Oligonucleotide modified vector as well as preparation method and application thereof

Abstract

The invention discloses an oligonucleotide modified carrier as well as a preparation method and application thereof, and particularly relates to a polymer, a polymer modified carrier, an oligonucleotide modified carrier as well as a preparation method and application thereof. In the oligonucleotide modified vector, oligonucleotide can be connected to the surface of the vector in a specific connection form, and the oligonucleotide is not easy to fall off and has good stability while the oligonucleotide load is relatively high, so that the oligonucleotide modified vector can be successfully applied to DNA template-free synthesis reaction and the like.

Description

Technical Field

[0001] The present invention relates to an oligonucleotide modified carrier and a preparation method and application thereof, and in particular to a polymer, a polymer modified carrier, an oligonucleotide modified carrier and a preparation method and application thereof. Background Art

[0002] Template-free DNA synthesis is a key technology in synthetic biology, used to construct DNA sequences with specific functions. However, traditional synthesis methods are limited in specificity and efficiency, especially when synthesizing complex sequences. Magnetic beads, as a sample processing and enrichment tool, can improve the efficiency and specificity of template-free synthesis.

[0003] In the field of oligonucleotide modification, surface polymer brush synthesis technology has gradually gained attention. Surface polymer brushes are a dense layer of polymer chains that form a uniform, ordered coating on the surface of magnetic beads. After oligonucleotide modification, these polymer brushes can achieve a high density of oriented oligonucleotides on the surface of the beads, thereby enhancing specific binding and synthesis efficiency. However, the application of this technology in the field of template-free DNA synthesis remains relatively limited and has not yet been fully developed and explored.

[0004] At present, the technical solutions for oligonucleotide-modified microspheres generally include active group modification, click chemistry reaction, biotin-avidin / streptavidin connection, and single-stranded DNA capture probe connection.

[0005] Active group modification: Active groups such as amino, hydroxyl or glycidyl groups are introduced on the surface of the microspheres, and then the oligonucleotides are covalently linked to these groups using cross-linkers or coupling reagents. Coupling reagents include polyethylene glycol succinimide active ester, glutaraldehyde, terephthalaldehyde, etc.

[0006] Click chemistry reaction: Using click chemistry reactions, such as copper-catalyzed alkyne-azide cyclization (CuAAC), alkyne or azide groups are introduced onto the surface of the microspheres, and click reactions occur with the corresponding alkyne or azide compounds to immobilize the oligonucleotides on the microspheres.

[0007] Biotin-avidin / streptavidin linkage: Biotin groups are modified on the surface of microspheres, and then avidin / streptavidin is modified onto the microspheres using the biotin-avidin interaction. The biotin-modified oligonucleotide molecules are then captured through the biotin-avidin interaction.

[0008] Single-stranded DNA capture probe ligation: A single-stranded DNA capture probe (such as polyT) is attached to the microsphere surface by a chemical bond ligation method, and the oligonucleotide sequence is captured by hybridization between the capture probe and the reverse complementary sequence in the oligonucleotide.

[0009] Among them, most microspheres currently used for template-free DNA synthesis rely on streptavidin-biotin interactions or polyT-modified microsphere surfaces as capture probes to capture the desired oligonucleotides. However, these non-covalent interactions can significantly dislodge attached oligonucleotides under certain conditions, such as heating or the presence of dissociating agents. Furthermore, streptavidin magnetic beads tend to stick together under conditions such as ultrasound, vigorous shaking, and heating, hindering the redispersion of the microspheres. Other attachment methods, such as succinimide-amino coupling, epoxy-amino coupling, and aldehyde-amino coupling, readily react with the amino groups of the oligonucleotide ribonucleotide bases, resulting in random oligonucleotide attachment patterns. This hinders oligonucleotide-enzyme binding during enzymatic template-free DNA synthesis, reducing reaction success and product accuracy. Traditional silane-based surface modification methods have limited modification density and relatively low oligonucleotide loading capacity. In template-free DNA synthesis, microspheres and attached oligonucleotides undergo multiple rounds of reaction, washing, separation, and redispersion. Therefore, stable attachment is a key requirement for oligonucleotide-modified microspheres.

[0010] Therefore, there is an urgent need for oligonucleotide-modified microspheres with high oligonucleotide loading capacity and good stability. Summary of the Invention

[0011] To address the shortcomings of prior art oligonucleotide-modified microspheres, such as the highly random oligonucleotide attachment morphology, poor stability, and low loading capacity, the present invention provides a polymer, a polymer-modified carrier, an oligonucleotide-modified carrier, and methods for preparing and using the same. The oligonucleotide-modified carrier based on this polymer can maintain the attachment morphology of the oligonucleotides on the carrier surface, while ensuring high loading capacity and being difficult to detach. It exhibits excellent stability and can be successfully used in template-free DNA synthesis reactions.

[0012] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0013] The present invention provides a polymer comprising a repeating unit A and a repeating unit B, wherein the repeating unit A is a fragment as shown in Formula 1, and the repeating unit B is a fragment as shown in one or more of Formulas 2-6:

[0014]

[0015]

[0016] The n is the degree of polymerization, which is independently 4-112.

[0017] In some embodiments, the molar ratio of the repeating unit A to the repeating unit B is 1:1-1:20, preferably 1:5-1:20, such as 1:9 or 1:19.

[0018] In some embodiments, in the fragments represented by formula 2-4, the n is independently 4-22.

[0019] In some embodiments, in the fragment represented by Formula 2-4, the n is independently 6-15.

[0020] In some specific embodiments, in the fragment represented by Formula 2, n is 6.

[0021] In some specific embodiments, in the fragment represented by Formula 3, n is 15.

[0022] In some specific embodiments, in the fragment represented by formula 4, n is 8.

[0023] In some embodiments, the average molecular weight of the fragments represented by Formulae 2-4 is independently 300-5000 Da, preferably 300-1000 Da.

[0024] In some specific embodiments, the average molecular weight of the fragment represented by Formula 2 is 336 Da.

[0025] In some specific embodiments, the average molecular weight of the fragment represented by Formula 3 is 878 Da.

[0026] In some specific embodiments, the average molecular weight of the fragment represented by Formula 4 is 438 Da.

[0027] In some specific embodiments, the average molecular weight of the fragment represented by Formula 2 is 400 Da.

[0028] In some specific embodiments, the average molecular weight of the fragment represented by Formula 4 is 480 Da.

[0029] The present invention also provides a method for preparing a polymer, which comprises the following steps:

[0030] The polymer is prepared by copolymerization of monomer A and monomer B; wherein,

[0031] The monomer A is glycidyl methacrylate;

[0032] The monomer B is one or more of polyethylene glycol acrylate, polyethylene glycol methacrylate, methoxy polyethylene glycol acrylate, sodium acrylate and vinyl acetate.

[0033] In some embodiments, the polymer is as described above.

