to any protein or analog that can bind to a polypeptide chain or analog and control its movement through a pore, such as an unfolding enzyme, etc. For examples of protein translocases, see Chinese Patent Application Publication No. CN104619854A.

[0049] Those skilled in the art can also easily understand that the "motor protein", "polynucleotide binding protein" or "protein translocase" of the present invention can be a combination of their variants, modifiers, motifs, functional regions or domains, as long as it can achieve binding with the biological molecule to be characterized and can control its relative movement.

[0050] As used herein, the term "helicase" or similar expressions may refer to an enzyme that unravels a nucleic acid base hydrogen bond. Helicases generally include members of the helicase superfamily 1 or superfamily 2. As non-limiting examples of members of the helicase superfamily 1, for example, members belonging to the Pif1-like family, the Upf1-like family, and the UvrD / Rep family can be cited, and more preferably, for example, members belonging to the RecD subfamily, the Upf1 subfamily, the PcrA subfamily, the Rep subfamily, and the UvrD subfamily can be cited. As non-limiting examples of members of the helicase superfamily 2, for example, members belonging to the Ski-like family, Rad3 / XPD family, NS3 / NPH-II family, DEAD family, DEAH / RHA family, RecG-like family, REcQ-like family, T1R-like family, Swi / Snf-like family and Rig-I-like family can be cited, and more preferably, members belonging to the Hel308 subfamily, Mtr4 subfamily, XPD subfamily, NS3 subfamily, Mss116 subfamily, Prp43 subfamily, RecG subfamily, RecQ subfamily, T1R subfamily, RapA subfamily and Hef subfamily can be cited. The helicase in the present invention includes DNA helicase and RNA helicase. In some embodiments, the DNA helicase can be selected from members belonging to the RecD subfamily, the PcrA subfamily, the Rep subfamily, the UvrD subfamily, the Hel308 subfamily, the XPD subfamily, the RecG subfamily, the RecQ subfamily, the T1R subfamily, and the RapA subfamily. In some embodiments, the RNA helicase can be selected from members belonging to the Upf1 subfamily, the Mtr4 subfamily, the NS3 subfamily, the Mss116 subfamily, the Prp43 subfamily, and the Hef subfamily. In a preferred embodiment, the helicase of the present invention can also be a Hel308 helicase, a RecD helicase, a Tral helicase, a TrwC helicase, an XPD helicase, a Dda helicase, a pif1-like helicase, a T4 helicase, an UvrD helicase, a rep protein, a PriA (n' protein), TraY / I, TF II-F, and TF II-H. In a more preferred embodiment, the helicase of the present invention is selected from Pif1-like helicase or Dda helicase. The helicase of the present invention can also be a functional variant of a helicase obtained by natural or artificial mutation or modification, or an artificial enzyme or enzyme analog having an enzymatic activity center of a helicase, or any combination of motifs, functional regions or domains having helicase activity.

[0051] As used herein, the term "nucleic acid analogue" or similar expressions refers to a nucleic acid-derived substance having similar properties to RNA and DNA. RNA and DNA are mainly composed of phosphate, pentose and bases, while nucleic acid analogs replace at least one of them with some other substances, such as peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), etc. The side chain region can be specifically set as ribonucleotides (RNA) and / or nucleic acid analogs to retain the original base specificity so that it can still bind to the linker while reducing the ability to bind to the motor protein (in some embodiments, the polynucleotide binding protein). Of course, the side chain region can also be a modified deoxyribonucleotide (DNA).

[0052] As used herein, the term "covalent binding" or similar expressions may refer to the intermolecular connection between two or more molecules by forming a covalent bond. As used herein, the term "non-covalent binding" or similar expressions may refer to the positional association between two or more molecules through non-covalent bonds (e.g., ionic bonds, affinity interactions, salt bridges, hydrogen bonds, or hydrophobic forces, etc.).

[0053] As used herein, the term "click chemistry" is a term first introduced by Kolb et al. in 2001 to describe a wider range of powerful, selective and modular building blocks that are reliably used in small-scale and large-scale applications (Kolb HC Finn, MG, Sharp less KB, click chemistry: diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021). They defined the following series of strict criteria for click chemistry: "The reaction must be modular, wide range, give very high yields, produce only harmless by-products that can be removed by non-chromatographic methods, and be stereospecific (but not necessarily enantioselective). Required process features include simple reaction conditions (ideally the method should be insensitive to oxygen and water), readily available starting materials and reagents, no solvent or use of solvents that are mild (e.g., water) or easily removed, and simple product isolation. Purification must be by non-chromatographic methods, such as crystallization or distillation, if necessary, and the product must be stable under physiological conditions." For example, free copper click chemistry or copper-catalyzed click chemistry can be used. Click chemistry is used in these applications due to its desirable properties and its scope for generating covalent linkages between a variety of building blocks. For example, it is fast, clean and non-toxic, producing only harmless byproducts. Suitable examples of click chemistry include, but are not limited to, the following: (a) 1,3 dipolar cycloaddition reactions of variants of free copper, where azide reacts with alkyne in a stress, such as in a cyclooctane ring; (b) reaction of an oxygen nucleophile on one linker with an epoxy or aziridine reactive moiety on the other linker; and (c) Staudinger ligation, where the alkyne moiety can be substituted with an aryl phosphine, resulting in a specific reaction with azide to give an amide bond. An exemplary click chemistry reaction is a Cu(I)-catalyzed 1,3 dipolar cycloaddition reaction between alkyne and azide, for example, the first group is an azide group and the second group is an alkyne group. The alkyne groups were commercially available from Berry Associates (Michigan, USA), and the azide groups were synthesized by ATDBio or IDT bio.

[0054] As used herein, the term "water-affinity group" or similar expressions may refer to an oleophobic group, an atomic group that is soluble in water or easily affinity with water, which may attract water molecules or dissolve in water, and the solid surface formed by such molecules is easily wetted by water. Exemplary water-affinity groups may include carboxylic acid groups, sulfonic acid groups and phosphoric acid groups, amino groups, quaternary ammonium groups, ether groups composed of oxygen-containing groups, hydroxyl groups and carboxylic acid esters, block polyethers, etc.

