A method for detecting a sugar chain monomer based on an aerolysin nanopore

Abstract

The application provides a method for detecting a sugar chain monomer by using an Aerolysin nanopore, which comprises the following steps: (1) labeling the sugar chain; (2) assembling a phospholipid bilayer, embedding Aerolysin protein to form a single nanopore; (3) adding the labeled sugar chain into a buffer of a nanopore detection device, connecting an instrument, and collecting a monomolecular signal of the sugar chain; and (4) statistically analyzing the monomolecular signal of the sugar chain to obtain a characteristic signal of the sugar chain, and based on the characteristic signal, different sugar chains are detected, analyzed and identified. The application can detect and identify different isomer or different length sugar chains at a monomolecular level, the method is simple in operation, high in sensitivity, and has important significance for analyzing and sequencing a fine structure of the sugar chain and development of glycomics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology in life sciences, specifically a method for detecting glycan chains at the single-molecule level using Aerolysin nanopores. Background Technology

[0002] As the most complex biopolymer discovered in nature, glycans play an important role in many life activities. Corresponding to the rich roles played by glycans is the complexity and diversity of their structures. The structural diversity of glycans mainly stems from: (1) a rich variety of monosaccharide building blocks (up to 120 monosaccharides have been discovered in nature according to literature statistics), with small structural differences and many being isomers; (2) diverse glycosidic bonds, where multiple hydroxyl groups on a monosaccharide can form glycosidic bonds with another monosaccharide, thus producing a variety of positional isomers; (3) glycans can be linear, or they can be bibranched, tribranched, or tetrabranched structures, and there are also many isomers. This complexity makes the structural analysis of glycans extremely difficult. In addition to complexity, glycans are fundamentally different from nucleic acids and proteins in that they are non-template-driven biopolymers, which cannot be amplified. Furthermore, the most valuable oligosaccharide chains (such as N-linked and O-linked glycans on glycoproteins) are numerous and extremely rare. All of this makes the sources of glycans extremely limited, further increasing the difficulty of glycan structural analysis. Therefore, glycan structure analysis and sequencing is a recognized global challenge that hinders scientists from exploring the mysterious world of glycobiology.

[0003] Nanopore single-molecule detection technology boasts advantages such as simplicity, speed, high sensitivity, and low cost, opening new doors for molecular biology research at the single-molecule level. This technology involves using an external voltage to allow molecules slightly smaller than the pore size to pass through a nanopore, monitoring the current changes generated as the molecule passes through, and analyzing the molecular characteristics. Nanopore single-molecule detection technology has been successfully applied to single-molecule DNA sequencing and is being explored for protein sequencing. Theoretically, nanopore-based single-molecule sensing technology provides an ideal tool for glycan structure analysis and even sequencing. However, current progress is limited to the detection of high-molecular-weight glycans or monosaccharides; no progress has been reported on the detection of more complex and diverse oligosaccharide chains. Summary of the Invention

[0004] This invention aims to address the aforementioned challenges by providing a method for detecting different glycan chains based on Aerolysin nanopores. This method enables ultrasensitive single-molecule detection of glycan chains, representing a crucial step towards the future development of single-molecule glycan sequencing. To achieve this objective, the invention employs the following technical solutions:

[0005] A single-molecule method for glycan chains based on Aerolysin nanopores, the main steps of which are as follows:

[0006] (1) Labeling the sugar chain. An aromatic ring compound with a primary amino group, hydrazine group, or hydroxylamine group is selected to react with the reduced-terminal hemiacetal of the sugar chain via reductive amination. For acidic sialic acid sugar chains, an aromatic ring compound containing a primary amino group, hydrazine group, or hydroxylamine group (abbreviated as Ar group) is directly used as the label. The labeling reaction is shown in the following formula:

[0007]

[0008] The Ar group can be one of the following groups:

[0009]

[0010] R1 can be a monosaccharide or an oligosaccharide, which together with the terminal reducing monosaccharide (as shown in the formula) forms a complete oligosaccharide chain;

[0011] The R2 substituent can be hydrogen, alkane (C1-C5), methoxy, nitro, or cyano.