[0034] Among them, the fragment represented by formula 1 can be derived from glycidyl methacrylate; the fragment represented by formula 2 can be derived from polyethylene glycol acrylate; the fragment represented by formula 3 can be derived from polyethylene glycol methacrylate; the fragment represented by formula 4 can be derived from methoxy polyethylene glycol acrylate; the fragment represented by formula 5 can be derived from sodium acrylate; and the fragment represented by formula 6 can be derived from vinyl acetate.

[0035] In some embodiments, the molar ratio of monomer A to monomer B is 1:1-1:20, preferably 1:5-1:20, for example 1:9 or 1:19.

[0036] In some embodiments, the average molecular weight of the polyethylene glycol acrylate, polyethylene glycol methacrylate, and methoxy polyethylene glycol acrylate is independently 300-5000 Da, preferably 300-1000 Da.

[0037] In some specific embodiments, the polyethylene glycol acrylate has an average molecular weight of 336 Da.

[0038] In some specific embodiments, the polyethylene glycol methacrylate has an average molecular weight of 878 Da.

[0039] In some specific embodiments, the average molecular weight of the methoxy polyethylene glycol acrylate is 438 Da.

[0040] In some specific embodiments, the polyethylene glycol acrylate has an average molecular weight of 400 Da.

[0041] In some specific embodiments, the average molecular weight of the methoxy polyethylene glycol acrylate is 480 Da.

[0042] In some embodiments, the copolymerization reaction is surface-initiated atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer polymerization (RAFT).

[0043] In some embodiments, when the copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction, the copolymerization reaction includes the step of conducting the reaction in a solvent in the presence of a catalyst and a ligand.

[0044] Wherein, the catalyst may be cuprous bromide and / or copper bromide.

[0045] Wherein, the ligand may be 2,2'-bipyridine.

[0046] Wherein, the solvent may be dimethylformamide.

[0047] Wherein, the copolymerization reaction can be carried out in a nitrogen atmosphere.

[0048] Wherein, the temperature of the copolymerization reaction can be room temperature.

[0049] In some embodiments, the catalyst is cuprous bromide and copper bromide in a molar ratio of 1:0.1.

[0050] In some embodiments, the molar ratio of the catalyst to the ligand is 1:2.

[0051] In some embodiments, the molar ratio of total monomers: the catalyst: the ligand is (50-200):1.1:2.2, for example 50:1.1:2.2, wherein the molar ratio of the total monomers is the sum of the molar ratios of the monomer A and the monomer B.

[0052] In some specific embodiments, the monomer A is glycidyl methacrylate, the monomer B is methoxy polyethylene glycol acrylate with an average molecular weight of 480 Da, the catalyst is cuprous bromide and cupric bromide, the ligand is 2,2'-bipyridine, and the molar ratio of each raw material is total monomer: cuprous bromide: cupric bromide: 2,2'-bipyridine = 50:1:0.1:2.2, wherein the molar ratio of the total monomer is the sum of the molar ratios of the monomer A and the monomer B.

[0053] The present invention also provides a polymer, which is prepared according to the polymer preparation method.

[0054] The present invention also provides a polymer-modified carrier, which comprises the polymer as described above and a carrier whose surface is modified with a connecting functional group A, wherein the polymer is covalently connected to the connecting functional group A.

[0055] In some embodiments, the linking functional group A is as shown in Formula 7:

[0056]

[0057] In some embodiments, the carrier is a conventional solid phase substrate in the art, generally selected from one of microspheres, silicon wafers, silicon dioxide sheets, and anodized aluminum films.

[0058] The microspheres may be magnetic microspheres (magnetic beads) or non-magnetic microspheres, preferably selected from silica microspheres, polystyrene microspheres, agarose microspheres, polyacrylamide microspheres, poly(glycidyl methacrylate) microspheres, silica-coated ferroferric oxide microspheres, polystyrene-coated ferroferric oxide microspheres, and agarose-coated ferroferric oxide microspheres, for example, silica-coated ferroferric oxide microspheres. The particle size of the microspheres may be 200 nm to 20 μm.

[0059] In some embodiments, after the polymer is covalently linked to the linking functional group A, it is wrapped on the surface of the carrier, and the thickness of the wrapping is preferably 25-150 nm, such as 98±23 nm.

[0060] The present invention also provides a method for preparing a polymer-modified carrier, which comprises the following steps:

[0061] The carrier with the surface modified with the connecting functional group A, the monomer A and the monomer B are prepared by copolymerization reaction; the carrier with the surface modified with the connecting functional group A, the monomer A and the monomer B are as described above.

[0062] In some embodiments, the polymer-modified carrier is as described above.

[0063] In some embodiments, the copolymerization reaction is surface-initiated atom transfer radical polymerization or reversible addition-fragmentation chain transfer polymerization.

[0064] In some embodiments, when the copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction, the copolymerization reaction includes the step of conducting the reaction in a solvent in the presence of a catalyst and a ligand.

[0065] Wherein, the catalyst may be cuprous bromide and / or copper bromide.

[0066] Wherein, the ligand may be 2,2'-bipyridine.

[0067] Wherein, the solvent may be dimethylformamide.

[0068] Wherein, the copolymerization reaction can be carried out in a nitrogen atmosphere.

[0069] Wherein, the temperature of the copolymerization reaction can be room temperature.

[0070] In some embodiments, the catalyst is cuprous bromide and copper bromide in a molar ratio of 1:0.1.

[0071] In some embodiments, the molar ratio of the catalyst to the ligand is 1:2.

[0072] In some embodiments, the molar ratio of total monomers: the catalyst: the ligand is (50-200):1.1:2.2, for example 50:1.1:2.2, wherein the molar ratio of the total monomers is the sum of the molar ratios of the monomer A and the monomer B.

[0073] In some specific embodiments, the monomer A is glycidyl methacrylate, the monomer B is methoxy polyethylene glycol acrylate with an average molecular weight of 480 Da, the catalyst is cuprous bromide and cupric bromide, the ligand is 2,2'-bipyridine, and the molar ratio of each raw material is total monomer: cuprous bromide: cupric bromide: 2,2'-bipyridine = 50:1:0.1:2.2, wherein the molar ratio of the total monomer is the sum of the molar ratios of the monomer A and the monomer B.

[0074] In some embodiments, when the copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction, the carrier with a surface modified with a connecting functional group A is prepared by reacting a carrier precursor and an initiator, and the connecting functional group A is, for example, the group shown in Formula 7.

[0075] The carrier precursor may be a product obtained by modifying the hydroxyl groups of an unmodified carrier through activation treatment, but before modification of the linking functional group A. The activation treatment step may include, for example, mixing a sodium hydroxide solution with the unmodified carrier and heating the mixture. The heating reaction temperature may be, for example, 80°C.

[0076] Wherein, the initiator may be 3-(triethoxysilyl)propyl 2-bromo-2-methylpropionate.

