Immobilized enzyme-based mRNA in-vitro transcription system and in-vitro transcription kit
Patent Information
- Application Number
- CN202510441585.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-01
AI Technical Summary
In the prior art, during mRNA in vitro transcription, T7RNA polymerase, inorganic pyrophosphatase and RNAse inhibitors are expensive and cannot be recycled, and at the same time, it is necessary to carry out expensive and time-consuming Oligo dT affinity chromatography purification steps.
The mRNA in vitro transcription system of immobilized enzymes is used to bind the enzyme with a magnetic solid-phase carrier and prevent enzyme dissociation through an ionic strength regulator to achieve the recovery and reuse of the enzyme, and avoid subsequent purification steps.
It reduces in vitro transcription costs, improves enzyme recovery, simplifies the operation process, and reduces purification steps.
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Figure CN120230813A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the technical field of in vitro transcription of mRNA, and specifically relates to an in vitro transcription system for mRNA based on immobilized enzymes and an in vitro transcription kit. Background Art
[0002] mRNA vaccines play a crucial role in disease prevention and treatment, mainly consisting of active mRNA molecules encoding the target protein and lipid delivery carriers. mRNA is mainly prepared by enzyme-catalyzed in vitro transcription. During the in vitro transcription process, three enzymes / proteins, namely RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, are used to transcribe mRNA using a linearized plasmid as a template. After transcription, DNaseI is used to digest the DNA template.
[0003] In the prior art, the three enzymes / proteins, namely T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, added for in vitro transcription of mRNA are costly and cannot be recycled. At the same time, in order to remove these three enzymes / proteins from the in vitro transcription reaction, an oligo dT affinity chromatography purification step is required. The oligo dT affinity chromatography packing is expensive and time-consuming and laborious. Summary of the Invention
[0004] The purpose of this application is to provide an in vitro transcription system for mRNA based on immobilized enzymes and an in vitro transcription kit, so as to solve the technical problems in the prior art that the three enzymes / proteins, namely T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, added for in vitro transcription are costly and cannot be recycled, and chromatography purification is required to remove the three enzymes, which is costly and time-consuming and laborious.
[0005] To achieve the above purpose, the first aspect of this application provides an in vitro transcription system for mRNA based on immobilized enzymes, including:
[0006] Immobilized enzymes, including a magnetic solid-phase carrier and enzymes bound to the magnetic solid-phase carrier, the enzymes including one or more combinations of RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, and nickel ions being chelated on the surface of the magnetic solid-phase carrier;
[0007] NTPs;
[0008] Linearized DNA template;
[0009] In vitro transcription buffer, including an ionic strength regulator for inhibiting the dissociation of the enzymes from the magnetic solid-phase carrier.
[0010] In one or more embodiments, the enzyme includes RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor. The concentration of the RNA polymerase in the system is 10 - 100 U / μL, the concentration of the inorganic pyrophosphatase in the system is 1 - 10 U / mL, and the concentration of the RNase inhibitor in the system is 0.2 - 2 U / μL.
[0011] In one or more embodiments, the ionic strength regulator is selected from one or more combinations of KCl, NaCl, CH3COONa, and (NH4)2SO4.
[0012] In one or more embodiments, the concentration of the ionic strength regulator in the system is 10 - 100 mM.
[0013] In one or more embodiments, the magnetic solid-phase carrier is one or more combinations of IDA-Ni magnetic beads, NTA-Ni magnetic beads, and TED-Ni magnetic beads.
[0014] In one or more embodiments, the concentration of the NTPs in the system is 20 - 40 mM.
[0015] In one or more embodiments, the concentration of the linearized DNA template in the system is 20 - 100 ng / μL.
[0016] In one or more embodiments, it further includes a cap structure analog, and the concentration of the cap structure analog in the system is 2 - 20 mM.
[0017] In one or more embodiments, the in vitro transcription buffer further includes a pH adjustment buffer salt, a magnesium salt, a reducing agent, a surfactant, and spermidine.
[0018] In one or more embodiments, the pH adjustment buffer salt is selected from one or more combinations of Tris-HCl, Hepes, and citrate, and the concentration of the pH adjustment buffer salt in the system is 10 - 60 mM.
[0019] In one or more embodiments, the magnesium salt is selected from one or two combinations of magnesium chloride and magnesium acetate, and the concentration of the magnesium salt in the system is 10 - 40 mM.
[0020] In one or more embodiments, the reducing agent is selected from one or two combinations of TCEP and DTT, and the concentration of the reducing agent in the system is 1 - 20 mM.