[0055] As used herein, the term "hydrophilic amino acid" or similar expressions may refer to natural amino acids, unnatural amino acids or amino acid analogs having a water solubility greater than 50 g / L at 25°C and standard atmospheric pressure. The term does not specifically refer to polar amino acids.

[0056] As used herein, the term "transmembrane nanopore" or similar expressions may refer to a structure that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other side of the membrane. A transmembrane nanopore is a polypeptide or a collection of polypeptides that allow hydrated ions (e.g., analytes) to flow from one side of the membrane to the other side of the membrane, which can form a hole that allows hydrated ions driven by an applied potential to flow from one side of the membrane to the other side. Transmembrane nanopores preferably allow analytes (e.g., nucleotides) to flow from one side of a membrane (e.g., a lipid bilayer) to the other side. Transmembrane protein pores allow polynucleotides (e.g., DNA or RNA) or polypeptides to move through the pores. Transmembrane nanopores may be monomers or oligomers. The pores are preferably composed of several repeating subunits (e.g., 6, 7, or 8 subunits). Transmembrane nanopores typically include barrels or channels through which ions can flow. Transmembrane nanopore subunits typically surround a central axis and provide chains for transmembrane β barrels or channels or transmembrane α-helical bundles or channels. The barrel or channel of a transmembrane nanopore typically contains amino acids that promote interaction with an analyte (e.g., a nucleotide, a polynucleotide or a nucleic acid, a polypeptide). These amino acids are preferably located near the constriction of the barrel or channel. Transmembrane nanopores typically contain one or more positively charged amino acids (e.g., arginine, lysine, or histidine) or aromatic amino acids (e.g., tyrosine or tryptophan). These amino acids typically promote interaction between the pore and a nucleotide or polynucleotide or a nucleic acid or a polypeptide.

[0057] As used herein, the term "spacer" or similar expressions may refer to an oligonucleotide region lacking a nucleobase and a sugar. For specific examples, synthesis methods, and composition forms, please refer to the prior art WO2014135838A1.

[0058] As used herein, the term "characterize" or similar expressions can refer to measuring one, two, three, four or five or more characteristics of a target biomolecule, for example, the length, identity, sequence, secondary structure of the biomolecule, whether the structural unit of the biomolecule is modified, etc. can be measured. The length of the biomolecule can be determined, for example, by determining the number of interactions of the target molecule with the pore, and the duration between interactions of the target biomolecule with the pore. The identity can be determined in conjunction with the determination of the sequence of the target biomolecule, or without the determination of the sequence of the target biomolecule. The former is direct; the biomolecule is sequenced and identified thereby. The latter can be accomplished in several ways. For example, the presence of a specific motif in a biomolecule can be determined (without determining the rest of the sequence of the molecule). Alternatively, a specific electrical and / or optical signal determined in the method can identify the target biomolecule from a specific source. The sequence can be determined as described above. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50):17961-72, and International Application WO 2000 / 28312. Secondary structure can be measured in a variety of ways. For example, if the method involves electrical measurements, secondary structure can be measured using changes in residence time across the pore or changes in current. This allows regions of single-stranded and double-stranded polynucleotides to be identified. The presence or absence of any modification can be determined. The method preferably includes determining whether the target polynucleotide is modified by methylation, oxidation, damage, with one or more proteins or one or more markers, tags or blocking strands, whether the target polypeptide is modified by glycosylation, acetylation, etc. Specific modifications will result in specific interactions with the pore, which can be determined using the methods described below. For example, cytosine and methylated cytosine can be distinguished based on the current passing through the pore during the interaction of the pore with each nucleotide.

[0059] As used herein, the term "biomolecule to be characterized" or similar expressions may refer to a nucleic acid chain or polypeptide chain whose sequence-related characteristics are to be determined, such as a library, a single-stranded / double-stranded DNA, RNA or a hybrid molecule thereof, etc. The term "target biomolecule" or similar expressions may refer to a nucleic acid molecule obtained by connecting a biomolecule to be characterized to a sequencing adapter.

[0060] Adapters and adapter complexes

[0061] The adapter provided by the present invention can be used to characterize biopolymers such as polynucleotides or polypeptides in nanopore sequencing. The adapter comprises at least two different components, namely, a main chain and a blocking band chain. Partial regions of the two components can be combined with each other in an easily opened (such as non-covalent) manner, thereby preventing or inhibiting the movement of the main chain relative to the blocking band chain or other elements combined therewith. The non-covalent binding can be in any manner known or unknown in the art, as long as the manner does not form a covalent connection between the main chain and the blocking band chain. In the characterization of polymers, when the non-covalent binding region is kept in an unopened state as required, the blocking band chain can prevent or limit the movement of other elements (such as polynucleotide binding proteins or protein translocases) connected thereto with regulatory molecule transpore activity relative to the main chain by the resistance generated by the unopened binding, thereby preventing the main chain, especially the molecules to be tested connected thereto, from undergoing undesirable transpore translocation movement. When necessary, the binding region can also be easily opened, thereby overcoming or removing the resistance to relative displacement, so that the main chain and the molecules to be tested (such as the biopolymers to be characterized) connected thereto can move through the nanopore, thereby completing the characterization. For example, in some exemplary embodiments, it is only necessary to apply an electric potential to either or both sides of the molecular membrane of the nanopore when characterization is desired, so that the electrostatic force of the molecules to be tested is opposite to the direction of the above resistance. When the magnitude of the electrostatic force exceeds the resistance, the resistance is overcome and the molecules to be tested pass through the nanopore for characterization; in other exemplary embodiments, non-covalent binding such as complementary pairing can be released by changing the ionic environment.

[0062] Thus, the main strand and the blocking band strand in the adapter of the present invention can be combined with the biopolymer to be characterized (e.g., polynucleotides such as dsDNA, ssDNA, or polypeptides, etc.) in any order. In some exemplary embodiments, the main strand and the blocking band strand can first form an adapter complex according to a certain ratio through, for example, mixing and annealing processes, and then the adapter is added to the molecule to be characterized as a whole by connecting the main strand and the biopolymer molecule to be characterized. In other exemplary embodiments, the main strand is first connected to the biopolymer molecule to be characterized, and then forms a non-covalent bond with the blocking band strand. In still other embodiments, the non-covalent bond between the main strand and the blocking band strand can occur simultaneously with its connection to the molecule to be characterized. In the present invention, the adapter and the molecule to be characterized can be connected in any manner known in the art, as long as it can keep the main strand connected to the molecule to be characterized before passing through the nanopore. For specific connection methods, see CN113736778A, the entire text of which is incorporated herein by reference.