[0077] The step of reacting the carrier precursor and the initiator may include: mixing the carrier precursor and the initiator in a solvent, and heating the mixture to react. The solvent is, for example, anhydrous ethanol. The temperature of the heating reaction is, for example, 80°C.

[0078] The present invention also provides a polymer-modified carrier, which is prepared according to the above-mentioned preparation method of the polymer-modified carrier.

[0079] The present invention also provides an oligonucleotide modified carrier, which comprises an oligonucleotide with a terminal modified linking functional group B and the aforementioned polymer modified carrier, wherein the linking functional group B and the polymer modified carrier are covalently linked;

[0080] Alternatively, the oligonucleotide modified carrier includes an oligonucleotide with a terminal modified linking functional group B, a linking functional group C and the polymer modified carrier as described above, and the two ends of the linking functional group C are covalently connected to the linking functional group B and the polymer modified carrier respectively.

[0081] In some embodiments, the oligonucleotide is an oligonucleotide whose 5' end is modified with a linking functional group B, for example, an oligonucleotide whose 5' end is modified with a thiol group.

[0082] In some embodiments, the oligonucleotide modified with a thiol group at the 5' end is linked to the repeating unit A of the polymer-modified carrier, for example, the thiol group is linked to the repeating unit A in the following manner:

[0083]

[0084] In some embodiments, the linking functional group C is as shown in Formula 8:

[0085]

[0086] In some embodiments, the oligonucleotide modified with a thiol group at the 5' end is connected to the repeating unit A of the polymer-modified carrier via the linking functional group C. For example, the thiol group and the repeating unit A are connected via the linking functional group C in the following manner:

[0087]

[0088] In some embodiments, the oligonucleotide modified vector comprises:

[0089] (1) A carrier with a surface modified with a connecting functional group A;

[0090] (2) a polymer covalently linked to the linking functional group A;

[0091] The polymer includes a repeating unit B and a repeating unit C; wherein the repeating unit B is a fragment as shown in one or more of Formulas 2-6, and the repeating unit C is a fragment as shown in Formula 9 or Formula 10:

[0092]

[0093] Wherein, the R is an oligonucleotide.

[0094] In some embodiments, the oligonucleotide is single-stranded DNA.

[0095] In some embodiments, the oligonucleotide is 12-25 bases in length.

[0096] In some specific embodiments, the sequence of the oligonucleotide is SH-5'-GCTACTAGGACGACTCGAATG-3' [SEQ ID NO: 1].

[0097] In some embodiments, the sequence of the oligonucleotide further contains an enzyme recognition cleavage site; the enzyme recognition cleavage site is preferably a uracil DNA glycosylase (UDG enzyme) cleavage site deoxyuridine (dU).

[0098] In some specific embodiments, the sequence of the oligonucleotide is SH-5'-AGT / ideoxyU / GCTACTAGGACGACTCGAATG-3' [SEQ ID NO: 2].

[0099] In some embodiments, the oligonucleotide content in the oligonucleotide-modified carrier is 0-3500 pmol / mg, and is not 0; for example, 850±24 pmol / mg or 604±31 pmol / mg.

[0100] The present invention also provides a method for preparing an oligonucleotide-modified vector, which comprises the following steps:

[0101] The oligonucleotide and the polymer modified carrier react to obtain the product;

[0102] Alternatively, the oligonucleotide and cystamine salt solution are mixed, a reducing agent is added, and then reacted with a polymer-modified carrier to obtain the oligonucleotide;

[0103] The oligonucleotide and the polymer-modified carrier are as described above.

[0104] In some embodiments, the oligonucleotide modified vector is as described above.

[0105] In some embodiments, the oligonucleotide is covalently linked to the polymer-modified carrier via a CS bond, wherein the CS bond is formed by the reaction of the linking functional group A (epoxy group) in the repeating unit A and the linking functional group C (thiol group) at the end of the oligonucleotide; alternatively, the oligonucleotide is covalently linked to the polymer-modified carrier via an SS bond, wherein the SS bond is formed by the reaction of the linking functional group A (epoxy group) in the repeating unit A and the cystamine group, followed by reduction of the disulfide bond by a reducing agent and then reaction with the linking functional group C (thiol group) at the end of the oligonucleotide.

[0106] In some embodiments, the reducing agent is TCEP.

[0107] In some embodiments, the ratio of the oligonucleotide to the polymer-modified carrier is 10 nmol:10 mg.

[0108] In some embodiments, the reaction steps are conventional in the art, such as stirring the reaction in a solvent at room temperature for 24 hours or 48 hours. When the reaction system contains a cystamine salt solution, the reaction steps can be stirring the reaction at room temperature for 48 hours.

[0109] The present invention also provides an oligonucleotide modified vector, which is prepared according to the above-mentioned method for preparing the oligonucleotide modified vector.

[0110] The present invention also provides an application of the oligonucleotide modified vector as described above in DNA template-free synthesis.

[0111] In some embodiments, the application comprises: mixing the oligonucleotide modified carrier with a DNA template-free synthesis reaction solution, and reacting to obtain the product.

[0112] The DNA template-free synthesis reaction solution can be conventional in the art, and generally includes a reaction buffer solution, terminal deoxynucleotidyl transferase (TdT enzyme) and deoxyribonucleotides.

[0113] Preferably, the reaction buffer solution comprises one or more of Tris-HCl, sodium chloride, cobalt chloride and Tween 20.

[0114] Preferably, the deoxyribonucleotide is a natural or 3'-end blocked deoxyribonucleotide, such as dideoxyguanosine 5'-triphosphate (ddGTP).

[0115] In some specific embodiments, the DNA template-free synthesis reaction solution includes 50 mM Tris-HCl, 100 mM sodium chloride, 0.25 mM cobalt chloride, 0.1% Tween 20, 1 U / μL terminal deoxynucleotidyl transferase and 0.25 mM dideoxyguanosine 5'-triphosphate.

[0116] The reaction steps may be conventional in the art, such as incubation at room temperature for 5 minutes.

[0117] The present invention also provides a use of the oligonucleotide modified carrier as described above in preparing DNA hybridization probe magnetic beads.

[0118] The present invention also provides a use of the oligonucleotide modified carrier as described above in the preparation of specific DNA / RNA capture magnetic beads.

[0119] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0120] The reagents and raw materials used in the present invention are commercially available.

[0121] The positive progress effect of the present invention is:

[0122] The polymer of the present invention is obtained by polymerizing two specific monomers. When the polymer-modified carrier is further covalently linked to an oligonucleotide to obtain an oligonucleotide-modified carrier and used in a DNA template-free synthesis reaction, it has the following advantages:

[0123] 1. The specificity of the covalent linking reaction is achieved through thiol-based chemical reactions such as epoxy-thiol addition / disulfide exchange, ensuring that the oligonucleotides on the carrier surface are in an upright connection form, which is conducive to improving the success rate of the synthesis reaction. It is not easy to fall off and has good stability, which is suitable for various synthesis scenarios and conditions;

[0124] 2. The polymer utilizes radial space to transform the oligonucleotide distribution from two-dimensional to three-dimensional on the carrier surface, which can increase the modification density of the oligonucleotide and reduce the carrier cost in synthesis.

