DNA microsphere, preparation method therefor, and use thereof

Abstract

Provided is a DNA microsphere, comprising: a microsphere body, a polymer connected to the surface of the microsphere body, and DNA combined with the polymer. The provided DNA microspheres can be stably used in an in-vitro protein synthesis reaction, significantly reducing the protein preparation cost and providing a potential method for large-scale industrial protein production, facilitating the research, development and production of protein drugs.

Description

DNA microsphere and its preparation method and application Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and in particular relates to a biological microsphere and a preparation method and application thereof. Background Art

[0002] DNA templates play a key role in in vitro gene expression systems, but their high cost and low utilization rate have hindered the industrialization of these technologies. Synthesis of DNA templates primarily relies on chemical synthesis or bacterial amplification, which is costly and difficult to produce on a large scale, failing to meet industrial needs. Furthermore, DNA templates are difficult to reuse in in vitro transcription and translation reactions, resulting in a waste of resources.

[0003] Reusable DNA templates can maximize their usefulness and achieve resource recycling. Therefore, the development of low-cost, reusable DNA templates is crucial. Advances in synthetic biology offer new avenues for constructing these novel templates. Compared to DNA looping techniques, linear template techniques, which connect DNA templates to microspheres or other solid supports, allow for template separation, elution, and reuse, offering advantages such as ease of use, low cost, and broad scalability.

[0004] An in vitro biosynthesis system refers to a system in which specific chemical molecules or biological macromolecules (DNA, RNA, proteins) are efficiently synthesized in vitro by adding exogenously encoded nucleic acids DNA, RNA, substrates, and energy sources into a lysis system of bacteria, fungi, plant cells, or animal cells.

[0005] Common in vitro biosynthesis systems include the in vitro protein synthesis system, which is a cell-free protein synthesis system. It uses exogenous mRNA or DNA templates and cell lysates to complete the rapid and efficient translation of exogenous recombinant proteins; another example is the in vitro RNA synthesis system (IVT).

[0006] Some in vitro protein synthesis systems are coupled in vitro transcription-translation systems (IVTT). Using a DNA template, RNA polymerase transcribes an mRNA intermediate, which then uses components such as amino acids and ATP to efficiently translate the exogenous protein in one step. Currently, common commercial in vitro protein expression systems include Escherichia coli extract (ECE), rabbit reticulocyte lysate (RRL), wheat germ extract (WGE), insect cell extract (ICE), and human systems.

[0007] Compared with traditional in vivo recombinant expression systems, in vitro cell-free protein synthesis systems have many advantages, such as the ability to express special proteins that are toxic to cells or contain non-natural amino acids (such as D-amino acids), the ability to directly use PCR products as templates to simultaneously synthesize multiple proteins in parallel, and the ability to carry out high-throughput drug screening and proteomics research.

[0008] Existing in vitro synthesis systems have the following limitations:

[0009] 1. Existing in vitro synthesis systems all rely on mRNA or DNA templates, and the template amount is often much larger than the final amount of protein synthesized. This limits the industrial application and cost reduction potential of in vitro protein synthesis technology. Currently, the cost of in vitro protein synthesis is still high, making it difficult to meet the needs of large-scale industrial production.

[0010] 2. Currently commercialized in vitro protein synthesis systems primarily include prokaryotic systems (such as E. coli) and eukaryotic systems (such as insect cells, mammalian cells, and yeast). Prokaryotic systems like E. coli are easier to culture and manipulate, resulting in higher yields, but the activity and accuracy of the proteins synthesized are lower than those produced by eukaryotic systems. Proteins synthesized by eukaryotic cell systems exhibit higher activity and post-translational modifications, but are expensive and difficult to produce on a large scale.

[0011] 3. The application scope of in vitro protein synthesis technology is relatively limited, mainly concentrated in the field of drug research and development, for the production of recombinant proteins for in vivo research or therapeutic protein drugs. The application potential in industrial production and other fields remains to be developed.

[0012] 4. Currently, the mainstream technology for industrial protein production worldwide is still based on cell fermentation. Compared with cell fermentation, in vitro protein synthesis technology offers advantages such as ease of operation, no risk of viral contamination, and high yields. However, it is relatively expensive and has a low level of industrialization, making it difficult to replace cell fermentation as the dominant method in industrial protein production.

[0013] In summary, while in vitro protein synthesis technology has great potential, its industrialization and large-scale application are currently limited, primarily due to high costs and strong template dependence. Breakthroughs in related technologies will facilitate the wider application of in vitro protein synthesis technology in protein drug development and industrial production.

[0014] Current status of DNA template technology:

[0015] 1. DNA templates are primarily used to guide in vitro transcription and translation reactions, synthesizing RNA and proteins. Commonly used DNA templates include plasmid DNA, linear DNA, and circular DNA. These DNA templates are obtained through bacterial transformation, PCR amplification, or synthesis.

[0016] 2. Currently, obtaining DNA templates primarily relies on chemical synthesis or bacterial transformation and amplification, which are costly and cumbersome, making them difficult to commercialize and apply on a large scale. While newer generation gene synthesis technologies, such as gene synthesis chips, have reduced costs, they remain prohibitively expensive given the demand for industrially produced DNA templates.

[0017] 3. In vitro transcription and translation systems typically require large amounts of DNA template (excessive mass of the final product), which limits the application of related technologies on an industrial scale. Methods that reuse or continuously utilize DNA templates can reduce costs and enable industrial production.

[0018] 4. In recent years, several new methods have enabled the reuse or continuous use of DNA templates, such as DNA recycling technology, linear expression template technology, and isothermal amplification. These new methods have provided new ideas for the large-scale use of DNA templates in industrial applications. However, the current level of industrialization of these technologies is still relatively low, and cost advantages need to be further improved.

[0019] Advantages and disadvantages of other DNA template reuse or continuous utilization technologies:

[0020] 1. Advantages of DNA recycling technology: DNA templates can be reused continuously, reducing the template demand; the operation is simple and easy to implement.

[0021] Disadvantages: The design of DNA cycling strategy is relatively complex, and different templates have different cycling functions and stabilities; cyclic DNA amplification is prone to introduce errors and requires highly sensitive detection methods.

[0022] 2. Advantages of linear expression template technology: Linear DNA is easy to obtain by chemical synthesis and has low cost; it has a certain template stability and can be used continuously.

[0023] Disadvantages: Linear DNA has low stability in in vitro transcription and translation reactions and its utilization efficiency is lower than that of DNA recycling; special treatment is required to prevent linear DNA from degrading during the reaction.

[0024] 3. Advantages of isothermal amplification: The reaction operation is simple, and PCR is used to automatically amplify DNA at a constant temperature, which is highly efficient.

[0025] Disadvantages: Requires dedicated primers and DNA polymerase, which is relatively expensive; the amplified DNA sequence needs to be designed to introduce looping or enzyme cutting sites, which is relatively cumbersome; the yield of amplified DNA is difficult to accurately control and there are certain differences.

[0026] 4. Advantages of CRISPR-Cas9 gene editing technology: It can modify DNA templates in a targeted manner and achieve reuse; it is relatively simple to operate and is based on the principle of enzymatic cleavage of DNA-RNA complexes.