[0063] The main chain and the blocking belt chain in the adapter can also be bound to the motor protein in any order. In some exemplary embodiments, the main chain and the blocking belt chain can first be mixed and annealed in a certain ratio to form an adapter complex, and then the motor protein is added to bind to the linker in the blocking belt chain. In other exemplary embodiments, the blocking belt chain can be formed into a non-covalent bond with the main chain after it has been bound to the motor protein through the linker. In still other embodiments, the binding of the linker of the blocking belt chain to the motor protein can occur simultaneously with the non-covalent binding of the blocking belt chain to the main chain. In the present invention, the binding of the linker to the motor protein can be carried out in any binding manner known in the art, such as covalent binding or non-covalent binding, as long as the motor protein can remain connected to the blocking belt chain during the characterization process. For covalent binding, it includes but is not limited to reversible or irreversible covalent linkers, click chemistry reaction groups, or covalent linking groups. For non-covalent binding, non-limiting examples can be found in Ivan Guryanov et al., Receptor-ligand interactions: Advanced biomedical applications, Materials Science and Engineering: C, Volume 68, 1 November 2016, Pages 890-903. Since covalent binding is generally more stable, the arresting belt chain of the present invention is preferably covalently bound to the motor protein through a covalent linker during the characterization process.

[0064] When considering the adaptor, the molecule to be characterized and the motor protein of the present invention at the same time, those skilled in the art can also understand that the main chain and the blocking band chain in the adaptor can be combined with the molecule to be characterized and the motor protein in any order, as long as the following complex can be obtained, that is, the blocking band chain forms a non-covalent bond with the main chain, so that the motor protein in a bound state does not move (stagnate) relative to the main chain, thereby inhibiting the main chain, especially the molecule to be characterized connected to it, from passing through the nanopore.

[0065] In one example, the adapter of the present invention can be used to characterize the constructed library. Optionally, after the library is end-repaired (converted to a flat-end DNA with 5'-phosphate and 3'-hydroxyl), it is connected to the adapter (flat end) by a ligase, or after the library is phosphorylated and repaired, it has sticky ends and is connected to an adapter with complementary sticky ends. The adapter can be connected to either side (for example, as shown in CN114134142A) or both sides of the molecule to be characterized.

[0066] In an optional embodiment, the adapter of the present invention may further include a connecting chain. The connecting chain is used to bind to a connector on a molecular membrane disposed on or near the nanopore protein so that the molecule to be characterized is close to the molecular membrane. To achieve the above purpose, the connecting chain generally includes a region capable of binding to the main chain and a region capable of binding to an external connector, and the binding is preferably non-covalent. For example, Chinese Patent Application Publication No. CN114134142A illustrates a connecting chain that can be used in the present invention. By further including a connecting chain, the adapter of the present invention can also enrich the molecules to be characterized connected thereto near the nanopore protein or the molecular membrane, increase the frequency of the pores of the molecules to be characterized, and improve the characterization efficiency. In addition, there is no special restriction on the regional position of the connecting chain in the main chain, as long as it does not overlap with the region where the main chain and the blocking band chain are non-covalently bound. In a preferred embodiment, along the direction in which the motor protein is expected to move, the connecting chain is bound to the downstream of the loading area in the main chain for accommodating the motor protein, which can play an auxiliary role in the positioning of the motor protein on the adapter and preventing undesirable pore movement.

[0067] It can be understood by those skilled in the art that in the present application, the length and modification of the main strand, the blocking band strand and the connecting strand can be set according to different sequencing platforms, different sequencing requirements, and different requirements for molecules to be characterized. Optionally, the main strand and the blocking band strand can contain 1 to 2000 binding units (for example, base complementary units) in the non-covalent binding region (for example, a binding unit can be 1 base of a nucleotide, or can be a G-quadruplex DNA base, or can be a cytosine-rich polypyrimidine PNA and a complementary polypurine DNA target, etc.), and can contain 1 to 2000, 2 to 2000, 2 to 1500, 2 to 1000, 3 to 500, 5 to 1500, 5 to 100, 10 to 1500, 11 to 1500, 12 to 1500, 13 to 1500,

[0068] 14 to 1500, 15 to 1500, 15 to 1000, 15 to 800, 15 to 600, 15 to 400, 15 to 300, 15 to 200, 15 to 100, 15 to 50 such binding units. Optionally, the connecting chain and the main chain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, 2000, 1500, 1000, 500, 300, 100, 50, 40, 30, 20 or less such binding units. Optionally, the connecting chain and the main chain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 such binding units. The modification process of each chain can be completed before annealing, and the obtained side chain region is modified by chemical synthesis, specifically by solid phase modification, and the specific solid phase method belongs to the prior art and is not described in detail here.

[0069] In an optional embodiment, the adapter of the present invention may also include a leader sequence in the main chain. The leader sequence is used to preferentially enter the nanopore, thereby facilitating the movement of the molecule to be characterized through the hole. The present invention may use any leader sequence suitable for achieving the above purpose. It is understood that in the adapter of the present invention, the leader sequence is generally located upstream of the main blocking element in the main chain along the direction in which the motor protein is expected to move.

[0070] The leader sequence generally includes a charged polymer. The polymer may be positively or negatively charged, preferably negatively charged. The charged polymer may be a polynucleotide, such as DNA or RNA, a modified nucleotide (e.g., a baseless DNA), PNA, LNA, polyethylene glycol (PEG), or a polypeptide such as a negatively charged polypeptide (e.g., a plurality of glutamine or asparagine residues) or a positively charged polypeptide (e.g., arginine, lysine), or other negatively charged synthetic monomers, such as dextran sulfate. The leader sequence preferably comprises a polynucleotide, more preferably a single-stranded polynucleotide. The leader sequence may be any length, but it is generally 10 to 150 nucleotides in length, such as 20 to 150 nucleotides in length. The length of the leader sequence generally depends on the transmembrane pore used in the method.