[0125] 3. The modification density of oligonucleotides in the obtained oligonucleotide-modified carrier is more uniform, and the distribution of oligonucleotide loading is more concentrated.

[0126] 4. The vector and oligonucleotide are connected in a reversible or irreversible manner. The reversible manner can easily replace the modified oligonucleotide primer, allowing the vector to be reused multiple times in the synthesis, further reducing the vector cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 Schematic diagram of the process of synthesizing polymers on the surface of magnetic beads according to the present invention.

[0128] Figure 2 Schematic diagram of the process of irreversibly linking oligonucleotides to the surface of polymer-modified magnetic beads according to the present invention.

[0129] Figure 3 Schematic diagram of the process of reversibly linking oligonucleotides to the surface of polymer-modified magnetic beads according to the present invention.

[0130] Figure 4 This is a particle size distribution diagram of the polymer-modified magnetic beads in Example 1 at different reaction times.

[0131] Figure 5 This is the standard curve for measuring the oligonucleotide loading in Example 2.

[0132] Figure 6 These are the electrophoresis results of DNA template-free synthesis products using oligonucleotide-modified magnetic beads obtained with different monomer ratios in Examples 5-8 in Example 1. DETAILED DESCRIPTION

[0133] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0134] Unless otherwise specified, in the following examples and effect examples, the storage buffer A is a pH 7.2 aqueous solution containing 50 mM Tris-HCl, 100 mM sodium chloride, and 0.1% Tween 20, and the percentages here are by mass.

[0135] Example 1 Preparation of polymer-modified magnetic beads

[0136] like Figure 1 FIG. 1 is a schematic diagram of the process of synthesizing a polymer on the surface of a magnetic bead according to the present invention, which specifically includes the following steps:

[0137] 1. Magnetic bead surface activation

[0138] Disperse 10 mg of 500 nm silica-coated ferroferric oxide magnetic beads in 10 mL of deionized water, separate the beads using a magnetic field, remove the supernatant, and repeat the wash three times. Rinse the beads and disperse them in 10 mL of 0.1 M sodium hydroxide solution. Add the solution to a three-necked flask, heat at 80°C, reflux, and stir for 30 minutes. After the reaction is terminated, separate the beads using a magnetic field, remove the supernatant, and rinse three times with 10 mL of deionized water and twice with anhydrous ethanol. Redisperse the beads in 1 mL of anhydrous ethanol to obtain a dispersion of activated magnetic beads.

[0139] 2. Initiator (containing bromine group) modification

[0140] Add 1 mL of the activated magnetic bead dispersion obtained in step 1 to 9 mL of anhydrous ethanol, add 0.2 mL of 3-(triethoxysilyl)propyl 2-bromo-2-methylpropionate, heat under reflux at 80°C, and stir to react for 24 hours. After the reaction, separate the magnetic beads using a magnetic field, remove the supernatant to remove unreacted 3-(triethoxysilyl)propyl 2-bromo-2-methylpropionate, wash with anhydrous ethanol three times, and wash with dimethylformamide twice. The washed magnetic beads are redispersed in 1 mL of dimethylformamide to obtain a dispersion of initiator-modified magnetic beads.

[0141] 3. Surface-initiated atom transfer radical polymerization

[0142] Add 9 mL of dimethylformamide to a three-necked flask, connect it to a Schlenk line, and perform three vacuum-nitrogen cycles to remove dissolved oxygen from the solvent. Weigh 4.5 mg of copper bromide (0.02 mmol), 28.7 mg of cuprous bromide (0.2 mmol), and 68.7 mg of 2,2'-bipyridine (0.44 mmol) into the three-necked flask. Perform three vacuum-nitrogen cycles to remove oxygen, and stir for 5 minutes to form a brown solution. Add two polymer monomers (total 10 mmol) at a molar ratio of 1:9 (glycidyl methacrylate and methoxypolyethylene glycol acrylate (average molecular weight approximately 480 Da, Aladdin P133111-250 mL) to the three-necked flask and stir for 30 minutes. The molar ratio of monomer to catalyst is 50:1:0.1:2.2:total monomer:cuprous bromide:copper bromide:2,2'-bipyridine.

[0143] Then, add 1 mL of the dispersion of initiator-modified magnetic beads from step 2 and react at room temperature under nitrogen for 72 hours with stirring. After the reaction, separate the beads using a magnetic field, remove the supernatant to remove unreacted monomer and catalyst, rinse three times with deionized water, and redisperse in 1 mL of deionized water to obtain a dispersion of polymer-modified magnetic beads.

[0144] Example 2 Preparation of polymer-modified magnetic beads

[0145] The other conditions were kept the same as those in Example 1, and only the ratio of the polymer monomers in step 3 was changed to a molar ratio of glycidyl methacrylate to methoxy polyethylene glycol acrylate of 1:1, to prepare a dispersion of polymer-modified magnetic beads of this example.

[0146] Example 3 Preparation of polymer-modified magnetic beads

[0147] The other conditions were kept the same as those in Example 1, and only the ratio of the polymer monomers in step 3 was changed to a molar ratio of glycidyl methacrylate to methoxy polyethylene glycol acrylate of 1:4, to prepare a dispersion of polymer-modified magnetic beads of this example.

[0148] Example 4 Preparation of polymer-modified magnetic beads

[0149] The other conditions were kept the same as those in Example 1, and only the ratio of the polymer monomers in step 3 was changed to a molar ratio of glycidyl methacrylate to methoxy polyethylene glycol acrylate of 1:19, to prepare a dispersion of polymer-modified magnetic beads of this example.

[0150] Example 5 Preparation of oligonucleotide-modified magnetic beads (irreversible linkage)

[0151] like Figure 2FIG. 1 is a flow chart of the process of irreversibly attaching oligonucleotides to the polymer-modified magnetic beads obtained in Example 1 to prepare oligonucleotide-modified magnetic beads according to the present invention, which specifically includes the following steps:

[0152] (1) 1 mL of the dispersion of polymer-modified magnetic beads obtained in Example 1 (containing 10 mg of magnetic beads) was used to separate the magnetic beads using a magnetic field, the supernatant was removed, and the beads were dispersed in 900 μL of PBS buffer solution with a pH of 6.8 to obtain a magnetic bead dispersion.