[0012] (2) For neutral sugar chains, sialic acid sugar chains are used as the "locomotive" to first react with compounds containing bis-primary amino groups, bis-hydrazine groups, or bis-hydroxylamine aromatic ring groups. The resulting derivative contains a residual primary amino group, hydrazine group, or hydroxylamine group, which serves as a complex tag to further undergo a reductive amination reaction with the hemiacetal hydroxyl group at the reducing end of the neutral sugar chain, thus achieving labeling. The labeling reaction is shown below:

[0013]

[0014] R1 can be a monosaccharide or an oligosaccharide, which together with the terminal reducing monosaccharide (as shown in the formula) forms a complete oligosaccharide chain.

[0015] (3) Assemble the phospholipid bilayer and embed Aerolysin protein to form individual nanopores. Prepare a phospholipid (diphytylphosphatidylcholine) solution (30 mg / mL) using n-decane as solvent. Use a phenolic resin detection cell (consisting of a resin cup inserted into a resin tank, with a cup volume of 1.5 mL and a remaining tank volume of 1.5 mL) (Warner Corporation, USA). The resin cup is the CIS cell, and the remaining resin tank space is the Trans cell. The resin cup wall (1 mm thick) has a 150 μm pore. Apply about 2 μL of phospholipid solution to a 5 mm diameter area around the pore using a No. 00 sable brush and let it dry. Add 1 mL of 10 mM Tris-HCl (containing 1 MCl, 1 mM EDTA, pH 8.0) electrolyte to each cell (ensuring the liquid level is above the pore). Form a phospholipid bilayer on the pore using the dip-coating method. Then add Aerolysin protein solution and wait for the pores to assemble and embed. Insert Ag / AgCl electrodes into the two cells. Connect the trans cell electrode to the positive terminal of the instrument headstage, and connect the cis cell electrode to the negative terminal of the headstage (grounded).

[0016] (4) Dissolve the labeled sugar chain in the electrolyte, and inject 1 μL of the sugar chain solution into the CIS cell electrolyte. The final concentration of the sugar chain in the electrolyte is 1 μM to 20 μM. Apply a voltage of +70 to +150 mV to the instrument, a sampling rate of 100 to 250 kHz, and a low-pass filter of 5 to 10 kHz. Begin acquiring the blocking current signal of the sugar chain.

[0017] (5) Analyze the glycan signal. The recorded blocking current pulse signal can be used to extract the current pulse size, pulse time, and pulse frequency. Based on the current size and time, a scatter fingerprint of the event can be generated, which enables the identification of different glycans.

[0018] The sugar chain described in step (1) comprises a neutral sugar chain consisting of 2 to 20 monosaccharides with reduced sugars at the ends, and an acidic sugar chain. The monosaccharides can be sialic acid, galactose, glucose, N-acetylgalactose, N-acetylglucose, fucose, mannose, xylose, glucuronic acid, or iduronic acid. The acidic and neutral sugar chains can be linear or bibranched sugar chains (the branch length can be 2 to 10 monosaccharides).

[0019] The benzene ring label mentioned in step (1) can be an aromatic ring compound containing a primary amine group, a hydrazine group, or a hydroxylamine group, wherein the aromatic ring group is benzene, diphenyl ether, biphenyl, naphthalene, anthracene, pyrene, or tetraphenylethylene and different neutral substitutes.

[0020] This invention enables the detection and identification of different isomorphic or different length glycan chains at the single-molecule level. The method is simple to operate, requires a small amount of sample, and has high sensitivity, which is of great significance for the analysis and sequencing of fine glycan structures. At the same time, the method is simple to operate and low in cost, and is expected to provide a single-molecule detection and analysis tool for glycomics. Attached Figure Description

[0021] Figure 1 A schematic diagram illustrating the principle of the method for detecting glycan chains based on Aerolysin nanopores in this invention;

[0022] Figure 2 . Labeled 6'-sialyl lactose (6SL), nanopore ion current blockage events and event distribution scatter plot;

[0023] Figure 3 .Scatter plot of nanopore ion current blockage events and event distribution of labeled 3'-sialyl lactose (3SL);

