Immobilized enzyme for catalyzing RNA ligation reaction, preparation method therefor, and use thereof

Abstract

Provided are an immobilized enzyme for catalyzing an RNA ligation reaction, a preparation method therefor, and use thereof. The immobilized enzyme comprises an immobilized carrier and an enzyme immobilized on the immobilized carrier. The immobilized carrier is an affinity carrier, and the enzyme is an enzyme that catalyzes the RNA ligation reaction. The present invention can solve the problem in the prior art that free enzymes capable of catalyzing RNA ligation reactions are unsuitable for industrial production, and is applicable to the field of immobilized enzyme preparation.

Description

Immobilized enzymes for catalyzing RNA ligation reactions, their preparation methods, and applications.

[0001] This application is based on and claims priority to Chinese application CN application number 202411797672X, filed on December 9, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of immobilized enzyme preparation, and more specifically, to an immobilized enzyme for catalyzing RNA ligation reactions, its preparation method, and its applications. Background Technology

[0003] Small nucleic acid drugs refer to nucleotide sequences of no more than 30 base pairs. Their mechanism of action involves specific nucleotide sequences acting on mRNA, thereby inhibiting the expression of target proteins and achieving therapeutic effects. Small nucleic acid drugs include various types of small nucleic acid molecules such as small interfering RNA (siRNA), microRNA (miRNA), mRNA, and RNA aptamers. Nucleic acid ligases can be classified into DNA ligases and RNA ligases. Current research has found that RNA ligases participate in the repair, splicing, and editing of damaged RNA molecules, or alteration of the primary structure of damaged RNA molecules. Therefore, the in vitro catalytic synthesis of small nucleic acid drugs using RNA ligases has significant clinical implications.

[0004] Enzyme immobilization technology is widely used worldwide. Compared with free enzymes, immobilized enzymes retain their high efficiency, specificity, and mild catalytic reaction characteristics while overcoming the shortcomings of free enzymes. They offer advantages such as high storage stability, easy separation and recovery, reusability, continuous and controllable operation, and simple processes. Therefore, immobilizing enzymes that catalyze RNA ligation reactions allows for enzyme reuse. Furthermore, after immobilization, washing and other steps ensure the target protein is fixed on the carrier, effectively purifying the enzyme and eliminating nuclease residues. Applying immobilized enzymes to continuous RNA synthesis reduces the steps of separating reaction products from the enzyme, lowers production costs, and increases production efficiency and capacity.

[0005] Currently, no reports have been found regarding this type of immobilized enzyme. If an immobilized enzyme with high enzyme activity recovery and good reusability can be developed for catalyzing RNA ligation reactions, not only can purification steps and removal of nucleases be eliminated, but costs can also be greatly reduced and production efficiency improved. Summary of the Invention

[0006] The main objective of this invention is to provide an immobilized enzyme for catalyzing RNA ligation reactions, its preparation method, and its application, in order to solve the problem that free enzymes capable of catalyzing RNA ligation reactions in the prior art are not suitable for industrial production.

[0007] To achieve the above objectives, according to a first aspect of the present invention, an immobilized enzyme for catalyzing RNA ligation reactions is provided, the immobilized enzyme comprising an immobilization carrier and an enzyme immobilized on the immobilization carrier, wherein the immobilization carrier is an affinity carrier and the enzyme is an enzyme that catalyzes RNA ligation reactions; the ligand of the immobilization carrier is one or more selected from nitrotriacetic acid, iminodiacetic acid, glutathione, Halo Tag, dextrin, or Strep-Tactin.

[0008] Furthermore, the matrix of the immobilization carrier is one or more of styrene, polystyrene, acrylic acid, polyacrylic acid, methacrylate, butyl methacrylate, silica, dextran, and agarose; preferably, the matrix of the immobilization carrier is agarose.

[0009] Furthermore, the connecting arms of the immobilized carrier are C6 to C14.

[0010] Further, the immobilization vector includes one or more of the following affinity vectors: EziG Amber, EziG Coral, EziG Opal, IB-HIS-1, IB-HIS-2, IMAC Chromstar FF, Ni Chromstar FF, Ni-IDA Purose 6FF, Ni-NTA Purose 6FF, AEC-9001, AEC-9002, AEC-9007, AEC-9008, AEC-9050, AEC-9051, AEC-9052, AEC-9060, AEC-9061, AEC-9062, AEC-9063, Glutathione Beads 4FF, Halo Link™ Resin, MBPseq Dextrin Agarose Resin 6FF, or Streptactin Agarose Resin 4FF; preferably, the immobilization vector includes the affinity vectors Ni Chromstar FF and Ni-IDA Purose. One or more of 6FF, Ni-NTA Purose 6FF, AEC-9002, AEC-9007, AEC-9008, AEC-9060 or AEC-9063; more preferably, the immobilization vector includes the affinity vector AEC-9007 or AEC-9008.