[0153] (2) Disperse 10 nmol of a 5'-terminus thiol-modified single-stranded DNA oligonucleotide in 90 μL of PBS buffer at pH 6.8 to obtain a single-stranded DNA oligonucleotide solution. Dissolve tris(carboxyethyl)phosphine hydrochloride in PBS buffer at pH 6.8 to obtain a 1 mM tris(carboxyethyl)phosphine hydrochloride solution. Add 10 μL of the tris(carboxyethyl)phosphine hydrochloride solution to the 90 μL single-stranded DNA oligonucleotide solution and shake at room temperature for 10 minutes to break any disulfide bonds formed between the oligonucleotides and expose the thiol groups.

[0154] (3) After the reaction of step (2) is completed, the single-stranded DNA oligonucleotide solution obtained by the reaction is mixed with the magnetic bead dispersion in step (1) to a total volume of 1 mL. With the help of a pH meter, the pH of the mixture is adjusted to about 9 using a 1 M potassium hydroxide solution. The solution is stirred at room temperature and reacted for 24 hours. After the reaction is completed, the magnetic beads are separated using a magnetic field, the supernatant is removed, the unreacted oligonucleotide is removed, the beads are washed three times with deionized water, and the beads are redispersed in magnetic bead storage buffer A to obtain an oligonucleotide-modified magnetic bead dispersion with a concentration of 10 mg / mL.

[0155] The oligonucleotide sequence used in this example is: SH-5′-AGT / ideoxyU / GCTACTAGGACGACTCGAATG-3′ [SEQ ID NO: 2].

[0156] To track the reaction conversion rate of the oligonucleotide in step (3), the present invention collected 100 μL of the mixture at 0, 1, 2, 4, 8, 12, and 24 hours, separated the magnetic beads using a magnetic field, collected the supernatant, and measured the concentration of unreacted oligonucleotide in the mixture using a UV spectrophotometer. The results are shown in Table 1. After 24 hours of reaction, the amount of oligonucleotide remaining in the mixture was <10%.

[0157] Table 1. Changes in the amount of oligonucleotide remaining in the reaction solution as a function of reaction time

[0158] Response time / hour Remaining oligonucleotide concentration in solution / μM 0 10±0 1 7.23±0.19 2 4.89±0.27 4 3.05±0.34 8 1.75±0.26 12 1.1±0.31 24 0.5±0.25

[0159] Example 6 Preparation of Oligonucleotide-Modified Magnetic Beads (Irreversible Linkage)

[0160] According to the conditions in Example 5, the polymer-modified microspheres in Example 2 were used to modify the oligonucleotides to prepare the oligonucleotide-modified magnetic bead dispersion of this example.

[0161] Example 7 Preparation of Oligonucleotide-Modified Magnetic Beads (Irreversible Linkage)

[0162] According to the conditions in Example 5, the polymer-modified microspheres in Example 3 were used to modify the oligonucleotides to prepare the oligonucleotide-modified magnetic bead dispersion of this example.

[0163] Example 8 Preparation of Oligonucleotide-Modified Magnetic Beads (Irreversible Linkage)

[0164] According to the conditions in Example 5, the polymer-modified microspheres in Example 4 were used to modify the oligonucleotides to prepare a dispersion of oligonucleotide-modified magnetic beads of this example.

[0165] Example 9 Preparation of Oligonucleotide-Modified Magnetic Beads (Reversible Linkage)

[0166] like Figure 3 FIG. 1 is a schematic diagram of a process for preparing oligonucleotide-modified magnetic beads by reversibly attaching oligonucleotides to the polymer-modified magnetic beads obtained in Example 2 of the present invention, which specifically includes the following steps:

[0167] (1) 1 mL of the dispersion of polymer-modified magnetic beads obtained in Example 2 (containing 10 mg of magnetic beads) was separated by magnetic field, the supernatant was removed, and the beads were dispersed in 1 mL of PBS buffer at a pH of 6.8 to obtain a magnetic bead dispersion. Cystamine hydrochloride was dissolved in a PBS buffer at a pH of 6.8 to obtain a 10 mM cystamine solution, and the pH of the solution was adjusted to about 7 using potassium hydroxide with the help of a pH meter. 100 μL of cystamine solution was added to the magnetic bead dispersion, and the reaction was shaken at room temperature for 3 hours. After the reaction was completed, the magnetic beads were separated by magnetic field, the supernatant and unreacted cystamine were removed, the beads were washed 3 times with deionized water, and the beads were redispersed in 1 mL of PBS buffer at a pH of 6.8 to obtain a magnetic bead dispersion.

[0168] (2) Dissolve tris(carboxyethyl)phosphine hydrochloride in PBS buffer at pH 6.8 to obtain a 100 mM tris(carboxyethyl)phosphine hydrochloride solution. Add 100 μL of tris(carboxyethyl)phosphine hydrochloride solution to 1 mL of the magnetic bead dispersion obtained in step (1), and shake the reaction at room temperature for 10 minutes to break the disulfide bond in cystamine and obtain exposed sulfhydryl groups. After the reaction, separate the magnetic beads using a magnetic field, remove the supernatant, rinse three times with deionized water, and redisperse in 900 μL of sodium bicarbonate buffer at pH 8 and 1 M to obtain a magnetic bead dispersion.

[0169] (3) 10 nmol of a 5'-terminus modified thiol single-stranded DNA oligonucleotide was dispersed in 100 μL of a 1 M sodium bicarbonate buffer solution at pH 8, and mixed with the magnetic bead dispersion obtained in step (2) to obtain a mixed solution. The mixture was stirred and reacted at room temperature for 48 hours. After the reaction, the magnetic beads were separated using a magnetic field, the supernatant was removed, and the unreacted oligonucleotide was removed. The beads were washed three times with deionized water and redispersed in magnetic bead storage buffer A to obtain oligonucleotide-modified magnetic beads at a concentration of 10 mg / mL.

[0170] The oligonucleotide sequence used in this example is: SH-5′-AGT / ideoxyU / GCTACTAGGACGACTCGAATG-3′ [SEQ ID NO: 2].

[0171] To track the reaction conversion rate of the oligonucleotide in step (3), the present invention collected 100 μL of the mixture at 0, 1, 2, 4, 8, 12, 24, and 48 hours, separated the magnetic beads using a magnetic field, collected the supernatant, and measured the concentration of unreacted oligonucleotide in the mixture using a UV spectrophotometer. As shown in Table 2, after 48 hours of reaction, the amount of oligonucleotide remaining in the mixture was <30%.

[0172] Table 2. Changes in the amount of oligonucleotide remaining in the reaction solution as a function of reaction time

[0173] Response time / hour Remaining oligonucleotide concentration in solution / μM 0 10±0 1 8.34±0.16 2 7.13±0.21 4 6.05±0.35 8 4.61±0.37 12 3.72±0.25 24 2.81±0.41 48 2.25±0.45

[0174] Effect Example 1 Characterization of relevant structural parameters of polymer-modified microspheres

[0175] To monitor the change in the length of the surface-initiated atom transfer radical polymerization reaction time of the polymer in Examples 1-4, the present invention took out 0.5 mL of the magnetic bead dispersion in progress or after the reaction at 0 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours, respectively. The magnetic beads were separated using a magnetic field, the supernatant was removed, and the beads were washed three times with deionized water. The beads were redispersed in 1 mL of deionized water, and the change in the magnetic bead particle size was measured using a dynamic light scattering particle size analyzer.