[0024] Figure 4 .Scatter plot of nanopore ion current blockage events and event distribution of labeled 3'-sialylgalactose (3SG);

[0025] Figure 5 Labeled sialyl lactose-N-tetrasaccharide a (LSTa), nanopore ion current blockage events, and scatter plot of event distribution;

[0026] Figure 6 Labeled lactose (Lac), nanopore ion current blockage events, and scatter plot of event distribution;

[0027] Figure 7 . Labeled 2'-fucosylated lactose (2FL), nanopore ion current blockage events and event distribution scatter plot;

[0028] Figure 8 . Labeled 3'-fucosylated lactose (3FL), nanopore ion current blockage events and event distribution scatter plot;

[0029] Figure 9 Labeled lactose-N-neotetrasaccharide (LNnT), nanopore ion current blockage events, and scatter plot of event distribution; Detailed Implementation

[0030] To make the content, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. These embodiments are only used to illustrate the present invention, and the present invention is not limited to the following embodiments.

[0031] Example 1: Labeling of sialylated sugar chains

[0032] (1) Labeling of acidic sialic acid sugar chains: 6'-sialyl lactose (6SL) was used as the acidic sugar chain, and 4-p-methoxyphenoxyaniline was selected to label the sugar chain. The reaction was a reductive amination reaction. 10 mg of 6'-sialyl lactose, 6.5 mg of 4-p-methoxyphenoxyaniline, and 4.8 mg of sodium cyanoborohydride were weighed and dissolved in 0.8 mL of anhydrous methanol. 0.2 mL of anhydrous dimethyl sulfoxide was added to aid dissolution, followed by 20 μL of acetic acid (2%, v / v). The solution was placed in a 5 mL brown bottle with a screw cap, and the cap was tightened. The bottle was partially placed in an oil bath and heated at 65 °C overnight.

[0033] The reaction mechanism of the reductive amination is as follows:

[0034]

[0035] (2) After the reaction was complete, 1 mL of pure water was added to quench the remaining sodium cyanoborohydride. After filtration through a 0.22 μm filter membrane, the obtained filtrate was separated by high-performance liquid chromatography (HPLC). A semi-preparative column (4.6 × 250 mm) packed with C18 silica gel stationary phase (silica gel particle size 5 μM) was selected; the mobile phase was an acetonitrile (0.1% trifluoroacetic acid) / water (0.1% trifluoroacetic acid) system, with binary gradient elution, a mobile phase ratio of 10%–60% acetonitrile, an elution time of 12 minutes, and a detection wavelength of 254 nm. The target product fraction was collected and lyophilized.

[0036] Q-TOF-MS characterization: 4-p-methoxyphenoxyaniline-labeled 6'-sialyl lactose (6SL) glycans: calculated mass-to-charge ratio [M+H] + The value is 833.3186, indicating a mass-to-charge ratio [M+H]. + The value is 833.3278.

[0037] (3) The labeling of other sialylated sugar chains is carried out in the same manner and under the same conditions as steps (1) and (2) above. The only difference is that different acidic sugar chains are used. For example, the labeling and separation methods of 3'-sialyl lactose (3SL), 3'-sialyl galactose (3SG), and sialyl lactose-N-tetrasaccharide a (LSTa) are the same as above.

[0038] Q-TOF-MS characterization: 4-p-methoxyphenoxyaniline labeled 3'-sialic acid lactose (3SL) glycans: calculated mass-to-charge ratio [M+H] + The value is 833.3186, indicating a mass-to-charge ratio [M+H]. + 833.3273; 4-p-methoxyphenoxyaniline-labeled 3'-sialic acid galactose (3SG) glycan: calculate mass-to-charge ratio [M+H] + The value is 671.2658, indicating a mass-to-charge ratio [M+H]. +671.2634; 4-p-methoxyphenoxyaniline-labeled sialyl lactose-N-tetrasaccharide a(LSTa) glycan: calculate mass-to-charge ratio [M+H] + The value is 1198.4508, indicating a mass-to-charge ratio [M+H]. + The value is 1198.4535.