[0027] Disadvantages: Requires gRNA design and synthesis, which is costly; it is difficult to completely avoid the production of a certain proportion of non-specific editing products.

[0028] Summary of the Invention

[0029] Given that existing in vitro protein synthesis systems are unable to meet the needs of daily biological research and development and industrialization, the present invention provides a reusable DNA template that can be stably used in in vitro protein synthesis reactions, significantly reducing protein preparation costs, providing a potential path for large-scale industrial protein production, and facilitating the research and development and production of protein drugs.

[0030] To this end, the present applicants have discovered that by connecting DNA templates to microspheres, a novel DNA template microsphere can be prepared. These DNA microspheres can be used as a reusable DNA template. This solution not only reduces the cost of DNA templates to enable industrial production, but also opens up new research directions in the field of artificial synthetic biology (such as directed cell evolution and functional regulation), bringing new opportunities for the widespread application of DNA template technology (such as gene therapy and synthetic biology research).

[0031] Specifically, we selected biocompatible microspheres as solid supports, chemically linked the linear template DNA, and optimized reaction conditions to achieve high ligation efficiency and template stability. Verification of these microspheres, including stability, reusability, and efficiency, demonstrated their potential for industrial production and cost-effective gene expression.

[0032] The first aspect of the present invention provides a DNA microsphere, characterized in that it comprises: a microsphere body, a polymer connected to the surface of the microsphere body, and DNA bound to the polymer.

[0033] Further preferably, the polymer binds to DNA via a functional group.

[0034] Further preferably, the functional group is selected from carboxyl, hydroxyl, amino, thiol, a salt form of carboxyl, a salt form of amino, an ester group, or a combination of the aforementioned functional groups.

[0035] Further preferably, the DNA is modified with the functional group.

[0036] Further preferably, the DNA is amplified by a DNA primer sequence.

[0037] Further preferably, the length of the DNA primer sequence is 6 to 30 bp; preferably 9 to 21 bp.

[0038] Further preferably, the amplification is performed after the DNA primer sequence is combined with the polymer via the functional group.

[0039] Further preferably, the DNA primer sequence is combined with the polymer via the functional group to perform the amplification and then connected to the surface of the microsphere body to obtain the DNA microsphere.

[0040] More preferably, the polymer has branches.

[0041] Further preferably, the polymer binds to DNA via a branched chain.

[0042] Further preferably, the polymer is directly or indirectly connected to the outer surface of the microsphere body through a connecting group.

[0043] More preferably, one end of the linking group is connected to the microsphere, and the other end is connected to the polymer by combining with an active group (such as an amino group, a carboxyl group, etc.) at the end of the polymer.

[0044] Further preferably, the outer surface of the microsphere body has at least one linear polymer with branches, one end of the linear polymer with branches is connected to the outer surface of the microsphere body, and the other part is free on the outer surface of the microsphere body; the branches of the linear polymer are connected to DNA.

[0045] Further preferably, the linear polymer is fixed to the outer surface of the microsphere body directly or indirectly through a linking group.

[0046] Further preferably, the linking group is selected from one or a combination of substituted or unsubstituted siloxy groups, amide groups, ester groups, carbonyl groups, alkenyl groups, alkynyl groups, alkylene groups, ether groups, and thioether groups.

[0047] Further preferably, the surface of the microsphere body contains silicon or silicon oxide.

[0048] More preferably, the material of the microsphere body is selected from glass, ceramic or magnetic material.

[0049] Further preferably, the microsphere bodies are magnetic microspheres.

[0050] Further preferably, the microspheres are selected from glass, ceramic or magnetic materials.

[0051] Further preferably, the chemical composition of the magnetic material includes any one of iron compounds, iron alloys, zinc oxides, manganese oxides, gadolinium oxides, cobalt compounds, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides, and chromium oxides, or any combination thereof.

[0052] Further preferably, the chemical composition of the magnetic material includes any one of iron oxide, zinc oxide, manganese oxide, gadolinium oxide, cobalt oxide, nickel oxide, manganese oxide, zinc oxide, gadolinium oxide, chromium oxide or any combination thereof.

[0053] Further preferably, the chemical composition of the magnetic material includes iron oxide, iron, cobalt, Co 2+ , iron nitride, Mn3O4, GdO, nickel, Ni 2+ Any one or any combination thereof; wherein, the iron oxide is preferably Fe3O4, γ-Fe2O3 or a combination thereof.

[0054] Further preferably, the chemical composition of the magnetic material includes any one of Fe3O4, γ-Fe2O3, iron nitride, Mn3O4, AlNi(Co), FeCr(Co), FeCrMo, FeAlC, AlNi(Co), FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), FeSiAl, BaO·6Fe2O3, SrO·6Fe2O3, PbO·6Fe2O3, GdO or any combination thereof.

[0055] Further preferably, the microspheres are selected from glass microspheres, ceramic microspheres or glass-ceramic microspheres.

[0056] Further preferably, the microspheres may be hollow.

[0057] Further preferably, the size of the microsphere body is selected from any one of the following particle size scales or the range between any two particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter sizes are average values.

[0058] More preferably, the diameter of the microsphere body is selected from 0.1 to 10 μm.

[0059] More preferably, the diameter of the microsphere body is selected from 0.2 to 6 μm.

[0060] More preferably, the diameter of the microsphere body is selected from 0.4 to 5 μm.

[0061] More preferably, the diameter of the microsphere body is selected from 0.5 to 3 μm.

[0062] More preferably, the diameter of the microsphere body is selected from 0.2 to 1 μm.

[0063] More preferably, the diameter of the microsphere body is selected from 0.5 to 1 μm.

[0064] Further preferably, the average diameter of the microsphere body is 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, with a deviation of ±20%, more preferably ±10%.

[0065] Further preferably, the main chain of the linear polymer is a polyolefin main chain or an acrylic polymer main chain.

[0066] Further preferably, the linear backbone of the polymer is a polyolefin backbone, and is provided by the backbone of an acrylic polymer.

[0067] Preferably, the average molecular weight (MW) of the polymer is 1,000 to 5,000,000; more preferably, the MW is 1,000 to 100,000; further preferably, the MW is 2,000 to 50,000; further preferably, the MW is 3,000 to 10,000.

[0068] More preferably, the monomer unit of the acrylic polymer is one of acrylic acid, acrylic acid salt, acrylic acid ester, methacrylic acid, methacrylic acid salt, methacrylic acid ester, or a combination thereof.

[0069] The second aspect of the present invention provides a method for preparing the DNA microspheres described in the first aspect of the present invention.

[0070] Further preferably, the preparation method includes the following steps: (1) chemically modifying the microsphere body using an aminosilane coupling agent to introduce amino groups into the outer surface of the microsphere body to form amino-modified microspheres A; (2) modifying the DNA primer sequence to obtain a modified DNA primer sequence; (3) combining the modified DNA primer sequence with a polymer and then amplifying it to obtain polymer-modified DNA; (4) reacting the polymer-modified DNA with the amino-modified microspheres A to obtain the DNA microspheres.

[0071] Further preferably, the amino silane coupling agent is selected from wherein R5 to R7 are each independently selected from C1-C 10 wherein m is 1-10.

[0072] More preferably, m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0073] More preferably, R5 to R7 are each independently selected from an alkyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0074] Further preferably, the amino silane coupling agent is selected from 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane.