[0176] like Figure 4 As shown in Table 3, the particle size of the magnetic beads in Example 1 (monomer molar ratio of 1:9) increased with reaction time, and the particle size distribution slightly broadened. After 72 hours of reaction, the resulting polymer thickness was approximately 98 ± 23 nm. Furthermore, as shown in Table 3, increasing the glycidyl methacrylate ratio (monomer molar ratios of 1:1 and 1:4) slowed the polymer growth rate and exhibited significant fluctuations in thickness. The polymer-modified magnetic beads with a monomer ratio of 1:1 exhibited significant aggregation and wall adhesion.

[0177] Table 3. Changes in particle size of polymer-modified magnetic beads obtained in Examples 1-4 as a function of reaction time

[0178] Reaction time <![CDATA[Particle size 1:1 > <![CDATA[Particle size 1:4 > <![CDATA[Particle size 1:9 > <![CDATA[Particle size 1:19 > 0h 507.85±11.9 504.85±12.9 502.85±13.9 501.85±11.9 8h 515.78±17.56 521.78±15.56 527.78±16.56 534.78±15.56 24h 533.63±20.87 542.63±19.87 557.63±18.87 577.63±16.87 48h 557.85±35.15 567.85±20.15 578.85±22.15 605.85±17.15 72h 579.35±49.35 589.35±23.35 600.35±22.35 625.35±19.35

[0179] Effect Example 2 Analysis of oligonucleotide loading capacity of oligonucleotide-modified microspheres

[0180] Take the oligonucleotide-modified magnetic beads in Examples 5-9 respectively, and perform oligonucleotide loading analysis according to the following steps:

[0181] Take 10 μL of oligonucleotide modified magnetic bead dispersion (containing 0.1 mg magnetic beads) with a concentration of 10 mg / mL in Example 5-9 respectively, add 100 μL of storage buffer A, separate the magnetic beads using a magnetic field, remove the supernatant, and redisperse the magnetic beads in 100 μL of storage buffer A to obtain a magnetic bead dispersion. 2 μL of UDG enzyme (Shanghai Biotech) with a concentration of 2 U / μL was added to the magnetic bead dispersion, and the mixture was incubated at 50°C for 5 minutes after pipetting. After the reaction was completed, the enzyme was inactivated by incubation at 95°C for 10 minutes. The magnetic beads were separated using a magnetic field and the supernatant was collected. A standard curve was established using an ultraviolet spectrophotometer with a 0.1-10 μM reference oligonucleotide (the same sequence as the modified oligonucleotide after cleavage) solution. Five groups of supernatants were tested, and the oligonucleotide concentration in the supernatant was calculated using the standard curve to deduce the oligonucleotide loading on the magnetic beads.

[0182] The reference oligonucleotide sequence is: 5'-GCTACTAGGACGACTCGAATG-3' [SEQ ID NO: 3]. The obtained standard curve is as follows Figure 5 The corresponding specific data are shown in Table 4.

[0183] Table 4. Specific values ​​of the standard curve for oligonucleotide loading determination

[0184]

[0185]

[0186] The results are as follows:

[0187] According to the UV spectrophotometer data, the oligonucleotide loading capacity of the oligonucleotide-modified magnetic beads (irreversibly linked) of Example 5 was measured to be 850±24 pmol / mg; the oligonucleotide loading capacity of the oligonucleotide-modified magnetic beads (reversibly linked) of Example 9 was measured to be 604±31 pmol / mg.

[0188] Furthermore, the saturated loading capacity of oligonucleotides on oligonucleotide-modified magnetic beads obtained with different monomer ratios in Examples 5-8 is shown in Table 5. The results show that when the ratio of the spacer monomer polyethylene glycol methacrylate is lower than 90%, the saturated oligonucleotide loading capacity of different batches of magnetic beads has large errors due to the inability of the surface epoxy groups to react completely.

[0189] Table 5. Comparison of saturated oligonucleotide loading capacity of oligonucleotide-modified magnetic beads obtained in Examples 5-8

[0190] Monomer ratio (GMA:PEGMA) Oligonucleotide loading (pmol / mg) 1:1 3378±347 1:4 1543±141 1:9 850±24 1:19 463±15

[0191] Furthermore, as shown in Table 6, compared with commercial oligonucleotide-modified magnetic beads, the oligonucleotide loading capacity of the polymer-modified magnetic beads of the present invention connected in an irreversible (Example 5) or reversible (Example 9) connection manner is better than that of commercial oligonucleotide-modified magnetic beads. Among them, compared with carboxyl magnetic beads, the oligonucleotide loading capacity of the oligonucleotide-modified magnetic beads of the present invention is about 4 to 6 times that of carboxyl magnetic beads.

[0192] The dT magnetic beads were from Thermo Fisher, Dynabeads Oligo(dT)25, and the captured DNA sequence was 5'-AGT / ideoxyU / GCTACTAGGACGACTCGAATGAAAAAAAAAAAAAAA-3' [SEQ ID NO: 4], which were prepared by incubation at 37°C for 1 hour. The streptavidin magnetic beads were from Thermo Fisher, Dynabeads M-270 streptavidin, and the captured DNA sequence was biotin-5'-AGT / ideoxyU / GCTACTAGGACGACTCGAATG-3' [SEQ ID NO: 5], which were prepared by incubation at 37°C for 1 hour. The carboxyl magnetic beads were from Thermo Fisher, Dynabeads M-270 carboxylic acid, and the captured DNA sequence was NH2-5'-AGT / ideoxyU / GCTACTAGGACGACTCGAATG-3' [SEQ ID NO: 6], which were ligated via an EDC / NHS reaction.