[0021] In one or more embodiments, the surfactant is selected from one or more of Triton X-100, polysorbate 20, polysorbate 80, sodium dodecyl sulfate, and poloxamer 188, and the volume fraction of the surfactant in the system is 0.01-0.1%.
[0022] In one or more embodiments, the concentration of spermidine in the system is 1-10 mM.
[0023] In one or more embodiments, the RNA polymerase is T7 RNA polymerase.
[0024] To achieve the above object, the second aspect of the present application provides an in vitro transcription kit, including the in vitro transcription system described in any of the above embodiments.
[0025] Different from the prior art, the beneficial effects of the present application are as follows:
[0026] On the one hand, the in vitro transcription system of the present application is based on immobilized enzymes for in vitro transcription and can be recovered by magnetic enrichment after transcription. On the other hand, by adding an ionic strength regulator to the in vitro transcription system, the ionic strength of the transcription system can be adjusted, effectively preventing the dissociation of the three enzymes / proteins, namely T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, from the magnetic solid-phase carrier, thereby ensuring the enzyme recovery rate, reducing the cost, and avoiding the subsequent Oligo dT affinity chromatography purification step. Description of the Drawings
[0027] To more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0028] Figure 1 It is the capillary electrophoresis diagram of the mRNA prepared in Example 1 of the present application;
[0029] Figure 2 It is the capillary electrophoresis diagram of the mRNA prepared in Comparative Example 1 of the present application. Detailed Embodiments
[0030] To enable those skilled in the art to better understand the technical solutions in this application, the following will clearly and completely describe the technical solutions in the embodiments of this application in conjunction with the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application.
[0031] The three enzymes / proteins, namely T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, added in in vitro transcription of mRNA are costly and cannot be recycled. At the same time, to remove these three enzymes / proteins from the in vitro transcription reaction, an OligodT affinity chromatography purification step is required. The Oligo dT affinity chromatography packing material is expensive and time-consuming and laborious.
[0032] To solve the above problems, the applicant has developed a new in vitro transcription system. This system uses immobilized enzymes, can effectively avoid enzyme dissociation, improve the recovery rate of enzymes, enable the enzymes to be reused, significantly reduce the cost of in vitro transcription, and avoid the subsequent affinity chromatography purification step, simplifying the in vitro transcription operation.
[0033] Specifically, the in vitro transcription system of this application includes immobilized enzymes, NTPs, linearized DNA templates, and in vitro transcription buffers.
[0034] Among them, the immobilized enzymes include magnetic solid carriers and enzymes bound to the magnetic solid carriers. The enzymes include one or more combinations of RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor.
[0035] In one embodiment, the RNA polymerase can be T7 RNA polymerase.
[0036] Among them, the T7 RNA polymerase is derived from T7 phage and is recombinantly expressed using hosts such as Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis;
[0037] The inorganic pyrophosphatase is derived from Saccharomyces cerevisiae and is recombinantly expressed using hosts such as Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis;
[0038] The RNase inhibitor is derived from mouse or human placenta and is recombinantly expressed using hosts such as Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis.
[0039] In one embodiment, the magnetic solid carrier can be one or a combination of two of IDA-Ni magnetic beads, NTA-Ni magnetic beads, and TED-Ni magnetic beads.
[0040] Since the three proteins, namely recombinant T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, are all produced by heterologous expression, and the N-terminus or C-terminus of the recombinant proteins has 6-10 His tags that can specifically bind to nickel ions, they can be bound to the magnetic solid-phase carrier chelated with nickel ions on the surface through metal ion chelation to achieve enzyme immobilization.
[0041] Of course, in other embodiments, other magnetic beads that can bind to proteins can also achieve the effects of this embodiment.
[0042] On the one hand, the magnetic solid-phase carrier can bind to the enzyme, and on the other hand, due to its magnetism, it can be enriched and recovered by a magnetic field to achieve the purpose of enzyme recovery.
[0043] In one embodiment, the enzymes bound to the magnetic solid-phase carrier can include three types: RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor.
[0044] Among them, the concentration of RNA polymerase in the system can be 10-100 U / μL, and preferably, it can be 50 U / μL.
[0045] The concentration of inorganic pyrophosphatase in the system can be 1-10 U / mL, and preferably, it can be 3 U / mL.
[0046] The concentration of RNase inhibitor in the system can be 0.2-2 U / μL, and preferably, it can be 1 U / μL.