[0075] Further preferably, in step (2), the 5' end of the DNA primer sequence is modified with the amino group; more preferably, the amino group is modified at the C2-C20 position of the 5' end; further preferably, the amino group is modified at the C5-C10 position of the 5' end; most preferably, the amino group is modified at the C6 position of the 5' end.

[0076] Further preferably, the length of the DNA primer sequence is 6 to 30 bp; preferably 9 to 21 bp.

[0077] Further preferably, the DNA primer sequence is GGCGTAGAGGAT.

[0078] Further preferably, the amplification is performed after the DNA primer sequence is combined with the polymer via the functional group.

[0079] Further preferably, the DNA primer sequence is combined with the polymer via the functional group to perform the amplification and then connected to the surface of the microsphere body to obtain the DNA microsphere.

[0080] Further preferably, the modification in step (2) is to react the DNA sequence with a polyolefin polymer.

[0081] Further preferably, the polyolefin polymer is selected from one or more of substituted or unsubstituted polypropylene, polyacrylic acid, polyacrylic acid salt, polyacrylic acid ester or polyacrylamide.

[0082] More preferably, the substituent is selected from C1-C20 alkyl groups.

[0083] Further preferably, the polyolefin polymer is selected from polyacrylic acid, polyacrylic acid salt, polymethacrylic acid or polymethacrylate.

[0084] Further preferably, in the step (4), before reacting the polymer-modified DNA with the microspheres A, the system containing the microspheres A is first subjected to ultrasonic vibration.

[0085] More preferably, the ultrasonic oscillation time is 1 to 30 min, more preferably 5 to 20 min, and particularly preferably 5 to 10 min.

[0086] The third aspect of the present invention provides the use of the DNA microspheres described in the first aspect of the present invention.

[0087] Further preferably, the DNA microspheres can be used as DNA templates.

[0088] More preferably, the DNA template is a DNA template of a target protein (or exogenous protein).

[0089] Further preferably, the target protein is selected from the group consisting of luciferin, or luciferase (such as firefly luciferase), green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutation, α-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, cytokine, interferon α2b, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, or a combination thereof.

[0090] The fourth aspect of the present invention provides use of the DNA microspheres described in the first aspect of the present invention as DNA templates in RNA transcription or protein synthesis.

[0091] Further preferably, the RNA transcription or protein synthesis is preferably in vitro RNA transcription or in vitro protein synthesis.

[0092] The fifth aspect of the present invention provides a cell-free in vitro synthesis system, comprising: (1) a cell extract; and (2) the DNA microspheres described in the first aspect of the present invention.

[0093] Further preferably, the cell-free in vitro synthesis system is a cell-free protein synthesis system.

[0094] Further preferably, the cell extract is derived from eukaryotic cells or prokaryotic cells.

[0095] Further preferably, the eukaryotic cell sources include but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell sources may include but are not limited to mouse sources (including rats, mice, guinea pigs, golden hamsters, hamsters, etc.), rabbit sources, monkey sources, human sources, pig sources, sheep sources, cattle sources, dog sources, horse sources, etc.

[0096] Further preferably, the yeast cell is selected from Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta, Ogataeaminuta, Pichia lindneri, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia g uercuum, Pichia apijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae. cerevisiae), brewer's yeast, sugarcane molasses yeast, Saccharomyces cerevisiae, Hansenula polymorpha, Candida utilis, Kluyveromyces cerevisiae, or a combination thereof.

[0097] Further preferably, the Kluyveromyces further includes: the Kluyveromyces includes: Kluyveromyces lactis (Kluyveromyces, K.lactis), Kluyveromyces marxianus, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii or a combination thereof.

[0098] The sixth aspect of the present invention provides a cell-free in vitro synthesis method, which is prepared using the system provided by the fifth aspect of the present invention.

[0099] Further preferably, the cell-free in vitro synthesis method is a cell-free protein synthesis method.

[0100] Compared with the prior art, the present invention has the following beneficial effects:

[0101] (1) Higher stability: Compared with free DNA templates, the DNA template attached to the surface of the microspheres is more stable, degrades more slowly under in vitro reaction conditions, and has a longer reaction life and a higher number of utilizations.

[0102] (2) The template concentration can be precisely controlled: the concentration of template DNA can be precisely controlled by controlling the microsphere loading capacity to meet the needs of different reactions.

[0103] (3) Low cost: Compared with PCR and gene editing technology, the cost of obtaining microsphere-linked DNA templates is lower and easy to achieve industrial production.

[0104] (4) Avoid product contamination: Compared with free DNA templates, connected DNA templates can be more easily removed from the reaction products by elution, reducing DNA contamination of the products. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] Figure 1 shows the activity data of the DNA magnetic microspheres prepared in Examples 1-3 of this application after IVTT. 75 μL of magnetic beads reacted with 200 μL of DNA treated with activation solution at room temperature (15°C). Groups MAG-1 to MAG-6 used identical pretreatment methods and reaction conditions to verify the reproducibility and stability of the reaction activity. PC represents the activity of DNA not linked to the magnetic microspheres.

[0106] Figure 2 shows the activity data of the supernatants from the DNA magnetic microspheres prepared in Examples 1-3 of this application after IVTT reaction. The MAG-1-S to MAG-6-S groups used identical pretreatment methods and reaction conditions to verify the reproducibility and stability of the reaction activity. PC represents the activity of DNA not linked to the magnetic microspheres.

[0107] Figure 3 compares the activity of the magnetic microspheres after repeated use with that after initial use, demonstrating the effectiveness of reusable DNA templates. Mag 1st refers to the activity of the DNA magnetic microspheres after initial use, mag 2nd refers to the activity of the DNA magnetic microspheres after washing (i.e., the second use), and 1, 2, and 3 refer to three replicate experiments.

[0108] Figure 4 shows a 6-fold scale-up of the magnetic microsphere reactivity validation. MAG 6X represents 1μm magnetic microspheres scaled 6x. 1, 2, and 3 represent parallel experiments conducted under identical conditions. -S represents the supernatant from the corresponding group, and PC represents the control. (The 6-fold scale-up refers to a 6-fold increase in the amount of magnetic microspheres, activation solution, and DNA added; this represents the overall scale-up of the experiment; the particle size and concentration of the magnetic microspheres remain unchanged.)

[0109] Figure 5 shows the activity data of the DNA magnetic microspheres and their supernatants prepared in Example 5 of the present application after IVTT reaction, wherein F refers to the forward primer (GGATAGCGA, 5' end C6NH2), and R refers to the reverse primer (GCGGTGGCA, 5' end C6NH2). As shown in the figure, 1F-supernatant refers to the first verification experiment of the DNA magnetic microsphere supernatant obtained by the forward primer, and 1F-beads refers to the first verification experiment of the DNA magnetic microspheres obtained by the forward primer. 1R-supernatant refers to the first verification experiment of the DNA magnetic microsphere supernatant obtained by the reverse primer, and 1R-beads refers to the first verification experiment of the DNA magnetic microspheres obtained by the reverse primer. The numbers in the figure represent the serial numbers of parallel reactions using exactly the same pre-treatment method and reaction conditions.