[0071] In an optional embodiment, the adapter of the present invention may also include a spacer in the main chain. The spacer is used to prevent the motor protein (in some embodiments, a polynucleotide binding protein) from passing through the spacer and further moving along the main chain and the molecule to be characterized connected thereto. The present invention may use any spacer suitable for achieving the above purpose, as an example, refer to the various spacers recorded in Chinese Patent Application Publication No. CN105209634A. It can be understood that in the adapter of the present invention, along the direction of the expected movement of the motor protein (in some embodiments, a polynucleotide binding protein), the spacer is generally located downstream of the loading area in the main chain, see, for example, CN105209634A, or the retardation zone as described in CN114457145A.

[0072] Motor proteins

[0073] According to some exemplary embodiments of the present application, a modified motor protein is provided, to which a blocking band chain is bound (for example, by click chemistry or covalent cross-linking). In one embodiment, the modified motor protein and the main chain and the connecting chain can be mixed and annealed in a certain proportion to form an adapter complex; the complex can be bound to the molecule to be characterized (for example, it can be connected to a nucleic acid library or a polypeptide library); and the connecting chain in the complex can be directly or indirectly connected to a molecular membrane or a nanopore protein, so that the molecule to be characterized and the motor protein are loaded at or near the nanopore.

[0074] Main chain and blocking belt chain

[0075] As mentioned above, the main chain and the blocking belt chain of the present invention respectively contain regions that can bind to each other in a non-covalent manner. The binding region in the main chain is called the main blocking element, and the corresponding binding region in the blocking belt chain is called the blocking ligand. The main blocking element and the blocking ligand can be any known or unknown paired binding elements that can form non-covalent binding. It can be understood that when one of the paired binding elements is selected as the main blocking element, the corresponding other one can naturally serve as a blocking ligand. The selection of the paired binding element has no limitation on its specific chemical structure, as long as it does not significantly hinder the use of nanopore technology to characterize biopolymers.

[0076] As a non-limiting example, the main blocking element can be selected from nucleotides or their analogs, amino acids or their analogs, polypeptides or their analogs, polysaccharides or their analogs, lipid molecules, receptors, ligands, antigens or antibodies, etc. For example, the main blocking element can include one or more consecutive nucleotides or nucleotide analogs, and the blocking ligand can include its reverse complementary sequence, and the two form a non-covalent bond according to the base complementary pairing rule. For another example, the main blocking element can include a natural or modified ligand such as streptavidin, and the blocking ligand can include a ligand capable of binding to the ligand such as biotin or desthiobiotin. Exemplarily, the main blocking element and the blocking ligand can be selected in pairs from the following ligands and their ligand combinations: (1) alpha-fetoprotein (AFP) / antibody; (2) human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), luteinizing hormone (hLH) / antibody; (3) oxidized low-density lipoprotein (oxLDL) / CD36 receptor; (4) vascular endothelial growth factor receptor (sVEGFR-1) / VEGF; (5) uric acid / uric acid enzyme; (6) folate receptor / folate; (7) SNAP-25 peptide / botulinum neurotoxin serotype A light chain (BoNT-LcA); (8) prostate-specific antigen (PSA), interleukin-6 (IL-6) / antibody; (9) HIV envelope protein gp120 / CD4 / antibody; (10) apurinic / apyrimidinic endonuclease 1 (APE-1) / antibody; (11) cardiac troponin I (cTnI) / antibody; (12) 8-hydroxy-2′-deoxy Guanosine (8-OHdG) / antibody; (13) carbohydrate antigen 15-3 (CA15-3) / antibody; (14) vascular endothelial growth factor (VEGF) / antibody; (15) thyroid hormone receptor (TRβ) mutant / agonist; (16) ferritin / antibody; (17) folate receptor / folate; (18) bacterial lipopolysaccharide (LPS) / anti-LPS peptide; (19) human epidermal growth factor receptor (HER-3) / antibody; (20) epidermal growth factor receptor ( EGFR) / GE11 peptide; (21) dengue virus / C-type lectin domain family 5 receptor; (22) IL-6, IL-8, VEGF, VEGF-C / antibody; (23) immunoglobulin A (IgA) / antibody; (24) glucose / glucose oxidase; (25) cholera toxin B (CTB) / ganglioside GM1; or (26) at least one pair of nucleotides that form complementary base pairs (e.g., adenylate / thymidine or guanylate, cytidine / guanylate). The main blocking elements and blocking ligands of the present invention can also be screened using methods known in the art for screening paired binding elements.

[0077] There is no particular restriction on the number of the main retardation elements in the main chain. For example, one main chain may include 1, 2, 3, 4, or 5 identical or different main retardation elements. On the other hand, one or more blocking ligands corresponding to these main retardation elements may be located in the same retardation belt chain or in different retardation belt chains, as long as they do not spatially affect the non-covalent binding of each retardation belt chain with the corresponding main retardation element.

[0078] In some embodiments, the main blocking element is directly connected to the loading region by a covalent bond. In other embodiments, the main blocking element is indirectly connected to the loading region through a spacer sequence. Herein, the loading region can be of any length as long as it is sufficient to accommodate the motor protein stalling. As an example, the length of the loading zone in the present invention can be selected from more than 1, more than 5, more than 10, more than 11, more than 12, more than 13, more than 14 or more than 15 nucleotides or amino acid residues. In a preferred embodiment, the length of the loading zone in the present invention can be 1 to 2000, 2 to 2000, 2 to 1500, 5 to 1500, 10 to 1500, 11 to 1500, 12 to 1500, 13 to 1500, 14 to 1500, 15 to 1500, 15 to 1000, 15 to 800, 15 to 600, 15 to 500, 15 to 400, 15 to 300, 15 to 200, 15 to 100, 15 to 50 nucleotides or amino acid residues.