[0047] In the system, NTPs refer to nucleoside triphosphates, including ATP, UTP, GTP, CTP, or modified nucleoside triphosphates such as N1-Me-Pseudo UTP, 5-Me-CTP, etc. In one embodiment, the concentration of NTPs in the system is 20-40 mM, and preferably, it can be 32 mM.
[0048] In the system, the linearized DNA template serves as the transcription reaction template and can be derived from the enzyme digestion and purification product of a circular plasmid or a PCR amplification product. In one embodiment, the concentration of the linearized DNA template in the system can be 20-100 ng / μL, and preferably, it can be 50 ng / μL.
[0049] To avoid the dissociation of the enzyme from the magnetic solid-phase carrier during the transcription reaction, the in vitro transcription buffer of the present application includes an ionic strength regulator, which adjusts the ionic strength of the transcription system and is beneficial to preventing the dissociation of the three enzymes / proteins, namely T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, from the magnetic solid-phase carrier.
[0050] In one embodiment, the ionic strength regulator can be selected from one or more combinations of (NH4)2SO4, KCl, NaCl, and CH3COONa. Preferably, the ionic strength regulator can be (NH4)2SO4.
[0051] In one embodiment, the concentration of the ionic strength regulator in the system can be 10 - 100 mM. Preferably, it can be 60 mM.
[0052] Specifically, T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor bind to the nickel ions of the magnetic beads through the His-tag to form coordination bonds. Due to the complexity of the components in the in vitro transcription system, the components in the system or the newly generated mRNA may interact with T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor due to the repulsive force between charges, resulting in the breakage of the coordination bonds. By increasing the ionic strength regulator, the ionic strength in the system can be increased, effectively shielding these repulsive forces between charges, thereby enhancing the binding stability between the His-tag and nickel ions.
[0053] To promote the capping of the 5' end of RNA and enhance stability, in one embodiment, the in vitro transcription system further includes a cap structure analog, and the concentration of the cap structure analog in the system can be 2 - 20 mM.
[0054] More preferably, the concentration of the cap structure analog can be 8 mM.
[0055] Furthermore, the in vitro transcription buffer of the present application further includes a pH adjustment buffer salt, a magnesium salt, a reducing agent, a surfactant, and spermidine.
[0056] Among them, the pH adjustment buffer is used to maintain the pH value in the transcription reaction system. In one embodiment, the pH adjustment buffer salt is selected from one or more combinations of Tris-HCl, Hepes, and citrate. Preferably, the pH adjustment buffer salt can be Tris-HCl.
[0057] In one embodiment, the concentration of the pH adjustment buffer salt can be 10 - 60 mM. More preferably, the concentration can be 20 mM.
[0058] The magnesium salt, as a cofactor of T7 RNA polymerase, can maintain the activity of the RNA-protein complex, stabilize the RNAP-DNA complex, promote the binding of NTP to RNAP, and is also related to the activity of inorganic pyrophosphatase. In one embodiment, the magnesium salt can be selected from one or two combinations of magnesium chloride and magnesium acetate, and the concentration of the magnesium salt in the system can be 10 - 40 mM.
[0059] More preferably, magnesium chloride can be selected as the magnesium salt, and its concentration can be 20 mM.
[0060] Reducing agents are used to prevent the oxidative inactivation of enzymes and play an important role in maintaining the activity of enzymes during the transcription process. In one embodiment, the reducing agent can be selected from one or a combination of two of TCEP and DTT, and the concentration of the reducing agent in the system is 1-20 mM.
[0061] More preferably, DTT can be selected as the reducing agent, and its concentration can be 10 mM.
[0062] Surfactants are used to promote the solubility of enzymes, maintain the stability of enzymes, and reduce the production of dsRNA. In one embodiment, the surfactant can be selected from one or more of TritonX-100, polysorbate 20, polysorbate 80, sodium dodecyl sulfate, and poloxamer 188, and the volume fraction of the surfactant in the system can be 0.01-0.1%.
[0063] More preferably, TritonX-100 can be selected as the surfactant, and the volume fraction can be 0.02%.
[0064] Spermidine plays an important role in the initiation of transcription, can dissociate RNA polymerase from the plasmid template, acts on the initiation and elongation stages of IVT, and stimulates the transcription reaction. In one embodiment, the concentration of spermidine in the system can be 1-10 mM. More preferably, the concentration of spermidine can be 2 mM.