[0110] Figure 6 shows the activity data of the DNA microspheres prepared in Examples 8-10 of this application after IVTT reaction. The activity of 100 μL of glass beads at room temperature (15°C) was tested against 200 μL of DNA treated with activation solution. 320 represents the mesh size of the glass beads (45 μm). Groups 1, 2, and 3 used identical pretreatment methods and reaction conditions to verify the reproducibility and stability of the reaction activity. PC represents the activity of DNA not linked to the glass microspheres.

[0111] Figure 7 shows the activity data of the supernatant corresponding to the DNA microspheres prepared in Example 12 of this application after IVTT reaction, used to verify the effect of ultrasonic vibration on the activity of glass beads. 320 and 2000 are the mesh sizes of the glass beads (corresponding to particle sizes of 45 μm and 7.5 μm), and -1 and -2 use exactly the same pretreatment method and reaction conditions to verify the repeatability and stability of the reaction activity. U indicates that the corresponding group was ultrasonically vibrated for 10 minutes before the reaction. DETAILED DESCRIPTION

[0112] The present invention will be further described below in conjunction with the specific embodiments and examples, and please refer to the accompanying drawings. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are preferably based on or with reference to the conditions indicated in the specific embodiments described above, and then can be based on conventional conditions, such as those described in "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)" and "Cell-free Protein Synthesis: Methods and Protocols [M], Edited by Alexander S. Spirin and James R. Swartz, Cell-free Protein Synthesis: Methods and Protocols [M], 2008," or according to the conditions recommended by the manufacturer.

[0113] Unless otherwise specified, percentages and parts mentioned in the present invention are percentages and parts by weight.

[0114] Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

[0115] Unless otherwise specified, the temperature units in this application are degrees Celsius (°C).

[0116] Nouns and terms

[0117] The following is an explanation or description of the meaning of some of the relevant "nouns" and "terms" used in the present invention, so as to better understand the present invention. The corresponding explanation or description applies to the full text of the present invention, both below and above. When the present invention involves references, the definitions of relevant terms, nouns, and phrases in the references are also quoted, but when they conflict with the definitions in the present invention, the definitions in the present invention shall prevail. When the definitions in the references conflict with the definitions in the present invention, it does not affect the cited components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., which are subject to the contents determined in the references.

[0118] In the description of the present invention, preferred embodiments such as “one of the preferred embodiments”, “one of the preferred modes”, “one of the preferred embodiments”, “one of the preferred examples”, “preferred example”, “in a preferred embodiment”, “some preferred examples”, “in some preferred modes”, “preferably”, “preferably”, “preferably”, “more preferably”, “more preferably”, “further preferably”, “most preferably”, and illustrative enumeration methods such as “one of the embodiments”, “one of the modes”, “example”, “specific example”, “for example”, “as an example”, “for example”, “such as”, etc., do not constitute any limitation on the scope of the invention and the scope of protection, and the specific features described in each mode are included in at least one specific embodiment of the present invention. In the present invention, the specific features described in each mode may be combined in a suitable manner in any one or more specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred mode may also be combined in any suitable manner.

[0119] In the present invention, “any combination thereof” means “greater than 1” in terms of quantity, and means a group consisting of the following situations in terms of coverage: “any one of them, or a group consisting of at least two of them”.

[0120] In the present invention, the descriptions of "one or more", "one or more" and "one or more" have the same meaning as "at least one", "at least one", "a combination thereof", "or a combination thereof", "and a combination thereof", "or any combination thereof", "and any combination thereof", etc., and can be used interchangeably to indicate that the quantity is equal to "1" or "greater than 1".

[0121] In the present invention, "or / and" and "and / or" are used to represent "optionally one of them or optional combination thereof", and also represent at least one of them.

[0122] The prior art means described in the present invention in terms of “usually”, “conventional”, “general”, “frequently”, “often”, etc. are also cited as references to the content of the present invention. Unless otherwise specified, they can be regarded as one of the preferred ways of some technical features of the present invention. It should be noted that they do not constitute any limitation on the scope of coverage and protection scope of the invention.

[0123] All documents mentioned in the present invention and documents directly or indirectly cited by these documents are incorporated by reference in this application as if each document was individually incorporated by reference.

[0124] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to the embodiments) can be combined with each other to form new or preferred technical solutions, as long as they can be used to implement the present invention. Due to space limitations, they will not be listed one by one.

[0125] Polymers, in the present invention, broadly include oligomers and high polymers, having at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be characterized by a suitable method, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.).

[0126] Acrylic monomer molecules: monomer molecules that can be used to synthesize acrylic polymers, having the basic structure of C(COO-)=C, for example, CH(COOH)=CH2, CH(COONa)=CH2, CH3C(COOH)=CH2, CH3C(COONa)=CH2, CH(COOCH3)=CH2, CH(COOCH2CH2OH)=CH2, CH3C(COOCH3)=CH2, CH3C(COOCH2CH2OH)=CH2, etc.

[0127] The "fixed" methods such as fixed, fixed to, fixed with, fixed in, etc. refer to covalent bonding methods.

[0128] The manner of “connection” / “binding” such as “carrying”, “connected”, “connected to”, “connected in”, “bound to”, “captured”, and “captured” is not particularly limited, and includes but is not limited to covalent manner, non-covalent manner, and the like.

[0129] Covalent bonding: direct bonding via covalent bonds. The covalent bonding includes but is not limited to dynamic covalent bonding, which refers to direct bonding via dynamic covalent bonds.

[0130] Covalent bonds: include common covalent bonds such as amide bonds and ester bonds, as well as dynamic covalent bonds with reversible properties.

[0131] The covalent bonds include dynamic covalent bonds. A dynamic covalent bond is a reversible chemical bond, including but not limited to an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof. Those skilled in the art of chemistry will understand its meaning.

[0132] "Modified" products, including but not limited to derivatives, modified products, genetically modified products, fusion products, etc. of the present invention, can maintain their original functions or properties, or optimize or change their functions or properties.

[0133] In vitro protein synthesis reaction refers to the reaction of synthesizing protein in an in vitro cell-free synthesis system, which at least includes the translation process. It includes but is not limited to IVT reaction (in vitro transcription reaction), IVTT reaction (in vitro transcription and translation reaction), and IVDTT reaction (in vitro replication transcription and translation reaction). In the present invention, IVTT reaction is preferred. IVTT reaction, corresponding to IVTT system, is the process of transcribing and translating DNA into protein (Protein) in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as D2P system, D-to-P system, D_to_P system, DNA-to-Protein system; the corresponding in vitro protein synthesis method is also called D2P method, D-to-P method, D_to_P method, DNA-to-Protein method.

[0134] "Cell-free system" refers to in vitro biosynthesis performed without secretory expression via intact cells. It should be noted that the in vitro synthesis system of the present invention allows for the addition of cellular components to facilitate the reaction, but the added cells are not primarily intended to secrete the exogenous target protein.

[0135] In addition, in the intact cell-free D2P system constructed under the guidance of the present invention, a small amount of intact cells are intentionally added (for example, the protein content provided by them does not exceed 30 wt% compared with the protein content provided by the cell extract). This "circumvention" method is also included in the scope of protection of the present invention.