[0079] In some embodiments, the main chain comprises a main retarding element and a loading region in sequence along the direction of movement of the motor protein. For example, when a polynucleotide binding protein is used as a motor protein, if the polynucleotide binding protein moves from the 5' to the 3' end during the characterization of the analyte, the main chain comprises a main retarding element and a loading region in sequence from the 5' to the 3' direction; conversely, if the polynucleotide binding protein moves from the 3' to the 5' end during the characterization of the analyte, the main chain comprises a main retarding element and a loading region in sequence from the 3' to the 5' direction.

[0080] In some embodiments, the main chain further comprises a connection region for binding to a molecule to be characterized, which region can be located at either end of the main chain. Via the connection region, the adapter of the present invention is bound to the biopolymer molecule to be characterized. In the present invention, the binding of the molecule to be characterized to the connection region is preferably covalent binding.

[0081] In some embodiments, the main chain may further include a spacer at an optional position. It should be noted that although the spacer can help promote the arrest of the motor protein, the present invention is still sufficient to effectively arrest the motor protein without the spacer.

[0082] In an optional embodiment, the main chain may further include a chemically modified group, such as a modified abasic group, in particular, a group that cannot form a base pair, such as an iSp18 group or an iSpC3 group, which may be referred to as an "abasic site" or "abasic nucleotide". The abasic group may be a nucleotide or nucleoside lacking a nucleobase at the 1' position of the sugar portion (see, for example, U.S. Pat. No. 5,998,203, Chinese Patent CN113462764B).

[0083] In some embodiments, the oligonucleotides in the main chain and the blocking band chain can be synthesized by solid phase synthesis. The solid phase synthesis method can refer to the "Molecular Cloning Guide". The sequence of the oligonucleotide can be randomly generated or artificially selected. Optionally, the blocking band chain can contain nucleic acid analogs such as PNA, or it contains nucleic acid analogs but does not contain natural nucleotides. Optionally, click chemistry groups can be modified on the nucleotides. The modification method can refer to CN114262735A.

[0084] In some embodiments, the linker in the blocking belt chain can use any linker known in the art or not yet known, such as but not limited to irreversible covalent linkers, reversible covalent linkers or click chemistry reaction groups. As non-limiting examples, irreversible covalent linkers, reversible covalent linkers, and click chemistry reaction groups containing azido, alkenyl, alkynyl, tetrazine, maleimide, and / or carboxyl groups can be cited. In a more preferred embodiment, the irreversible covalent linker, reversible covalent linker, and click chemistry reaction group in the blocking belt chain include cyclooctenyl (such as trans-cyclooctenyl (TCO)), cyclooctyne (such as dibenzocyclooctyne (DBCO)), difluorinated cyclooctyne (DIFO), bicyclononynyl (BCN), 1,2,3,4-tetrazine, 1,2,3,5-tetrazine, 1,2,4,5-tetrazine, maleimide, and / or carboxyl. In a more preferred embodiment, the irreversible covalent linker, reversible covalent linker, click chemistry reactive group in the blocking band chain comprises DBCO or maleimide group. In a most preferred embodiment, the linker in the blocking band chain is DBCO.

[0085] In some embodiments, the linker in the blocking belt chain is bound to the amino acid side chain of the motor protein, so that the motor protein can be blocked in the loading area of ​​the main chain by binding between the blocking belt chain and the main chain. In a preferred embodiment, the linker in the blocking belt chain is covalently linked to the side chain of Cys, Tyr, and Lys of the motor protein. In a more preferred embodiment, the linker in the blocking belt chain is bound to the motor protein by covalent crosslinking between DBCO and Tyr or its derivatives, by covalent crosslinking between DBCO and covalently crosslinked Tyr-Tyr or its derivatives, by covalent crosslinking between maleimide group and Cys, Tyr or Lys, by covalent linkage between ester bond or thioester bond and Tyr or Cys, or by covalent linkage with Lys through amide bond.

[0086] Connection Chain

[0087] In the characterization of biological molecules, the more free biological molecules to be tested are near the transmembrane nanopore, the more efficient the biological molecules to be tested are through the transmembrane nanopore, and thus the higher the detection efficiency is. Therefore, by providing a connector at or near the transmembrane nanopore and connecting it to the connector through a connecting chain, the concentration of the biological molecules to be tested enriched around the transmembrane nanopore and the efficiency of passing through the transmembrane nanopore can be increased.

[0088] The connecting strand is complementary paired with a part of the main strand through the complementary region it contains. The complementary paired region can be of any length as long as it can enrich the main strand bound to the biomolecule to be detected around the nanopore to a concentration that allows effective sequencing.

[0089] If the molecular membrane is an amphiphilic layer, such as a copolymer membrane or a lipid bilayer, the connector may include a polypeptide anchor and / or a hydrophobic anchor embedded in or on the surface of the molecular membrane, and a tether connected to the polypeptide anchor and / or the hydrophobic anchor. The tether is a polynucleotide chain with certain specificity, and the tether can be connected to the connector by complementary base pairing with the side chain region in the connecting chain.

[0090] It can be understood by those skilled in the art that cholesterol with a tether can be set in an external matrix / connector (for example, a nanopore protein or a molecular membrane near it), and a part of the region in the connecting chain in the adapter can be combined with the tether (for example, base complementary pairing), and can be connected to the molecular membrane of the transmembrane nanopore based on the cholesterol with a tether. The region in the connecting chain that is combined with the tether can be a charge-dense region, which can wander near the transmembrane nanopore. When it is combined with the tether (for example, base complementary pairing), under the action of the external potential, the biomolecule to be measured connected to the adapter enters the transmembrane nanopore, so as to achieve the characterization of the biomolecule to be measured through the change of the electrical signal.

[0091] In an exemplary embodiment, the main chain: the blocking band chain: the connecting chain can form a mixed solution according to a certain molar ratio (e.g., 1:1.1:1.1), and the temperature is slowly lowered (e.g., lowered to 25°C at a rate not higher than 0.1°C / s) by increasing the temperature to a certain temperature (e.g., 96°C) to form an adapter; and then a certain amount of motor protein (e.g., 2 to 10 times the molar amount of helicase relative to the main chain) and a buffer are added to load the protein on the adapter. In an optional embodiment, the main chain: the connecting chain can form a mixed solution according to a certain molar ratio (e.g., 1:1.1), and the temperature is slowly lowered (e.g., lowered to 25°C at a rate not higher than 0.1°C / s) by increasing the temperature to a certain temperature (e.g., 96°C) to form an annealing product, and then a modified motor protein (coupled with a blocking band chain) is added to the annealing product (or its purified product) and incubated at a certain temperature (e.g., 30°C) to form an adapter complex.