[0039] Example 2. Labeling of neutral sugar chains

[0040] (1) For neutral sugar chains, a composite label is used, with a shorter sialic acid sugar chain as the "locomotive" and an aromatic ring compound as the intermediate unit. 6'-sialyl lactose (6SL) is used as the "locomotive," and 4,4'-diaminodiphenyl ether is selected as the aromatic ring intermediate unit to react with 6SL first. The reaction is a reductive amination reaction. 100 mg of 6'-sialyl lactose, 305 mg of 4,4'-diaminodiphenyl ether, and 50 mg of sodium cyanoborohydride are weighed and dissolved in 4 mL of anhydrous methanol. 4 mL of anhydrous dimethyl sulfoxide is added to aid dissolution, followed by 160 μL of acetic acid (2%, v / v). The solution is placed in a 10 mL brown bottle with a screw cap, the cap is tightened, and the solution is partially placed in an oil bath and heated at 65 °C overnight.

[0041] (2) The separation and purification process after the reaction is completed is the same as step (2) in Example 1.

[0042] (3) The 6SL derivative obtained in the above steps serves as a composite tag (referred to as 6SL-diphenyl ether). The residual primary amino group in its structure can react with the reducing end hemiacetal hydroxyl group of the neutral sugar chain to achieve labeling. Taking lactose as the neutral sugar chain as an example, the reaction employs a reductive amination reaction. Weigh 3 mg of lactose, 10 mg of the composite tag, and 2.8 mg of sodium cyanoborohydride, dissolve them in 0.5 mL of anhydrous methanol, add 0.5 mL of anhydrous dimethyl sulfoxide to aid dissolution, and then add 20 μL of acetic acid (2%, v / v). Place the solution in a 5 mL brown bottle with a screw cap, tighten the cap, and place the solution portion of the bottle in an oil bath. Heat at 65°C and react overnight.

[0043] (4) After the reaction is completed, the separation and purification process is the same as step (2) of Example 1.

[0044] Mass spectrometry Q-TOF-MS characterization: lactose glycans labeled with the complex tag 6SL-diphenyl ether: calculated mass-to-charge ratio [M+H] + The value is 144.4402, indicating the mass-to-charge ratio [M+H]. + The value is 1144.4292.

[0045] The reaction mechanism of the two-step reductive amination is as follows:

[0046]

[0047] (5) The labeling of other neutral sugar chains is carried out in the same manner and under the same conditions as steps (1)-(4) above. The only difference is that the neutral sugar chains used are different. For example, the labeling and separation methods of 2'-fucosylated lactose (2FL), 3'-fucosylated lactose (3FL), and lactose-N-neotetrasaccharide (LNnT) are the same as above.

[0048] Q-TOF-MS characterization: 2'-fucosyllactose (2FL) glycans labeled with the complex tag 6SL-diphenyl ether: calculated mass-to-charge ratio [M+H] + The value is 1290.4982, indicating the mass-to-charge ratio [M+H] was detected. + 1290.5014; 3'-fucosyllactose (3FL) glycans labeled with the complex tag 6SL-diphenyl ether: calculated mass-to-charge ratio [M+H] + The value is 1290.4982, indicating the mass-to-charge ratio [M+H] was detected. + 1290.5041; Compound tag 6SL-diphenyl ether labeled lactose-N-neotetrasaccharide (LNnT) glycan: calculated mass-to-charge ratio [M+2H] 2+ The value was 755.2899, and the mass-to-charge ratio [M+2H] was measured. 2+ It is 755.2935.