[0136] Exogenous protein: The target expression product of the in vitro protein synthesis system of the present invention is not synthesized by host cell secretion, but is synthesized in vitro based on an exogenous nucleic acid template, and therefore can also be called a target protein. The exogenous protein can be a protein, a fusion protein, a mixture of protein-containing molecules or fusion protein molecules; it also broadly includes polypeptides. The product obtained after the in vitro protein synthesis reaction based on the nucleic acid template encoding the exogenous protein can be a single substance or a combination of two or more substances. "Exogenous protein", "target protein", "target protein", "target translation product" have the same meaning and can be translated as "objective protein", "interested protein", "objective translated product", "interested protein product", etc., and can be used interchangeably in the present invention.

[0137] D2P, DNA-to-Protein, refers to the process from DNA template to protein product. For example, D2P technology, D2P system, D2P method, D2P kit, etc.

[0138] "The expression system of the present invention", "the in vitro expression system of the present invention", "the in vitro cell-free expression system", and "the in vitro cell-free expression system" are used interchangeably and all refer to the in vitro expression system of the present invention. Other descriptions may also be used, such as: in vitro protein synthesis system, in vitro protein synthesis system, cell-free system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. According to the reaction mechanism, it may include an in vitro transcription system (which may be abbreviated as an IVT system, a mR2P system), an in vitro transcription-translation system (which may be abbreviated as an IVTT system, a D2P system), an in vitro replication-transcription-translation system (which may be abbreviated as an IVDTT system, a D2P system), etc. In the present invention, the IVTT system is preferred. We also refer to the in vitro protein synthesis system as a "protein synthesis factory" ("Protein Factory" or "proteinfactory" or "Proteinfactory"). The in vitro protein synthesis system provided by the present invention adopts an open description method for its components. The cell-free protein synthesis system of the present invention uses exogenous DNA, mRNA or a combination thereof as the nucleic acid template for protein synthesis, and achieves in vitro synthesis of the target protein by artificially controlling the addition of substrates and transcription- and translation-related protein factors required for protein synthesis.

[0139] One of the specific embodiments of the in vitro protein synthesis system, further includes but is not limited to, for example, the cell-free protein synthesis system based on Escherichia coli described in WO2016005982A1. Other cited documents of the present invention, directly and indirectly cited documents including but not limited to the in vitro cell-free protein synthesis system based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus, are also included in the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, the document “Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45” includes but is not limited to the in vitro cell-free protein synthesis system (In vitro cell-free protein synthesis system) recorded in the cited documents on pages 27-28 of “2.1 Systems and Advantages”, which can be used as the in vitro protein synthesis system for implementing the present invention.For example (unless they conflict with the present invention, the following documents and their references are cited in their entirety and for all purposes), documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN110093284A, CN110408635A, CN110408636A, CN110551745 A. CN110551700A, CN110551785A, CN110819647A, CN110845622, CN110938649A, CN110964736A, CN111378706 A. CN111378707A, CN111378708A, CN111718419A, CN111748569A, CN2019107298813, CN2019112066163, CN20 18112862093, CN2019114181518, CN2020100693833, CN2020101796894, CN202010269333X, CN2020102693382, CN2020113574616 and the references thereto, and the methods for constructing and amplifying DNA templates thereof can all be used as the in vitro protein synthesis system and the methods for constructing and amplifying DNA templates thereof for implementing the present invention.

[0140] In the present invention, "protein" and "protein" have the same meaning and can be used interchangeably.

[0141] In the present invention, “system” and “system” are both translated as system and can be used interchangeably.

[0142] In the present invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

[0143] In the present invention, cell extract, cell extract, cell lysate, cell disrupted product and cell lysate have the same meaning and can be used interchangeably. In English, cell extract, cell lysate and the like can be used as descriptions.

[0144] In the present invention, energy system, energy system, and energy supply system have the same meaning and can be used interchangeably. Energy regeneration system and energy regeneration system have the same meaning and can be used interchangeably. The energy regeneration system is a preferred embodiment or component of the energy system.

[0145] The in vitro synthesis system of the present invention further comprises one or more components selected from the group consisting of a substrate for protein synthesis, a substrate for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, a buffer, an energy regeneration system, polyethylene glycol (PEG) or its analogs, dithiothreitol (DTT), and optionally a solvent, wherein the solvent is water or an aqueous solvent. Furthermore, the cell extract is free of long-chain nucleic acid molecules endogenous to yeast.

[0146] Furthermore, the substrate for synthesizing RNA includes: nucleoside monophosphate, nucleoside triphosphate or a combination thereof.

[0147] Furthermore, the substrates for synthesizing proteins include 20 natural amino acids and unnatural amino acids.

[0148] Furthermore, the magnesium ions are derived from a magnesium ion source, and the magnesium ion source is selected from the following group: magnesium acetate, magnesium glutamate, or a combination thereof.

[0149] Furthermore, the potassium ions are derived from a potassium ion source, and the potassium ion source is selected from the following group: potassium acetate, potassium glutamate, or a combination thereof.

[0150] Furthermore, the energy regeneration system is selected from the group consisting of a creatine phosphate / creatine phosphate enzyme system, one of the glycolysis pathway intermediate energy systems, sucrose, or a combination thereof.

[0151] Furthermore, the buffer is selected from the following group: 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0152] Furthermore, the protein synthesis system contains polyethylene glycol (PEG) or its analog. The concentration of the PEG or its analog is not particularly limited. Typically, the concentration (w / v) of the PEG or its analog is 0.1-8%, preferably 0.5-4%, and more preferably 1-2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of PEG 3000, PEG 3350, PEG 6000, and PEG 8000, or a combination thereof.

[0153] Furthermore, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc. Preferably, the polyethylene glycol has a molecular weight of 3000-10000.

[0154] An optional scheme is that the protein synthesis system provided by the present invention includes: yeast cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNA polymerase, polyethylene glycol, and sucrose.

[0155] In the present invention, the cell extract does not contain intact cells. A typical cell extract includes ribosomes, transfer RNA, aminoacyl-tRNA synthetases, initiation factors and elongation factors required for protein synthesis, and termination and release factors. In addition, the cell extract also contains some other proteins derived from the cell cytoplasm, especially soluble proteins.

[0156] In the present invention, the protein content of the cell extract is 20-100 mg / ml, preferably 50-100 mg / ml. The protein content is determined by Coomassie Brilliant Blue assay.

[0157] In the present invention, the preparation method of the cell extract or cell lysate is not limited. A preferred preparation method includes the following steps:

[0158] (i) providing cells;

[0159] (ii) washing the cells to obtain washed cells;

[0160] (iii) performing cell disruption treatment on the washed cells to obtain a crude cell extract;

[0161] (iv) performing solid-liquid separation on the crude cell extract to obtain a liquid portion, which is the cell extract.

[0162] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0163] In the present invention, the centrifugation conditions are not particularly limited. A preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0164] In the present invention, the centrifugation time is not particularly limited. A preferred centrifugation time is 0.5 min-2 h, preferably 20 min-50 min.

[0165] In the present invention, the centrifugation temperature is not particularly limited. Preferably, the centrifugation is performed at 1-10°C, more preferably, at 2-6°C.