[0065] Based on the in vitro transcription system of the above embodiments, on the one hand, in vitro transcription is carried out based on immobilized enzymes, and after the transcription is completed, it can be recovered by magnetic enrichment. On the other hand, by adding an ionic strength regulator to the in vitro transcription system, the ionic strength of the transcription system can be adjusted, effectively preventing the dissociation of the three enzymes / proteins, namely T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor, from the magnetic solid-phase carrier, thus ensuring the enzyme recovery rate, reducing the cost, and also avoiding the subsequent OligodT affinity chromatography purification step.
[0066] The effects of the technical solutions of the present application will be further elaborated in detail below with specific examples.
[0067] Example 1:
[0068] An in vitro transcription method uses immobilized enzymes based on TED-Ni magnetic beads for in vitro transcription. The steps include:
[0069] Step 1: Preparation of immobilized enzymes
[0070] Mix the TED-Ni magnetic beads well, pipette 20 μL of the suspension into a 1.5 mL centrifuge tube, place it on a magnetic stand, let it stand for 3 - 5 min, and pipette away the storage solution; then add equilibration Buffer (20 mM PBS, 0.5 M NaCl, pH 7.4 ± 0.1) with a volume 10 times that of the TED-Ni magnetic beads, pipette to mix well, place it on the magnetic stand, let it stand for 3 - 5 min, and pipette away the equilibration solution; repeat the above steps 3 times, and then add the TED-Ni magnetic beads to the equilibration Buffer containing 50 KU of T7 RNA polymerase, 3 U of inorganic pyrophosphatase, and 1000 U of RNase inhibitor, mix in a rotary mixer for 30 min, then place it on the magnetic stand, pipette away the supernatant, and wash 2 - 3 times with in vitro transcription buffer diluted to 1× to obtain the immobilized enzyme;
[0071] Step 2: In vitro transcription
[0072] Add NTPs, cap structure analog, linearized DNA template, in vitro transcription buffer, and enzyme-free and sterile water to the centrifuge tube containing the above immobilized enzyme to prepare an in vitro transcription system. The components of the in vitro transcription system are shown in Table 1. Place the centrifuge tube in a rotary mixer and react at 35 °C for 3 h;
[0073] After the reaction, place the centrifuge tube on the magnetic stand, let it stand for 3 - 5 min, pipette the supernatant into another centrifuge tube. The TED-Ni magnetic beads at the bottom of the centrifuge tube can be used for the next IVT reaction;
[0074] Add DNase I to the centrifuge tube containing the supernatant to a final concentration of 50 U / mL, react at 35 °C for 30 min, and then use a 100 kDa ultrafiltration tube to change the solution to remove DNase I and small molecule impurities to obtain purified mRNA.
[0075] Table 1 In vitro transcription system of Example 1
[0076]
[0077]
[0078] Example 2:
[0079] An in vitro transcription method, the steps are basically the same as those in Example 1, the difference is that:
[0080] In this example, an immobilized enzyme based on IDA-Ni magnetic beads is used for transcription, and the selection and concentration of each component in the in vitro transcription system are different. The in vitro transcription system of Example 2 is shown in Table 2.
[0081] Table 2 In vitro transcription system of Example 2
[0082]
[0083]
[0084] Example 3:
[0085] An in vitro transcription method, the steps are basically the same as those in Example 1, except that:
[0086] In this example, an immobilized enzyme based on NTA-Ni magnetic beads was used for transcription, and the selection and concentration of each component in the in vitro transcription system were different. See Table 3 for the in vitro transcription system of Example 3.
[0087] Table 3 In vitro transcription system of Example 3
[0088]
[0089]
[0090] Comparative Example 1:
[0091] An in vitro transcription method, the steps are basically the same as those in Example 1, except that:
[0092] In the in vitro transcription system of Comparative Example 1, the in vitro transcription buffer does not contain the ionic strength regulator (NH4)2SO4.
[0093] Effect Example:
[0094] Capillary electrophoresis was performed on the mRNA prepared in Example 1 and Comparative Example 1 to obtain Figure 1 and Figure 2 data, Figure 1 is the capillary electrophoresis diagram of the mRNA prepared in Example 1 of this application, Figure 2 is the capillary electrophoresis diagram of the mRNA prepared in Comparative Example 1 of this application.
[0095] As Figure 1 and Figure 2 shown, the target mRNA was successfully prepared in both Example 1 and Comparative Example 1, and the addition of the ionic strength regulator does not affect the integrity of the mRNA.