[0049] Example 3. Assembly of Aerolysin nanopores

[0050] (1) Assembly of Aerolysin nanopores Reference: Cao, C., Liao, DF., Yu, J. et al. Construction of an aerolysin nanopore in a lipid bilayer for single-oligonucleotide analysis. Nat. Protoc. 12, 1901–1911 (2017). Specifically, a phospholipid (diphytylphosphatidylcholine) solution (30 mg / mL) was prepared using n-decane as the solvent. A phenolic resin detection cell (consisting of a resin cup with an open top embedded in a resin tank with an open top, wherein the cup has a volume of 1.5 mL and the remaining volume of the resin tank after filling the cup is 1.5 mL) (Warner Corporation, USA) was used. The inner cavity of the resin cup served as the CIS cell, and the remaining cavity in the resin tank after filling the cup served as the trans cell. There was a small hole with a diameter of 150 μm in the lower middle part of the 1 mm thick wall of the resin cup. The phospholipid solution (about 2 μL) was applied to a circular area with a diameter of 5 mm around the small hole on both sides of the small hole (centered on the center of the small hole) using a No. 00 sable brush and allowed to dry. 1 mL of 10 mM Tris-HCl (containing 1 M KCl, 1 mM EDTA, pH 8.0) electrolyte was added to each of the two cells (CIS cell and trans cell) (ensuring that the liquid level is higher than the position of the small hole). Ag / AgCl electrodes were inserted into both cells. The trans cell electrode was connected to the positive electrode of the instrument headstage, and the cis cell electrode was connected to the negative electrode of the headstage (grounded). The single-channel current amplifier Axon200B and analog-to-digital converter 1550B were turned on. A 1 mL syringe was inserted into the bottom of the cis cell, and a phospholipid bilayer film was formed on the pores by slowly drawing out the solution (observing when the instrument current value was 0) and then slowly injecting the solution (observing the current; the current became 0 at the instant of film formation). Then, 0.5 μL of Aerolysin protein solution (20 μg / mL) was added to the cis cell near the pores, and a voltage of +200 mV was applied to wait for the pores to assemble and embed. Once a single Aerolysin nanopore was assembled, the observed ionic current became about 50 pA.

[0051] Example 4. Detection of labeled sugar chains using Aerolysin nanopores

[0052] (1) The glycan sample was dissolved in 10mM Tris-HCl buffer (containing 1M KCl, 1mM EDTA, pH 8.0) to a concentration of 2mM. 1μL of the glycan solution was added to the CIS cell electrolyte to achieve a final concentration of 2μM. The instrument was set to a voltage of +100mV, a sampling rate of 250kHz, and a low-pass filter of 5kHz to begin acquiring the glycan blocking current signal.

[0053] (2) Extract the pulse current magnitude and time from the recorded blocking current pulse signal to obtain the blocking current ratio (Iblocking current). b / I0, where I b The blocking current is represented by I0, the open-circuit current (i.e., the constant baseline current obtained at +100mV before the addition of the glycan sample), and the dwell time is represented by the dwell time. A scatter plot is then created with the dwell current ratio on the x-axis and the logarithm of the dwell time on the y-axis. For the dwell current ratio and dwell time, Gaussian distribution fitting is performed using OriginPro 2021 (OriginLab, Northampton, Massachusetts, USA) to obtain the average dwell current and average dwell time specific to the glycans. Where:

[0054] The characteristic blocking current ratio of 4-p-methoxyphenoxyaniline-labeled 6'-sialic acid lactose (6SL) glycan was 0.423, and the blocking time was 0.927 ms.

[0055] The mean blocking current ratio of 4-p-methoxyphenoxyaniline-labeled 3'-sialic acid lactose (3SL) glycans was 0.542, and the mean blocking time was 0.787 ms.

[0056] The mean blocking current ratio of 4-p-methoxyphenoxyaniline-labeled 3'-sialic acid galactose (3SG) glycans was 0.676, and the mean blocking time was 0.918 ms.

[0057] The characteristic blocking current ratio of 4-p-methoxyphenoxyaniline-labeled sialyl lactose-N-tetrasaccharide a (LSTa) glycan was 0.455, and the average blocking time was 0.901 ms.

[0058] The characteristic blocking current ratio of the lactose sugar chain labeled with the composite tag 6SL-diphenyl ether was 0.400, and the blocking time was 0.876 ms.

[0059] The characteristic blocking current ratio of the 2'-fucosyl lactose (2FL) glycan labeled with the complex tag 6SL-diphenyl ether was 0.277, and the blocking time was 0.916 ms.

[0060] The characteristic blocking current ratio of the 3'-fucosyl lactose (3FL) glycan labeled with the compound tag 6SL-diphenyl ether was 0.346, and the blocking time was 0.909 ms.