[0092] Reagent test kit

[0093] In an optional embodiment, the present application also provides a kit for characterizing biological molecules, comprising the main chain, the blocking band chain and the connecting chain as described above, and optionally further comprising a helicase. In an optional embodiment, the present application also provides a kit for characterizing biological molecules, comprising the main chain and the connecting chain as described above, and a helicase covalently bound to the blocking band chain. In an optional embodiment, the present application also provides a kit for characterizing biological molecules, comprising one or more adapters and one or more helicases. In the kit of the present application, the above-mentioned components can be physically separated and independently contained in different containers, or they can be combined in any way or all contained in the same container.

[0094] The kit of the present application may also include any of the nanopores and nanopore proteins discussed above. The kit may also include the components of the membrane, such as the phospholipids such as lipid bilayers required to form the amphiphilic molecular layer. The kit may additionally include one or more other reagents or instruments that enable any of the above-mentioned embodiments to be implemented. Such reagents or instruments include one or more of the following reagents or instruments: a suitable buffer (aqueous solution), a tool for obtaining a sample from a subject (e.g., a container or instrument comprising a needle), a tool for amplifying and / or expressing biomolecules, a membrane or pressure clamp or patch clamp device defined above. The reagents may be present in a dry state in the kit so that the fluid sample resuspends the reagent.

[0095] Optionally, the kit may also include instructions for using the kit in the method of the invention, or detailed information on which patients the method can be used for. The kit may optionally include the necessary components to promote the movement of the helicase (e.g., ATP and Mg 2+).

[0096] The present invention is further described below by way of specific embodiments, but this is not intended to limit the present invention. Those skilled in the art may make various modifications or adjustments based on the teachings of the present invention without departing from the spirit and scope of the present invention.

[0097] Example

[0098] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0099] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0100] Experimental materials, reagents, instruments and experimental methods

[0101] Unless otherwise specified, the reagents, instruments, genes, and enzymes used in the present invention are all commercially available, and the relevant nucleic acid chains, genes, and enzymes can also be obtained through conventional molecular biology experimental techniques based on information in public databases.

[0102] Wherein, the amino acid sequence of helicase T4 Dda-E94Y / / N365Y is shown in SEQ ID NO: 1;

[0103] The nucleic acid modification method can use conventional methods known in the art;

[0104] The PNA synthesis method can be performed using conventional methods known in the art;

[0105] The main chain modification method can be found in CN114134142A, the entire text of which is incorporated herein by reference.

[0106] Unless otherwise expressly stated herein, other reagents, consumables and methods used herein are specifically described in the instruction manual of the QNome-3841 sequencer (or its equivalent, updated or upgraded model sequencer), as well as the instructions for the dedicated library construction kit, sequencing kit and sequencing chip specified in the instruction manual of the QNome-3841 sequencer (or its equivalent, updated or upgraded model sequencer).

[0107] Example 1: Preparation of arrest complex

[0108] The main chain S1, the blocking chain PNA and the connecting chain YB were synthesized by chemical synthesis method.

[0109] The main chain S1 is composed of:

[0110]

[0111] The blocking band PNA is composed of (from N-terminus to C-terminus):

[0112] DBCO-OO- CGACTTCTACCGTGA -RR;

[0113] The connecting chain YB is composed of:

[0114]

[0115] Among them, the multiple 3s at the 5' end of the main chain S1 represent iSpC3 modification; DBCO in the blocking band chain PNA is a click chemistry group, R is an arginine residue, and O is an O-linker; i2OMe m (m is A, G, C, U) in the connecting chain YB represents a nucleotide residue with 2'-O-methylation modification on the ribose.

[0116] The main chain, blocking band chain and connecting chain are mixed in a ratio of 1:1.1:1.1, and then slowly cooled from 95°C to 25°C, with the cooling amplitude not exceeding 0.1°C / s, for annealing to form a linker.

[0117] 7.5 times the amount of helicase T4 Dda-E94Y / / N365Y (SEQ ID No.: 1), 0.1U HRP, and 100uM H2O2 were added to 1uM adapter, mixed and incubated at 30°C for 18h to form a stalled complex. The enzyme cross-linking was detected by running TBE PAGE gel at 160V for 40min, and the results are shown in Figure 2. In Figure 2, the left lane represents the nucleic acid molecule marker, and the right lane represents the stalled complex. It can be observed from Figure 2 that the stalled complex formed is basically pure and has a high formation efficiency.

[0118] Example 2: Purification of the arrest complex

[0119] The product prepared in Example 1 was added to a DNAPac PA200 oligonucleotide HPLC column and eluted and purified to remove the enzyme that was not bound to the adapter and other impurities.

[0120] The nucleic acid protein complex was then eluted with a mixture of 10 column volumes of buffer A (20 mM Na-CHES, 250 mM NaCl, 4% (W / V) glycerol, pH 8.6) and buffer B (20 mM Na-CHES, 1 M NaCl, 4% (W / V) glycerol, pH 8.6). The main elution peak was then collected, its concentration was measured, and a TBE PAGE gel was run at 160 V for 40 minutes. The elution results are shown in FIG. Figure 3 shown.

[0121] collect Figure 3 The elution peaks were detected by TBE PAGE gel at 160V for 40min. Figure 4 shown.

[0122] Identification Figure 4 The E3 elution peak in contains the target stalled complex.

[0123] Example 3: Quality control of the arrest complex

[0124] 0.2 pmol of the complex prepared in Example 2 was added to a system containing 500 mM KCl, 100 mM MgCl2, and 10 mM, 25 mM, 50 mM, and 100 mM ATP, respectively, and ddH2O was added to a total volume of 10 ul. The mixture was allowed to stand at 40°C for 30 minutes. TBE PAGE gel was then run at 160 V for 40 minutes for detection. Figure 5 As shown, the gray value ratio of the double strands and complexes produced by enzyme shedding was analyzed, and the results were as follows Figure 6 shown. Figure 6 AMX in the figure represents a component of the prior art product Ligation Sequencing Kit SQK-LSK110 of Oxford Nanopore, which is an adapter of the spacer region stalled helicase.