[0193] Table 6. Comparison of oligonucleotide loading capacity of oligonucleotide-modified magnetic beads obtained in the present invention and commercially available magnetic beads

[0194] Oligonucleotide loading (pmol / mg) dT magnetic beads 70 Streptavidin magnetic beads 600 Carboxyl magnetic beads 150 Polymer-modified magnetic beads—reversible 650 Polymer-modified magnetic beads—irreversible 850

[0195] Effect Example 3 Stability Analysis of Oligonucleotide Modified Microspheres

[0196] To verify the stability difference between the oligonucleotide-modified magnetic beads of Examples 5 and 9 of the present invention and commercial oligonucleotide-modified magnetic beads, the present invention compared the retention of oligonucleotides loaded in different ways on the magnetic beads at different temperatures. The specific steps are as follows:

[0197] Oligonucleotide-modified magnetic beads (100 pmol oligonucleotide) were washed, diluted, and diluted to 100 μL. The beads were incubated at different temperatures (60°C, 70°C, 80°C, and 90°C) with shaking for 5 min. The beads were separated by a magnetic field, and the supernatant was removed. The beads were washed once with deionized water and redispersed in 100 μL of deionized water. 2 μL of 2 U / μL UDG enzyme was added, mixed thoroughly, and incubated at 50°C for 5 minutes. After the reaction was complete, the beads were incubated at 95°C for 10 minutes to inactivate the enzyme. The beads were separated by a magnetic field, and the supernatant was collected. A standard curve was established using a UV spectrophotometer using 0.1-10 μM reference oligonucleotide solutions (with the same sequence as the modified oligonucleotide after cleavage). Five supernatants were tested. The oligonucleotide concentration in the supernatant was calculated using the standard curve, and the oligonucleotide loading on the beads was deduced.

[0198] As shown in Table 7, beads attached to oligonucleotides via non-covalent linkages (dT, streptavidin) exhibited significantly decreased oligonucleotide retention after high-temperature treatment, particularly at temperatures above 70°C, where retention was less than 60%. In contrast, beads attached to oligonucleotides via covalent linkages (carboxyl groups, polymers) exhibited greater than 98% oligonucleotide retention after high-temperature treatment. These results demonstrate that covalently linked oligonucleotides exhibit greater stability than oligonucleotides attached via non-covalent linkages (e.g., DNA sequence hybridization, streptavidin-biotin).

[0199] Table 7. Comparative results of the stability of the oligonucleotide modified magnetic beads obtained in the present invention and commercially available magnetic beads

[0200]

[0201] Application Example 1: Template-free DNA synthesis based on oligonucleotide-modified magnetic beads

[0202] Dispersions of the four oligonucleotide-modified magnetic beads from Examples 5-8 were prepared. The amount of dispersion required to achieve a 0.1 nmol oligonucleotide loading was calculated based on the oligonucleotide loading capacity. The beads were then dispersed in 100 μL of storage buffer A. The beads were separated using a magnetic field, and the supernatant was removed. 100 μL of the following template-free synthesis reaction solution (using water as the solvent) was added to each dispersion.

[0203] Table 8 Component information of template-free synthesis reaction solution

[0204] Reagents concentration Tris-HCl 50mM Sodium chloride 100mM Cobalt chloride 0.25mM TdT enzyme (NEB) 1U / μL dideoxyguanosine 5'-triphosphate (ddGTP) 0.25mM Twain 20 0.1％

[0205] After mixing by pipetting, the beads were incubated at room temperature for 5 minutes. After the reaction was complete, the beads were separated using a magnetic field, the supernatant was removed, and the beads were washed twice with storage buffer A. The beads were then redispersed in 100 μL of storage buffer A. 2 μL of 2 U / μL UDG enzyme (Shanghai Biotechnology) was added to the magnetic bead dispersion, mixed by pipetting, and incubated at 50°C for 5 minutes. After the reaction was complete, the enzyme was inactivated by incubation at 95°C for 10 minutes. The beads were separated using a magnetic field, and the supernatant was collected.

[0206] As a control group, 12.5 μL of the oligonucleotide-modified magnetic bead dispersion from Example 5 was dispersed in 100 μL of storage buffer A. The magnetic beads were separated using a magnetic field, the supernatant removed, and the beads were washed twice with storage buffer A. The beads were then redispersed in 100 μL of storage buffer A. 2 μL of 2 U / μL UDG enzyme was added to the magnetic bead dispersion, mixed by pipetting, and incubated at 50°C for 5 minutes. After the reaction was complete, the enzyme was incubated at 95°C for 10 minutes to inactivate the enzyme. The magnetic beads were separated using a magnetic field, and the supernatant collected.

[0207] 10 μL of the supernatant was taken for product analysis, 10 μL of 2X TBE-Urea Loading Buffer (Biyuntian) was added, mixed, and loaded onto TBE-urea gel for comparison.

[0208] like Figure 6 As shown, the results in TBE-urea gels indicate that the oligonucleotides loaded on the oligonucleotide-modified magnetic beads of Examples 5 and 8 (monomer ratios of 1:9 and 1:19) can serve as primers for enzymatic DNA template-free synthesis, and no unextended oligonucleotides remain. The oligonucleotides loaded on the oligonucleotide-modified magnetic beads of Examples 6 and 7 (monomer ratios of 1:1 and 1:4) cannot be completely reacted, resulting in obvious unreacted bands.

Claims

1. A polymer, characterized in that The polymer includes a repeating unit A and a repeating unit B; wherein the repeating unit A is a fragment as shown in Formula 1, and the repeating unit B is a fragment as shown in one or more of Formulas 2-6: The n is the degree of polymerization, which is independently 4-112.

2. The polymer according to claim 1, wherein The molar ratio of the repeating unit A to the repeating unit B is 1:1-1:20, preferably 1:5-1:20, for example 1:9 or 1:19; and / or, in the repeating unit B, the average molecular weight of the fragments represented by formula 2-4 is independently 300-5000 Da; Preferably, the n is independently 4-22; more preferably, the n is independently 6-15; And / or, in the repeating unit B, the average molecular weight of the fragments represented by formula 2-4 is independently 300-1000 Da.

3. A method for preparing a polymer, characterized in that: The preparation method of the polymer comprises the following steps: The polymer is prepared by copolymerization of monomer A and monomer B; wherein, The monomer A is glycidyl methacrylate; The monomer B is one or more of polyethylene glycol acrylate, polyethylene glycol methacrylate, methoxy polyethylene glycol acrylate, sodium acrylate and vinyl acetate.

4. The method for preparing a polymer according to claim 3, wherein: The polymer as claimed in claim 1 or 2; and / or, the molar ratio of monomer A to monomer B is 1:1-1:20, preferably 1:5-1:20, for example 1:9 or 1:19; And / or, the average molecular weight of the polyethylene glycol acrylate, polyethylene glycol methacrylate and methoxy polyethylene glycol acrylate is independently 300-5000 Da, preferably 300-1000 Da; And / or, the copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction or a reversible addition-fragmentation chain transfer polymerization reaction.

5. The method for preparing a polymer according to claim 4, wherein: The copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction, which includes the steps of reacting in a solvent in the presence of a catalyst and a ligand; Preferably, the catalyst is cuprous bromide and / or copper bromide; and / or, the ligand is 2,2'-bipyridine; and / or, the solvent is dimethylformamide; And / or, the copolymerization reaction is carried out in a nitrogen atmosphere; And / or, the copolymerization reaction temperature is room temperature; More preferably, the catalyst is cuprous bromide and copper bromide in a molar ratio of 1:0.1; and / or, the molar ratio of the catalyst to the ligand is 1:2; Further preferably, the molar ratio of total monomers: the catalyst: the ligand is (50-200):1.1:2.2, for example 50:1.1:2.2, wherein the molar ratio of the total monomers is the sum of the molar ratios of the monomer A and the monomer B.