[0096] Furthermore, in the experimental steps of Examples 1 to 3 and Comparative Example 1, after adsorbing the magnetic beads with a magnetic stand, the supernatant was aspirated, and NanoOrange TM Protein Quantification Kit was used to detect the total protein residues in Examples 1 to 3 and Comparative Example 1. The results are shown in Table 4. The results show that the total protein residue in Example 1 is 0.49 μg / mL, the total protein residue in Example 2 is 1.21 μg / mL, the total protein residue in Example 3 is 0.54 μg / mL, and the total protein residue in Comparative Example 1 is 6.08 μg / mL.
[0097] Since the total concentration of immobilized and entrapped T7 RNA polymerase, inorganic pyrophosphatase, and RNase inhibitor is approximately 135 μg / mL, in the solutions of Examples 1 to 3, the protein dissociation ratios are 0.36%, 0.94%, and 0.40% respectively, and in the solution of Comparative Example 1, the protein dissociation ratio is 4.50%. Therefore, adding an ionic strength regulator to the in vitro transcription buffer can significantly inhibit the dissociation of enzymes on the magnetic solid support, which is beneficial to the reuse of the immobilized enzymes.
[0098] Table 4 Protein Residue in the IVT Reaction Solution after Magnetic Bead Recovery
[0099]
[0100] For those skilled in the art, it is obvious that this application is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of this application, this application can be implemented in other specific forms. Therefore, from any perspective, the embodiments should be regarded as exemplary and non-limiting. The scope of this application is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within this application. Any reference signs in the claims should not be construed as limiting the claimed rights.
[0101] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.
Claims
1. An mRNA in vitro transcription system based on immobilized enzyme, characterized in that: include: An immobilized enzyme comprises a magnetic solid phase carrier and an enzyme bound to the magnetic solid phase carrier, wherein the enzyme comprises one or more combinations of RNA polymerase, inorganic pyrophosphatase and RNA enzyme inhibitor, and the surface of the magnetic solid phase carrier is chelated with nickel ions; NTPs; Linearize DNA template; The in vitro transcription buffer comprises an ionic strength regulator for inhibiting the enzyme from dissociating from the magnetic solid phase carrier.
2. The in vivo mRNA transcription system according to claim 1, characterized in that: The enzymes include RNA polymerase, inorganic pyrophosphatase and RNA enzyme inhibitor. The concentration of the RNA polymerase in the system is 10-100 U / μL, the concentration of the inorganic pyrophosphatase in the system is 1-10 U / mL, and the concentration of the RNA enzyme inhibitor in the system is 0.2-2 U / μL.
3. The mRNA in vitro transcription system according to claim 1, characterized in that The ionic strength regulator is selected from one or more combinations of (NH4)2SO4, KCl, NaCl, CH3COONa; and / or, The concentration of the ionic strength regulator in the system is 10-100 mM.
4. The mRNA in vitro transcription system according to claim 1, characterized in that The magnetic solid phase carrier is one or more combinations of IDA-Ni magnetic beads, NTA-Ni magnetic beads and TED-Ni magnetic beads.
5. The mRNA in vitro transcription system according to claim 1, characterized in that: The concentration of the NTPs in the system is 20-40 mM; and / or, The concentration of the linearized DNA template in the system is 20-100 ng / μL.
6. The mRNA in vitro transcription system according to claim 1, characterized in that: Also included is a cap structure analogue, and the concentration of the cap structure analogue in the system is 2-20 mM.
7. The mRNA in vitro transcription system according to claim 1, characterized in that: The in vitro transcription buffer further comprises a pH adjusting buffer salt, a magnesium salt, a reducing agent, a surfactant and spermidine.
8. The mRNA in vitro transcription system according to claim 7, characterized in that: The pH adjusting buffer salt is selected from one or more combinations of Tris-HCl, Hepes and citrate, and the concentration of the pH adjusting buffer salt in the system is 10-60 mM; and / or, The magnesium salt is selected from one or a combination of magnesium chloride and magnesium acetate, and the concentration of the magnesium salt in the system is 10-40 mM; and / or, The reducing agent is selected from one or a combination of TCEP and DTT, and the concentration of the reducing agent in the system is 1-20 mM; and / or, The surfactant is selected from one or more of Triton X-100, polysorbate 20, polysorbate 80, sodium lauryl sulfate, and poloxamer 188, and the volume fraction of the surfactant in the system is 0.01-0.1%; and / or, The concentration of spermidine in the system is 1-10 mM.
9. The mRNA in vitro transcription system according to any one of claims 1 to 8, characterized in that: The RNA polymerase is T7 RNA polymerase.
10. An in vitro transcription kit, characterized in that: The invention comprises the mRNA in vitro transcription system described in any one of claims 1 to 9.