[0125] Depend on Figure 6 It can be seen that when the polynucleotide sequence prepared by the adapter of the present invention is used for nanopore sequencing, the stability and blocking efficiency of the arrest complex formed by the adapter of the present application are basically unchanged under low ATP concentration (10, 25, 50 mM); unexpectedly, under high ATP concentration (100 nM), the present application can achieve a blocking effect at least substantially the same (non-inferior) as that of AMX as a control. This shows that the adapter of the present application can efficiently form a stable arrest complex.

[0126] Example 4: Nanopore sequencing using stalled complexes

[0127] A 10 kb library was prepared by end repair, and the library was ligated with the arrest complex prepared in Example 2 (Qi Carbon Technology, library construction kit QLK-V1.1.1). Then, QNome-3841 (Qi Carbon Technology Co., Ltd.) was used for sequencing, and the sequencing signal obtained was as follows: Figure 7 shown.

[0128] Figure 7 It is shown that the sequencing process can be effectively completed by using the adapter of the present invention. It is shown that the blocking band chain can block the relative displacement of the helicase before the start of sequencing, and release the blocking of the helicase after the start of sequencing, so as to achieve accurate control of the start time of nanopore sequencing. Figure 7 The lower part is a local enlarged diagram of the current signal at a specific time period in the sequencing process. As shown therein, the peaks are relatively uniform in time sequence, and there are no dense peak areas or sparse peak areas in time sequence, indicating that the relative movement of the helicase on the sequence to be tested is effectively controlled, achieving the effect of speed control.

Claims

1. An adapter for characterizing an analyte, characterized in that: The adaptor comprises a main chain for connecting to an analyte and a blocking chain for arresting the motor protein, wherein The main chain comprises a main arresting element and a loading region for accommodating the motor protein in a stalled state, The blocking band chain comprises a blocking ligand and a linker, The arresting belt chain forms a non-covalent bond between the arresting ligand and the main arresting element, and a bond between the linker and the motor protein, so that the motor protein is arrested in the loading area of ​​the main chain.

2. The adapter according to claim 1, wherein: The adaptor further comprises a connecting strand, which comprises a complementary region that is complementary to a partial region in the main strand and a side chain region for combining with an external connector.

3. The adapter according to claim 1 or 2, wherein: The non-covalent binding of the main blocking element to the blocking ligand can be selected from one or more of ionic bonds, affinity interactions, salt bridges, hydrogen bonds, van der Waals interactions, polar interactions, Coulomb attraction, hydrophobic interactions, and close molecular packing, preferably selected from hydrogen bonds or affinity interactions. Optionally, the hydrogen bond can be selected from base complementary pairing between DNA and DNA, DNA and RNA, DNA and PNA, PNA and PNA, Optionally, the affinity can be selected from the affinity of a ligand and a ligand, the affinity of an antigen and an antibody, and the affinity of a nucleic acid aptamer and a ligand.

4. An adapter according to any preceding claim, wherein: The main blocking element is selected from any one of nucleotides or their analogs, amino acids or their analogs, polypeptides or their analogs, or any combination thereof, and the blocking ligand is selected from any one of nucleic acids, peptide nucleic acids (PNA), glycerol nucleic acids (GNA), threose nucleic acids (TNA), closed-loop nucleic acids (LNA), and bridge nucleic acids (BNA), or any combination thereof.

5. An adapter according to any preceding claim, wherein: The main blocking element and the blocking ligand are selected from any of the following combinations in pairs: (1) alpha-fetoprotein (AFP) / antibody; (2) human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), luteinizing hormone (hLH) / antibody; (3) oxidized low-density lipoprotein (oxLDL) / CD36 receptor; (4) vascular endothelial growth factor receptor (sVEGFR-1) / VEGF; (5) uric acid / uricase; (6) thrombin / antibody; (7) urea / antibody; (8) splenic fibrosis / antibody; (9) urea / antibody; (10) uric acid / uricase; (11) urea / antibody; (12) urea / antibody; (13) oxidized low-density lipoprotein (oxLDL) / CD36 receptor; (14) vascular endothelial growth factor receptor (sVEGFR-1) / VEGF; (15) uric acid / uricase; (16) urea / antibody; (17) urea / antibody; (18) urea / antibody; (19) urea / antibody 6) folate receptor / folate; (7) SNAP-25 peptide / botulinum neurotoxin serotype A light chain (BoNT-LcA); (8) prostate-specific antigen (PSA), interleukin-6 (IL-6) / antibody; (9) HIV envelope protein gp120 / CD4 / antibody; (10) apurinic / apyrimidinic endonuclease 1 (APE-1) / antibody; (11) cardiac troponin I (cTnI) / antibody; (12) 8-hydroxy-2′- deoxyguanosine (8-OHdG) / antibody; (13) carbohydrate antigen 15-3 (CA15-3) / antibody; (14) vascular endothelial growth factor (VEGF) / antibody; (15) thyroid hormone receptor (TRβ) mutant / agonist; (16) ferritin / antibody; (17) folate receptor / folate; (18) bacterial lipopolysaccharide (LPS) / anti-LPS peptide; (19) human epidermal growth factor receptor (HER-3) / antibody; (20) epidermal growth factor receptor (HER-3) / antibody; (21) elongation factor receptor (EGFR) / GE11 peptide; (21) dengue virus / C-type lectin domain family 5 receptor; (22) IL-6, IL-8, VEGF, VEGF-C / antibody; (23) immunoglobulin A (IgA) / antibody; (24) glucose / glucose oxidase; (25) cholera toxin B (CTB) / ganglioside GM1; (26) streptavidin / biotin; or (27) at least one pair of nucleotides that form complementary base pairs.

6. An adapter according to any preceding claim, wherein: The main body chain comprises the main body blocking element and the loading region in sequence along the direction in which the motor protein moves when characterizing an analyte.