[0166] In the present invention, the washing treatment method is not particularly limited. A preferred washing treatment method is to use a washing liquid at a pH of 7-8 (preferably 7.4). The washing liquid is not particularly limited. Typically, the washing liquid is selected from the following group: potassium 4-hydroxyethylpiperazineethanesulfonate, potassium acetate, magnesium acetate, or a combination thereof.

[0167] In the present invention, the cell disruption treatment method is not particularly limited. A preferred cell disruption treatment includes high-pressure disruption and freeze-thaw (such as liquid nitrogen low-temperature) disruption.

[0168] The nucleoside triphosphate mixture in the protein synthesis system is adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine triphosphate. In the present invention, the concentration of each mononucleotide is not particularly limited, and the concentration of each mononucleotide is generally 0.5-5mM, preferably 1.0-2.0mM.

[0169] The amino acid mixture in the protein synthesis system can include natural or unnatural amino acids, including D- or L-amino acids. Representative amino acids include (but are not limited to) the 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is typically 0.01-0.5 mM, preferably 0.02-0.2 mM, such as 0.05, 0.06, 0.07, or 0.08 mM.

[0170] In a preferred embodiment, the in vitro cell-free protein synthesis system further contains sucrose, and the concentration of the sucrose is 0.03-40 wt%, preferably 0.08-10 wt%, and more preferably 0.1-5 wt%, based on the total weight of the protein synthesis system.

[0171] A particularly preferred in vitro cell-free protein synthesis system contains the following components in addition to yeast cell extract: 22 mM 4-hydroxyethylpiperazineethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg / mL creatine phosphokinase, 1%-4% polyethylene glycol, 0.5%-2% sucrose, and 0.027-0.054 mg / mL T7 RNA polymerase.

[0172] Cell extracts

[0173] In the present invention, cell extract and cell lysate have the same meaning, and both are substances obtained after cell disruption.

[0174] Example 1 Modification of magnetic microspheres

[0175] 1-150 g of silica-coated ferroferric oxide magnetic microspheres were weighed and washed 2-5 times with anhydrous ethanol. 5-500 mL of an ethanol solution (5%-20%, v / v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the washed magnetic microspheres. The mixture was reacted for 12-72 hours. The magnetic microspheres were then washed 3-5 times with anhydrous ethanol and distilled water to obtain amino-modified magnetic microspheres A.

[0176] Example 2: Preparation of DNA samples

[0177] (1) Polyacrylic acid solution a configuration

[0178] a. Take 4 ml of polyacrylic acid solution (molecular weight 3000, mass fraction 50%), add ddH2O to 40 mL, add 1 mol / L NaOH dropwise to adjust the pH to 7.5, and then add purified water to 50 mL;

[0179] b. Transfer 230 μL of the solution to a test tube, add ddH2O to make up to 10 mL, and shake thoroughly.

[0180] (2) Add 318 μL of polyacrylic acid solution to the DNA primer (specific sequence: GGCGTAGAGGAT, amino modified at the C6 position of the 5' end during synthesis), shake well on a vortex for 5 minutes, and let it stand for 25 minutes.

[0181] (3) Use the modified DNA primer sequence (polyacrylic acid modified DNA primer sequence) to amplify (Ampi) to obtain the green fluorescent protein DNA sample (DNA template).

[0182] Example 3: Preparation of DNA magnetic microspheres.

[0183] (1) Take 75 μL of magnetic microspheres A (particle size 1 μm, containing liquid) and transfer them to a 2 mL centrifuge tube a.

[0184] (2) Take 200 μL of DNA sample and transfer it to another centrifuge tube b.

[0185] (3) Prepare the activation solution. Weigh 0.300 g of EDC and 0.150 g of NHS into a 15 mL centrifuge tube. Add ddH2O to 10 mL and shake thoroughly to dissolve the drugs completely.

[0186] (4) Transfer 100 μL of activation solution to centrifuge tube b, shake well on a vortex for 1 min, and let it stand for 15 min.

[0187] (5) Transfer all the mixed liquid in centrifuge tube b to centrifuge tube a, and shake centrifuge tube a manually to fully mix the contents.

[0188] (6) Place centrifuge tube a on a rotator and react at room temperature (preferably 18°C) for 24 hours to obtain a solution containing DNA magnetic microspheres.

[0189] Example 4: Activity Verification

[0190] (1) Remove centrifuge tube a and place it on a magnetic test tube rack to wait for the DNA magnetic microspheres to separate from the liquid.

[0191] (2) Transfer the supernatant to centrifuge tube c.

[0192] (3) Add ddH2O to centrifuge tube a for cleaning and aspirate the supernatant.

[0193] (4) Centrifuge tube a and centrifuge tube c were sent for inspection, and the target protein was synthesized in the IVTT system.

[0194] The IVTT system includes the following components (final concentrations): 9.78 mM Tris-HCl (pH 8.0), 80 mM potassium acetate, 5 mM magnesium acetate, 1.8 mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate, each at a concentration of 1.8 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan), and 1.8 mM daptomycin. The mixture is prepared from 1% 4% 4-amino-3-dextrose (1% 4-amino-3-dextrose), 1 ...

[0195] The Kluyveromyces lactis extract includes endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified by: using a modified strain based on the Kluyveromyces lactis strain ATCC8585; integrating the gene encoding T7 RNA polymerase into the Kluyveromyces lactis genome using the method described in CN109423496A to obtain a modified strain that endogenously expresses T7 RNA polymerase; culturing cells from the modified strain, and then preparing a cell extract. The Kluyveromyces lactis cell extract is prepared using conventional techniques, with reference to the method described in CN109593656A. In summary, the preparation steps include: providing an appropriate amount of fermented Kluyveromyces lactis cells, quick-freezing the cells with liquid nitrogen, disrupting the cells, and collecting the supernatant by centrifugation to obtain a cell extract. The resulting Kluyveromyces lactis cell extract has a protein concentration of 20 to 40 mg / mL.

[0196] IVTT reaction: Add the components of centrifuge tubes a and c to the in vitro protein synthesis system to perform an in vitro protein synthesis reaction. Synthesize the protein to obtain an IVTT reaction solution containing the protein. Fluorescent protein activity assay: Place the plate in an Envision 2120 multi-function microplate reader (Perkin Elmer) and measure the fluorescence signal. Relative fluorescence units (RFUs) are used as activity units (see Figures 1-2 for details).

[0197] As shown in Figures 1-2, there is very little free template DNA in the centrifuge, and its activity is almost negligible, indicating that the template DNA is almost entirely linked to the magnetic microspheres, and the resulting DNA magnetic microspheres possess high biological activity. Furthermore, the activity data for MAG-1 to MAG-6 show that the activities of the six groups of magnetic beads (RFU4607, RFU4587, RFU4332, RFU4418, RFU4695, and RFU4652) produced under identical reaction conditions all exceeded those of the control group (MAG-3 had the lowest activity of 102.07% of PC activity, while MAG-5 had the highest activity of 110.63%). The maximum difference in activity between groups was RFU363 (8.38%), meeting the activity requirements. This demonstrates that the DNA magnetic microspheres maintain high activity as template DNA even after repeated use.