[0011] Furthermore, the enzyme used to catalyze the RNA ligation reaction is any one or more of RNA ligase families 1, 2, and 3.

[0012] Furthermore, the enzyme used to catalyze the RNA ligation reaction is derived from pure enzyme solution or crude enzyme solution.

[0013] To achieve the above objective, according to a second aspect of the present invention, a method for preparing the enzyme for catalyzing RNA ligation reaction is provided, the method comprising: mixing an immobilization carrier with an enzyme for catalyzing RNA ligation reaction and performing immobilization incubation to obtain an immobilized enzyme.

[0014] Further, the above preparation method includes: S1, washing the immobilization carrier for the first time; S2, mixing the washed immobilization carrier with an enzyme that catalyzes the RNA ligation reaction and incubating for immobilization; S3, washing the incubated immobilized enzyme for the second time to obtain the immobilized enzyme used to catalyze the RNA ligation reaction.

[0015] Further, S1 includes: performing two first washes on the immobilized carrier using a first buffer solution, the first buffer solution comprising 0.05-0.2M PB, 0.2-1M NaCl, 20-100mM imidazole, pH 7.0-8.0; preferably, the first buffer solution comprises 0.1M PB, 0.5M NaCl, 50mM imidazole, pH 7.0-8.0.

[0016] Further, the immobilization incubation in S2 includes: mixing the cleaned immobilized carrier with an enzyme that catalyzes the RNA ligation reaction, stirring and incubating at 20°C for 4 to 48 hours, followed by filtration; preferably, the enzyme that catalyzes the RNA ligation reaction includes an enzyme solution, and the volume ratio of the enzyme solution to the cleaned immobilized carrier is 2-6:1, preferably 4:1; more preferably, the enzyme solution includes crude enzyme solution or pure enzyme solution; even more preferably, the protein concentration in the crude enzyme solution is 20 to 50 mg / mL, and the protein concentration in the pure enzyme solution is 1 to 5 mg / mL; preferably, the stirring time is 4 to 16 hours.

[0017] Further, S3 includes: performing a second wash 3 to 4 times on the incubated immobilized enzyme using a second buffer, the second buffer comprising 0.05-0.2M PB, 10-50mM imidazole, pH 7.0-8.0; preferably, the second buffer comprises 0.1M PB, 30mM imidazole, pH 7.0-8.0.

[0018] To achieve the above objectives, according to a third aspect of the present invention, the immobilized enzyme described above for catalyzing RNA ligation reactions, or the immobilized enzyme prepared by the above-described method for preparing immobilized enzymes for catalyzing RNA ligation reactions, is provided for use in RNA synthesis.

[0019] Further applications include setting immobilized enzymes used to catalyze RNA ligation reactions in a continuous flow apparatus to achieve continuous RNA synthesis.

[0020] By applying the technical solution of the present invention, the immobilized enzyme using the above-mentioned immobilization carrier as an affinity carrier can be reused for catalyzing RNA ligation reactions, achieving efficient enzyme immobilization, improving enzyme utilization and stability, and reducing the purification pressure of enzymes before the reaction. This includes, but is not limited to, applications in continuous RNA synthesis, which will reduce the steps of separating reaction products from enzymes, reduce production costs, and improve production efficiency and capacity. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 shows the UPLC results according to Embodiment 8 of the present invention.

[0023] Figure 2 shows a schematic diagram of a single-feed continuous reaction apparatus according to Embodiment 13 of the present invention.

[0024] Figure 3 shows a schematic diagram of a continuous reaction apparatus with split-feed according to Embodiment 13 of the present invention.