6. A polymer, characterized in that The polymer is prepared according to the method for preparing the polymer as claimed in any one of claims 3 to 5.

7. A polymer-modified carrier, characterized in that The polymer-modified carrier comprises the polymer according to any one of claims 1, 2 and 6 and a carrier whose surface is modified with a connecting functional group A, wherein the polymer is covalently connected to the connecting functional group A.

8. The polymer-modified carrier according to claim 7, wherein The connecting functional group A is shown in Formula 7: And / or, the carrier is selected from one of microspheres, silicon wafers, silicon dioxide sheets and anodized aluminum films; preferably, the microspheres are magnetic microspheres or non-magnetic microspheres, more preferably selected from one of silica microspheres, polystyrene microspheres, agarose microspheres, polyacrylamide microspheres, polyglycidyl methacrylate microspheres, silica-coated ferroferric oxide microspheres, polystyrene-coated ferroferric oxide microspheres and agarose-coated ferroferric oxide microspheres, for example, silica-coated ferroferric oxide microspheres; preferably, the particle size of the microspheres is 200 nm-20 μm; And / or, after the polymer is covalently linked to the linking functional group A, it is wrapped on the surface of the carrier, and the thickness of the wrapping is 25-150 nm, for example, 98±23 nm.

9. A method for preparing a polymer-modified carrier, characterized in that: The preparation method of the polymer-modified carrier comprises the following steps: It is prepared by copolymerization of a carrier with a surface modified with a connecting functional group A, a monomer A and a monomer B; The monomer A and monomer B are as described in claim 3 or 4; The carrier whose surface is modified with a connecting functional group A is as described in claim 7 or 8.

10. The method for preparing a polymer-modified carrier according to claim 9, wherein: The polymer-modified carrier is as described in claim 7 or 8; And / or, the copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction or a reversible addition-fragmentation chain transfer polymerization reaction.

11. The method for preparing a polymer-modified carrier according to claim 10, wherein: The copolymerization reaction is a surface-initiated atom transfer radical polymerization reaction, which includes the steps of reacting in a solvent in the presence of a catalyst and a ligand; Preferably, the catalyst is cuprous bromide and / or copper bromide; and / or, the ligand is 2,2'-bipyridine; and / or, the solvent is dimethylformamide; And / or, the copolymerization reaction is carried out in a nitrogen atmosphere; And / or, the copolymerization reaction temperature is room temperature; More preferably, the catalyst is cuprous bromide and copper bromide in a molar ratio of 1:0.1; and / or, the molar ratio of the catalyst to the ligand is 1:2; Further preferably, the molar ratio of total monomers: the catalyst: the ligand is (50-200):1.1:2.2, for example 50:1.1:2.2, wherein the molar ratio of the total monomers is the sum of the molar ratios of the monomer A and the monomer B.

12. A polymer-modified carrier, characterized in that: The polymer-modified carrier is prepared according to the preparation method of any one of claims 9 to 11.

13. An oligonucleotide modified vector, characterized in that The oligonucleotide-modified carrier comprises an oligonucleotide with a terminal-modified linking functional group B and a polymer-modified carrier according to any one of claims 7, 8 and 12, wherein the linking functional group B and the polymer-modified carrier are covalently linked; Alternatively, the oligonucleotide-modified carrier comprises an oligonucleotide with a terminal-modified linking functional group B, a linking functional group C, and a polymer-modified carrier as described in any one of claims 7, 8 and 12, wherein both ends of the linking functional group C are covalently linked to the linking functional group B and the polymer-modified carrier, respectively.

14. The oligonucleotide modified vector according to claim 13, wherein The oligonucleotide is an oligonucleotide whose 5' end is modified with a linking functional group B, for example, an oligonucleotide whose 5' end is modified with a thiol group; And / or, the linking functional group C is as shown in Formula 8: Preferably, the oligonucleotide modified with a thiol group at the 5' end is connected to the repeating unit A of the polymer-modified carrier, for example, the thiol group is connected to the repeating unit A in the following manner: And / or, the oligonucleotide modified with a thiol group at the 5' end is connected to the repeating unit A of the polymer-modified carrier via the linking functional group C, for example, the thiol group and the repeating unit A are connected via the linking functional group C in the following manner: More preferably, the oligonucleotide modified vector comprises: (1) A carrier with a surface modified with a connecting functional group A; (2) a polymer covalently linked to the linking functional group A; The polymer includes a repeating unit B and a repeating unit C; wherein the repeating unit B is a fragment as shown in one or more of Formulas 2-6, and the repeating unit C is a fragment as shown in Formula 9 or Formula 10:

15. The oligonucleotide modified vector according to claim 13 or 14, characterized in that: The oligonucleotide is a single-stranded DNA; and / or, the oligonucleotide is 12-25 bases in length; And / or, the oligonucleotide content in the oligonucleotide-modified vector is 0-3500 pmol / mg, and is not 0; for example, 850±24 pmol / mg or 604±31 pmol / mg.

16. A method for preparing an oligonucleotide-modified vector, characterized in that: The preparation method comprises the following steps: The oligonucleotide and the polymer modified carrier react to obtain the product; Alternatively, the oligonucleotide and cystamine salt solution are mixed, a reducing agent is added, and then reacted with a polymer-modified carrier to obtain the oligonucleotide; The polymer-modified carrier is as described in any one of claims 7, 8 and 12; The oligonucleotide is as described in any one of claims 13-15.

17. The method for preparing an oligonucleotide-modified vector according to claim 16, wherein: The oligonucleotide modified vector is as described in any one of claims 13 to 15; and / or, the reducing agent is TCEP; And / or, the usage ratio of the oligonucleotide and the polymer-modified carrier is 10 nmol:10 mg.

18. An oligonucleotide modified vector, characterized in that It is prepared according to the preparation method of the oligonucleotide modified vector as claimed in claim 16 or 17.

19. Use of the oligonucleotide modified carrier according to any one of claims 13 to 15 and 17 in DNA template-free synthesis, DNA hybridization probe magnetic beads, heterosexual DNA capture magnetic beads or heterosexual RNA capture magnetic beads.

20. The use according to claim 19, characterized in that The application comprises: mixing the oligonucleotide modified carrier with a DNA template-free synthesis reaction solution, and reacting to obtain the product; Preferably, the DNA template-free synthesis reaction solution comprises a reaction buffer solution, terminal deoxynucleotidyl transferase and deoxyribonucleotides; More preferably, the reaction buffer solution comprises one or more of Tris-HCl, sodium chloride, cobalt chloride and Tween 20; And / or, the deoxyribonucleotide is a natural or 3'-end blocked deoxyribonucleotide, such as dideoxyguanosine 5'-triphosphate.

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