[0061] The characteristic blocking current ratio of the 6SL-diphenyl ether-labeled lactose-N-neotetrasaccharide (LNnT) glycan chain was 0.280, and the blocking time was 0.938 ms.

[0062] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for detecting single-molecule glycan chains based on Aerolysin nanopores, characterized in that: (1) The sugar chain is labeled with a tag containing an aromatic ring group; (2) Assemble a phospholipid bilayer and embed Aerolysin protein to form a single nanopore; (3) Add the labeled sugar chains to the buffer solution of the nanopore detection device, connect the instrument, and collect the single-molecule current signal of the sugar chains; (4) Statistical analysis of single-molecule sugar signals is performed to obtain sugar characteristic signals, and different sugar chains are detected and analyzed based on these signals; The sugar chain in step (1) comprises one or more of the following: neutral sugar chains or acidic sugar chains composed of 2 to 20 monosaccharides with reduced sugars at the ends. The monosaccharides are one or more of the following: sialic acid, galactose, glucose, N-acetylgalactose, N-acetylglucose, fucose, mannose, xylose, glucuronic acid, and iduronic acid. The acidic sugar chain or the neutral sugar chain is either a straight-chain type without branches or a bibranched type with one branch. The label containing aromatic ring groups refers to a label containing one or more of the following aromatic ring groups: primary amine, hydrazine or hydroxylamine. The labeling method of the sugar chain in step (1) is a reductive amination reaction, that is, using one of the primary amine, hydrazine or hydroxylamine groups connected by the aromatic ring groups to react with the hemiacetal hydroxyl group at the reducing end of the sugar chain, and then reducing it with sodium cyanoborohydride. The aromatic ring group is one of benzene, diphenyl ether, biphenyl, naphthalene, anthracene, pyrene, or tetraphenylethylene and various neutral substitutes.

2. The method for detecting single sugar molecules according to claim 1, characterized in that: The branch length of a bibranched sugar chain is 1 to 9 monosaccharides.

3. The method for detecting single sugar molecules according to claim 1, characterized in that: (1) Targeting acidic sialic acid sugar chains, The specific structural formulas for the labeled reaction and the label are as follows: ； R1 is a monosaccharide or oligosaccharide, which forms an oligosaccharide chain with the terminal reducing monosaccharide; The R2 substituent is one or more of hydrogen, C1-C5 alkane group, methoxy group, nitro group or cyano group; Or, (2) For neutral sugar chains, sialic acid sugar chains are used as the "locomotive" to react with compounds containing aromatic ring groups, such as those containing bis-primary amines, dihydrazides, or dihydroxyamines. The resulting derivatives are used as a composite tag to undergo a reduction amination reaction with the hemiacetal hydroxyl group at the reducing end of the neutral sugar chain to achieve labeling. The specific structural formulas for the labeled reaction and the label are as follows: ； R1 is a monosaccharide or oligosaccharide, which together with the terminal reducing monosaccharide forms a complete oligosaccharide chain.

4. The method for detecting single sugar molecules according to claim 3, characterized in that: The specific reaction of the labeling reaction is reductive amination. The reaction process involves dissolving the sugar chain molecule, the tag molecule and sodium cyanoborohydride in anhydrous methanol, heating at 65°C overnight, and then separating by liquid chromatography to obtain the target product.

5. The method for detecting single sugar molecules according to claim 1, characterized in that: The phospholipid used in step (2) is diphytylphosphatidylcholine, and the protein channels used are assembled from Aerolysin.

6. The method for detecting single sugar molecules according to claim 1, characterized in that: The detection device in step (3) trans The Ag / AgCl electrode in the cell electrolyte is connected to the headstage probe of an Axon 200B instrument. cis The Ag / AgCl electrode in the electrolyte is connected to the ground terminal; labeled sugar molecules are added to... cis In the pool; The instrument applies a voltage of +70 ~ +150 mV, a sampling rate of 100 ~ 250 kHz, and a low-pass filter of 5 ~ 10 kHz.

7. The method for detecting single sugar molecules according to claim 1, characterized in that, The single-molecule signal of the sugar chain in step (4) includes blocking current and blocking time, wherein different sugar chains correspond to different blocking current magnitudes and different blocking times.

Images