[0025] The above figures include the following reference numerals: A: substrate system; B: peristaltic pump; C: packed bed reactor; D: post-reaction system; A1: substrate 1 system; A2: substrate 2 system; A3: substrate 3 system; A4: substrate 4 system; B1: substrate 1 peristaltic pump; B2: substrate 2 peristaltic pump; B3: substrate 3 peristaltic pump; B4: substrate 4 peristaltic pump; C1: packed bed reactor; D1: post-reaction system; E: mixer. Detailed Implementation

[0026] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0027] Currently, the synthesis of small RNA molecules in vitro mainly relies on in vitro transcription and solid-phase synthesis. However, existing solid-phase synthesis methods primarily utilize primer synthesis, which limits the scale of synthesis and makes it difficult to scale up to production. Furthermore, due to the inherent characteristics of solid-phase synthesis, increasing chain length leads to decreased purity and yield, resulting in problems such as reduced coupling efficiency, decreased carrier physical stability, and increased branched impurities. Currently, using enzymes to catalyze RNA ligation reactions for in vitro RNA synthesis requires enzyme purification or secondary purification to remove nucleases before use; otherwise, the oligonucleotide substrate is easily degraded, making the reaction difficult and introducing impurities such as endotoxins. The purification process involves affinity resins, purification equipment, and manual operation, and the enzymes are unstable and easily inactivated. Purified enzymes used for RNA synthesis can only be used once and cannot be reused. All these factors combined result in high costs for in vitro RNA synthesis. In this application, the inventors attempt to develop an immobilized enzyme for catalyzing RNA ligation reactions, and thus propose a series of protective schemes.

[0028] In a first typical embodiment of this application, an immobilized enzyme for catalyzing RNA ligation reaction is provided. The immobilized enzyme includes an immobilization carrier and an enzyme immobilized on the immobilization carrier. The immobilization carrier is an affinity carrier, and the enzyme is an enzyme that catalyzes RNA ligation reaction. The ligand of the immobilization carrier is one or more of nitrilotriacetic acid, iminodiacetic acid, glutathione, Halo Tag, dextrin, or Strep-Tactin.

[0029] In this application, the inventors unexpectedly discovered that using an affinity carrier containing the aforementioned ligand as an immobilization carrier for enzymes used in catalyzing RNA ligation reactions can form a stable binding with minimal impact on enzyme activity. This binding not only facilitates the purification of the ligase but also allows for its reuse, thereby improving its economic efficiency and environmental friendliness in the catalytic synthesis of non-natural RNA.

[0030] The Halo Tag ligand is a chloroalkane-PEG compound that is itself a genetically modified substrate for the bacterial dehalogenase dehalogenase (DhaA from Rhodococcus sp.), enabling it to covalently bind efficiently to the dehalogenase under physiological conditions. The Strep-Tactin ligand is a specially modified streptavidin derivative that binds efficiently and specifically to the short peptide tag Strep-tag.

[0031] Nitrotriacetic acid (NTA) and iminodiacetic acid (IDA) can react with Ni 2+Metal ions form stable chelate structures, exhibiting good recognition of proteins containing His tags. Affinity carriers modified with ligands such as glutathione, Halo Tag, dextrin, or Strep-Tactin also possess specific recognition capabilities for proteins with specific tags. When these ligands are covalently bound to carrier materials such as resin and agarose, the recognition and binding of protein sequences enables better immobilization of enzymes catalyzing RNA ligation reactions, resulting in immobilized enzymes with superior performance for RNA ligation.

[0032] Using carriers with the aforementioned ligands as enzyme immobilization carriers can effectively immobilize enzymes that catalyze RNA ligation reactions. Compared to other ligands, such as primary amines, secondary amines, epoxy groups, and cyano groups, it can avoid modification of amino acid residues near the active site, and can directionally immobilize the enzyme onto the carrier, eliminating the influence of contaminating enzymes, retaining high enzyme activity, and improving the stability of the immobilized enzyme, thus enabling the enzyme to be reused.

[0033] In a preferred embodiment, the matrix of the immobilization carrier is one or more of styrene, polystyrene, acrylic acid, polyacrylic acid, methacrylate, butyl methacrylate, silica, dextran, and agarose; preferably, the matrix of the immobilization carrier is agarose.

[0034] In a preferred embodiment, the connecting arms of the immobilized carrier are C6 to C14.

[0035] The relationship between the carrier matrix and the linker arms primarily lies in how they work synergistically to achieve enzyme immobilization, thereby improving enzyme stability and reusability during the synthesis of non-natural RNA. The carrier matrix is ​​typically a solid material with high surface area and good mechanical stability, providing a physical platform for enzyme attachment. The linker arms, acting as a bridge between the carrier matrix and the enzyme molecule, influence the spatial arrangement and activity of the enzyme molecule.