[0198] Example 5 Repeated testing after washing the DNA magnetic microspheres

[0199] After washing, the DNA magnetic microspheres from the above example were retested with IVTT activity, and parallel tests were performed using the same testing conditions as in Example 4. The results of three parallel tests showed that the activity of the washed and reused beads (RFU5396-RFU5845) was 98.83%-107.05% of the initial activity (RFU5460). The maximum decay rate was 1.17%. Assuming a 1.17% decrease in activity with each repetition, the activity after 20 repetitions should be 79.03%. If the PC activity is RFU5460, the activity should be RFU4315, which still meets the activity requirements (see Figure 3).

[0200] Example 6 Expanded Experimental Verification

[0201] The above experiment was repeated with the addition of magnetic microspheres A, activation solution, and DNA increased sixfold. As shown in Figure 4, the activities of MAG 6X 1, 2, and 3 (RFU3366, RFU3239, and RFU3610, respectively) were all above 100% of the PC control. The highest activity was 118% for MAG 6X 3, while the lowest was 105.8% for MAG 6X 2. This demonstrates that even with the reaction conditions scaled up several times, nearly all DNA sequences were linked to the microspheres, and the DNA magnetic microspheres maintained high activity.

[0202] Example 7 (Comparative Example): The DNA primer sequence was linked to the magnetic microspheres and amplified, and the activity of the magnetic microspheres was verified.

[0203] (1) Take 75 μL of magnetic microspheres A (particle size 1 μm, containing liquid) and transfer them to a 2 mL centrifuge tube a.

[0204] (2) Take 200 μL of amino-modified DNA primer sequence to another centrifuge tube b.

[0205] (3) Prepare the activation solution. Weigh 0.300 g of EDC and 0.150 g of NHS into a 15 mL centrifuge tube. Add ddH2O to 10 mL and shake thoroughly to dissolve the drugs completely.

[0206] (4) Transfer 100 μL of activation solution to centrifuge tube b, shake well on a vortex for 1 min, and let it stand for 15 min.

[0207] (5) Transfer all the mixed liquid in centrifuge tube b to centrifuge tube a, and shake centrifuge tube a manually to fully mix the contents.

[0208] (6) Place centrifuge tube a on a rotator and react at room temperature for 24 hours.

[0209] (7) Remove centrifuge tube a and place it on a magnetic test tube rack to wait for the magnetic beads to separate from the liquid.

[0210] (8) Take the supernatant and place it in centrifuge tube c.

[0211] (9) Add ddH2O to centrifuge tube a and wash three times, then aspirate the supernatant.

[0212] (10) The magnetic beads and DNA sequence linkers in centrifuge tube a and the free DNA sequence in centrifuge tube c are sent for amplification (green fluorescent protein DNA template).

[0213] (11) After amplification, the samples in centrifuge tubes a and c were subjected to fluorescent protein activity assay (the method is the same as in Example 4): the samples were placed in an Envision 2120 multifunctional microplate reader (Perkin Elmer) to detect the intensity of the fluorescent signal, and the relative fluorescence unit (RFU) was used as the activity unit (as shown in FIG5 ).

[0214] As shown in Figure (5), after the DNA sequence is linked to the magnetic beads and then amplified, the magnetic beads are washed and compared with the supernatant for IVTT reaction. The washed magnetic beads have almost no activity, while the supernatant has relatively high activity. This indicates that after the DNA primer sequence is linked to the magnetic beads and then amplified, the activity of the DNA sequence linked to the magnetic beads is extremely low. This proves that the DNA magnetic beads prepared by the method of the present application have high activity.

[0215] Example 8: Microsphere modification

[0216] 1-50 g of glass microspheres were weighed and ultrasonically cleaned with distilled water for 1-3 times. 5-250 mL of 1-2 mol / L NaOH solution was added to the cleaned glass microspheres, reacted at 70° C. for 1-2 h, and washed with distilled water for 5-10 times. 5-200 mL of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) ethanol solution (5%-20%, v / v) was added to the cleaned glass microspheres, reacted for 12-72 hours, and then washed with anhydrous ethanol and distilled water for 3-5 times to obtain modified glass microspheres A.

[0217] Example 9: Preparation of DNA samples

[0218] (1) Polyacrylic acid solution a configuration

[0219] a. Take 4 ml of polyacrylic acid solution (molecular weight 3000, mass fraction 50%), add ddH2O to 40 mL, add 1 mol / L NaOH dropwise to adjust the pH to 7.5, and then add purified water to 50 mL;

[0220] b. Transfer 230 μL of the solution to a test tube, add ddH2O to make up to 10 mL, and shake thoroughly.

[0221] (2) Add 318 μL of polyacrylic acid solution to the DNA primer sequence (the experiment used artificially synthesized DNA, the specific sequence is GGCGTAGAGGAT, and the amino group was modified at the C6 position of the 5' end during synthesis), shake it in a vortex for 5 minutes, and let it stand for 25 minutes.

[0222] (3) Use the modified DNA primer sequence amplification (Ampi) to obtain green fluorescent protein DNA sample.

[0223] Example 10: Preparation of DNA glass microspheres.

[0224] (1) MES-KOH buffer solution preparation

[0225] ①Put 1.952g of MES in a beaker;

[0226] ② Add about 60 mL of ddH2O to the beaker and stir to dissolve MES.

[0227] ③ Add 1 mol / L KOH solution into the beaker and adjust the pH to 5.6.

[0228] ④ Transfer the liquid in the beaker to a 100mL volumetric flask and adjust the volume.

[0229] (2) Wash glass microspheres A (320 mesh, 45 μm) to reduce the pH to 7-8.

[0230] (3) Take 100 μl of the washed glass microspheres A (without liquid) into centrifuge tube a, wash them three times with MES-KOH buffer, and then centrifuge to remove the supernatant.

[0231] (4) Add 100 μl of MES-KOH buffer to centrifuge tube a.

[0232] (5) Take 200 μl of green fluorescent protein DNA sample and transfer it to another centrifuge tube b.

[0233] (6) Prepare the activation solution by weighing 0.300 g of EDC and 0.150 g of NHS into a 15 mL centrifuge tube, adding MES-KOH buffer to 10 mL, and shaking thoroughly until the drugs are completely dissolved.

[0234] (7) Transfer 100 μl of activation solution to centrifuge tube b, shake well on a vortexer for 1 min, and let it stand for 15 min.

[0235] (8) Transfer all the mixed liquid in centrifuge tube b to centrifuge tube a and shake centrifuge tube a manually to fully mix the contents.

[0236] (9) Place centrifuge tube a on a rotator and react at room temperature (preferably 15°C) for 24 hours.

[0237] Example 11: Activity Verification

[0238] (1) Remove centrifuge tube a and place it on a test tube rack to wait for the microspheres to separate from the liquid.

[0239] (2) Transfer the supernatant to centrifuge tube c.

[0240] (3) Add ddH2O to centrifuge tube a and wash it three times, then aspirate the supernatant.

[0241] (4) Centrifuge tube a and centrifuge tube c were sent for inspection, and the target protein was synthesized in the IVTT system (same as in Example 4).

[0242] As shown in Figure 6, three parallel experiments were conducted on 320-mesh microspheres. The activities of groups 1, 2, and 3 of DNA microspheres were measured, respectively: RFU3699, RFU4532, and RFU3893. Group 2 had the highest activity, reaching 106% of the PC activity. Even Group 1, which had the lowest activity, had an activity of 86.5% of the PC activity. This demonstrates that the DNA glass microspheres of the present invention exhibit good reactivity as DNA templates, along with good reproducibility and stability.