7. An adapter according to any preceding claim, wherein: The linker can be selected from an irreversible covalent linker, a reversible covalent linker, and a click chemistry reactive group; preferably, the irreversible covalent linker, the reversible covalent linker, and the click chemistry reactive group comprise an azide group, an alkenyl group, an alkynyl group, a tetrazine group, a maleimide group, and / or a carboxyl group; more preferably, the irreversible covalent linker, the reversible covalent linker, and the click chemistry reactive group comprise a cyclooctenyl group (such as trans-cyclooctenyl (TCO)), a cyclooctyne group (such as dibenzocyclooctyne group (DBCO)), a difluorinated cyclooctyne group (DIFO), a bicyclononynyl group (BCN), a 1,2,3,4-tetrazine group, a 1,2,3,5-tetrazine group, a 1,2,4,5-tetrazine group, a maleimide group, and / or a carboxyl group; more preferably, it is a DBCO or a maleimide group; most preferably, it is DBCO; Optionally, the linker is bound to an amino acid side chain of the motor protein; Optionally, the linker is covalently linked to the side chain of Cys, Tyr, or Lys of the motor protein, preferably through covalent crosslinking between DBCO and Tyr or its derivatives, through covalent crosslinking between DBCO and covalently crosslinked Tyr-Tyr or its derivatives, through covalent crosslinking between a maleimide group and Cys, Tyr, or Lys, through a covalent bond between an ester bond or a thioester bond and Tyr or Cys, or through a covalent bond to Lys through an amide bond.

8. An adapter according to any preceding claim, wherein: The blocking belt chain also contains a water-affinity group for increasing the solubility of the blocking belt chain. Optionally, the water-affinity group is a hydrophilic amino acid, or a dipeptide, oligopeptide or polypeptide composed thereof, Optionally, the hydrophilic amino acid is selected from the group consisting of arginine, alanine, glycine, proline, serine, cysteine, lysine, and threonine; preferably selected from the group consisting of arginine and proline; more preferably a dipeptide consisting of arginine and proline.

9. An adapter according to any preceding claim, wherein: The motor protein is a polynucleotide binding protein or a protein translocase, Optionally, the polynucleotide binding protein is selected from at least one of a DNA polymerase, an RNA polymerase, a helicase, an endonuclease, an exonuclease, a DNA protease, a topoisomerase, a nucleic acid translocase, a nucleic acid nicking enzyme, or any fusion protein thereof, Optionally, the protein translocase is an unfoldase, preferably an NTP-driven unfoldase, more preferably an AAA+ enzyme, most preferably a ClpX unfoldase, a VAT unfoldase, a PAN unfoldase, an AMA unfoldase, an MBA unfoldase, a SAMP unfoldase, a ClpA unfoldase, a ClpC unfoldase or a ClpE unfoldase, or a functional mutant thereof.

10. An adapter according to any preceding claim, wherein: The main body chain further comprises a linking region for binding an analyte to be characterized, and the linking region is preferably located at either end of the main body chain.

11. An adapter according to any preceding claim, wherein: The main chain does not include a spacer region for blocking the relative movement of the motor protein thereon.

12. An adapter according to any preceding claim, wherein: The main chain also comprises a spacer region for blocking the relative movement of the motor protein thereon.

13. An adapter according to any preceding claim, wherein: The main strand also includes a leader sequence.

14. A construct comprising an analyte to be characterized and an adaptor according to any preceding claim covalently bound thereto.

15. An adaptor protein complex comprising: The adapter according to any one of claims 1 to 13, and Motor proteins, in, The motor protein is covalently bound to the linker of the arresting strand in the adaptor and optionally arrested at the loading zone in the main strand.

16. A construct protein complex comprising: The construct according to claim 14, and Motor proteins, in, The motor protein is covalently bound to the linker of the arresting strand in the adaptor and optionally arrested at the loading zone in the main strand.

17. A modified motor protein, wherein the motor protein is covalently linked to the arresting belt chain according to any one of claims 1 to 13.

18. A method for characterizing an analyte, the method comprising the following steps (a) and (b): (a) using the adaptor of any one of claims 1 to 13, the construct of claim 14, the adaptor-protein complex of claim 15, the construct-protein complex of claim 16, and the modified motor protein of claim 17, to prepare a characterization mixture comprising a complex formed by the adaptor, an analyte, and a motor protein; (b) contacting the characterization mixture of step (a) with a pore, allowing the analyte to pass through and move relative to the pore, and obtaining one or more measurements representing one or more characteristics of the analyte, Optionally, the pore is selected from a nanopore, a transmembrane pore, a biological pore, a solid-state pore, or a biological and solid-state hybrid pore; The biological pore is preferably derived from hemolysin, leukocidin, CsGG, Mycobacterium smegmatis porin A (MspA), porin B, porin C, porin D, outer membrane porin F, outer membrane porin G, outer membrane phospholipase A, Neisseria autotransporter, and WZA; The solid-state pores are preferably derived from graphene nanopores, MoS2 nanopores, BN nanopores or PA63 nanopores. Optionally, the analyte is selected from a polynucleotide, a polypeptide, a polysaccharide, or a lipid.

19. The method according to claim 18, characterized in that In step (a), the motor protein is arrested at the loading region of the main chain via the arresting belt chain or via the arresting belt chain and the spacer region.

20. The method according to claim 18 or 19, characterized in that In step (b), the motor protein is released from the loading zone by eliminating the non-covalent binding between the main strand and the arresting band strand, thereby allowing the analyte to pass through and move relative to the transmembrane nanopore.

21. A kit for characterizing an analyte, comprising: The adapter according to any one of claims 1 to 13, the construct according to claim 14, the adapter protein complex according to claim 15, the construct protein complex according to claim 16, or the modified motor protein according to claim 17.

22. Use of the adaptor of any one of claims 1 to 13, the construct of claim 14, the adaptor protein complex of claim 15, the construct protein complex of claim 16, the modified motor protein of claim 17, or the kit of claim 21 for characterizing a biopolymer or controlling the movement of a biopolymer, or for preparing a product for characterizing a biopolymer or controlling the movement of a biopolymer.

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