[0243] Example 12: Verify the effect of ultrasonic vibration on the activity of DNA microspheres.

[0244] The remaining steps were similar to those in Example 10, except that step (4-1) was added between steps (4) and (5) of Example 10: the glass microspheres, after washing and resuspending in the buffer solution, were placed in an ultrasonic oscillator and oscillated for 10 minutes. The remaining steps were similar to those in Example 10. The activity of the DNA microspheres was then verified in the same manner as in Example 11, and the effect of ultrasonic oscillation on the activity of DNA microspheres of different particle sizes was also verified. Multiple parallel experiments were performed for effective verification (see Figure 7 for details).

[0245] As shown in Figure 7, the activity of DNA microspheres linked after ultrasonic treatment, whether 320 mesh or 2000 mesh (7.5μm), was higher than that of DNA microspheres without ultrasonic treatment, and this conclusion remained true after repeated experimental verification. The group with the largest improvement was 2000U2-1, with an RFU increase of 81.7%, and the lowest was 2000U2-2, with an RFU increase of 2.3%. The above experiments prove that ultrasonic vibration of the glass microsphere suspension before linking the glass microspheres to DNA can significantly improve the reactivity of the DNA microspheres, and is applicable to microspheres of different particle sizes while still maintaining good repeatability and stability.

[0246] Based on the above-mentioned ideal embodiments of this application, and in accordance with the above description, relevant personnel can make various changes and modifications without departing from the technical scope of this application. The technical scope of this application is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A DNA microsphere, characterized in that: include: Microsphere body, polymer connected to the surface of the microsphere body, and DNA bound to the polymer.

2. The DNA microsphere according to claim 1, characterized in that: The polymer binds to DNA via a functional group; the functional group is selected from carboxyl, hydroxyl, amino, sulfhydryl, a salt form of a carboxyl, a salt form of an amino, an ester group, or a combination of the aforementioned functional groups.

3. The DNA microsphere according to claim 1 or 2, characterized in that: The DNA is modified with the functional group.

4. The DNA microsphere according to any one of claims 1 to 3, characterized in that: The polymer is directly or indirectly connected to the outer surface of the microsphere body through a connecting group.

5. The DNA microsphere according to claim 4, characterized in that: The linking group is selected from one or a combination of substituted or unsubstituted siloxy groups, amide groups, ester groups, carbonyl groups, alkenyl groups, alkynyl groups, alkylene groups, ether groups, and thioether groups.

6. The DNA microsphere according to any one of claims 1 to 5, characterized in that: The surface of the microsphere body contains silicon or silicon oxide; preferably, the material of the microsphere body is selected from glass, ceramic or magnetic material. When the microsphere body is a magnetic microsphere, its surface is coated with silicon dioxide.

7. The DNA microsphere according to any one of claims 1 to 6, characterized in that: The size of the microsphere body is selected from any one of the following particle size scales or the range between any two particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm , 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter size is the average value; In one preferred embodiment, the diameter of the microsphere body is selected from 0.1 to 10 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.2 to 6 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.4 to 5 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.5 to 3 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.2 to 1 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 0.5 to 1 μm; In one preferred embodiment, the diameter of the microsphere body is selected from 1 μm to 1 mm; In one preferred embodiment, the average diameter of the microsphere body is 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, with a deviation of ±20%, more preferably ±10%.

8. The DNA microsphere according to any one of claims 1 to 7, characterized in that: The polymer is one or more of polyolefin, PEG, polyacrylic acid, polyamide or polyester.

9. The DNA microsphere according to any one of claims 1 to 8, characterized in that: The average molecular weight MW of the polymer is 1000-5000000; more preferably, the MW is 1000-100000; further preferably, the MW is 2000-50000; further preferably, the MW is 3000-10000.

10. The DNA microsphere according to any one of claims 1 to 9, characterized in that: The DNA is obtained by amplifying a DNA primer sequence. Preferably, the length of the DNA primer sequence is 6 to 30 bp; preferably, 9 to 21 bp. Furthermore, preferably, the DNA primer sequence is combined with the polymer through the functional group before the amplification is performed. Furthermore, the DNA primer sequence is combined with the polymer through the functional group for the amplification and then connected to the surface of the microsphere body to obtain the DNA microsphere.

11. The DNA microsphere according to claim 10, characterized in that: in, The DNA primer sequence is modified with the functional group; further, the functional group is an amino group, and the 5' end of the DNA primer sequence contains an amino group; preferably, it contains an amino group at the C2-C20 position of the 5' end; more preferably, it contains an amino group at the C5-C10 position of the 5' end; most preferably, it contains an amino group at the C6 position of the 5' end.

12. A method for preparing the DNA microsphere according to any one of claims 1 to 11, characterized in that: The method comprises the following steps: (1) chemically modifying the microsphere body by using an amino silane coupling agent to introduce an amino group into the outer surface of the microsphere body to form an amino-modified microsphere A; (2) modifying a DNA primer sequence to obtain a modified DNA primer sequence; (3) combining the modified DNA primer sequence with a polymer, and then amplifying the DNA to obtain a polymer-modified DNA; and (4) reacting the polymer-modified DNA with the amino-modified microsphere A to obtain the DNA microsphere.

13. The method for preparing DNA microspheres according to claim 13, characterized in that: In step (2), the 5' end of the DNA primer sequence is modified with the amino group; preferably, the amino group is modified at the C2-C20 position of the 5' end; more preferably, the amino group is modified at the C5-C10 position of the 5' end; most preferably, the amino group is modified at the C6 position of the 5' end.

14. The method for preparing DNA microspheres according to claim 12 or 13, wherein the length of the DNA primer sequence is 6 to 30 bp; preferably 9 to 21 bp, and further, the DNA primer sequence is GGCGTAGAGGAT.

15. The method for preparing DNA microspheres according to any one of claims 12 to 14, characterized in that: The polymer is a polyolefin polymer; preferably, the polyolefin polymer is selected from one or more of substituted or unsubstituted polypropylene, polyacrylic acid, polyacrylic acid salt, polyacrylic acid ester or polyacrylamide.

16. The method for preparing DNA microspheres according to any one of claims 12 to 15, characterized in that: In step (4), before reacting the polymer-modified DNA with microsphere A, the system containing microsphere A is first subjected to ultrasonic vibration. 17 . The method for preparing DNA microspheres according to claim 16 , wherein the ultrasonic oscillation time is 1 to 30 min, preferably 5 to 20 min; more preferably 5 to 10 min.

18. Use of the DNA microsphere according to any one of claims 1 to 11, wherein the DNA microsphere is used as a DNA template.

19. Use of the DNA microsphere according to any one of claims 1 to 11 as a DNA template in RNA transcription or protein synthesis.

20. The use according to claim 20, wherein the RNA transcription or protein synthesis is in vitro RNA or in vitro protein synthesis.

21. A cell-free in vitro synthesis system, comprising: (1) Cell extracts; (2) The DNA microsphere according to any one of claims 1 to 11.

22. A cell-free in vitro synthesis method, comprising preparing the system according to claim 21.

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