[0036] The linker arm length of the immobilization carrier used in this application is preferably C6 to C14, indicating that the linker arm is a molecule composed of 6 to 14 carbon atoms. Linker arms within the above length range can effectively immobilize the enzyme on the carrier, without affecting catalytic efficiency, and ensure a stable connection between the enzyme and the carrier.

[0037] In a preferred embodiment, the immobilized vector is one or more of the following affinity vectors: EziG Amber, EziG Coral, EziG Opal, IB-HIS-1, IB-HIS-2, IMAC Chromstar FF, Ni Chromstar FF, Ni-IDA Purose 6FF, Ni-NTA Purose 6FF, AEC-9001, AEC-9002, AEC-9007, AEC-9008, AEC-9050, AEC-9051, AEC-9052, AEC-9060, AEC-9061, AEC-9062, AEC-9063, Glutathione Beads 4FF, Halo Link™ Resin, MBPseq Dextrin Agarose Resin 6FF, or Streptactin Agarose Resin 4FF.

[0038] Preferably, the immobilization support includes one or more of the following: Ni Chromstar FF, Ni-IDA Purose 6FF, Ni-NTA Purose 6FF, AEC-9002, AEC-9007, AEC-9008, AEC-9060, or AEC-9063; more preferably, the immobilization support includes the nickel affinity support AEC-9007 or AEC-9008.

[0039] The affinity vectors applicable to this application are shown in Tables 1 and 2, and the vectors in Table 2 are all commercially available vectors.

[0040] Table 1

[0041] Table 2

[0042] Using the aforementioned affinity carriers, immobilized enzymes for catalyzing RNA ligation reactions can be prepared. However, due to differences in carrier structure and other factors among different carrier types, the activity, stability, and reusability of the immobilized enzymes also vary. Among the enzymes used for catalyzing RNA ligation reactions, the immobilized enzymes prepared using AEC-9007 or AEC-9008 resins exhibit the best activity and stability.

[0043] In a preferred embodiment, the enzyme catalyzing the reverse RNA ligation is any one or more of RNA ligase families 1, 2, and 3.

[0044] In a preferred embodiment, the enzyme is derived from pure enzyme solution or crude enzyme solution.

[0045] In a second typical embodiment of this application, a method for preparing the above-mentioned immobilized enzyme for catalyzing RNA ligation reaction is provided. The method includes: mixing an immobilization carrier with an enzyme for catalyzing RNA ligation reaction and performing immobilization incubation to obtain the immobilized enzyme.

[0046] In a preferred embodiment, the preparation method includes: S1, washing the immobilization carrier for the first time; S2, mixing the washed immobilization carrier with an enzyme that catalyzes RNA ligation reaction and incubating for immobilization; S3, washing the incubated immobilized enzyme for the second time to obtain the immobilized enzyme.

[0047] In the above preparation method, an optimized process is proposed for the preparation of immobilized enzymes catalyzing RNA ligation reactions, aiming to improve enzyme immobilization efficiency, purity, and activity retention, thereby ensuring its performance in non-natural RNA synthesis. The following is a detailed analysis of the principles and effects of each step:

[0048] In the first washing step, the immobilization carrier may adsorb residual chemicals, microorganisms, or their metabolites during storage or transportation. These impurities can not only affect the enzyme's adsorption efficiency but also introduce non-specific reactions or contamination in subsequent catalytic reactions. Therefore, the first washing step, using a buffer solution, effectively removes these impurities, creating a clean environment for enzyme immobilization. Through this first washing, the carrier surface is cleaned, reducing non-specific adsorption that may occur during subsequent immobilization and improving the purity and specific adsorption rate of the ligase. This step is crucial for the subsequent enzyme activity and stability, contributing to improved RNA synthesis efficiency and yield.

[0049] In the second washing step, unbound enzyme molecules or chemical components from the washing solution may remain on the surface of the immobilized enzyme after incubation. These residues may affect the enzyme's catalytic activity or the stability of the reaction system. Therefore, the second washing step aims to thoroughly remove these residues and ensure the purity of the immobilized enzyme. This second washing step further improves the purity of the immobilized enzyme by removing residues that may inhibit enzyme activity or cause nonspecific reactions. Using a buffer containing an appropriate concentration of imidazole for washing avoids enzyme desorption caused by over-washing, while ensuring the purity of the reaction system, which is beneficial for improving the efficiency of RNA synthesis and the purity of the product.

[0050] The aforementioned method for preparing immobilized RNA ligases effectively avoids the complex steps and costs associated with enzyme purification. Furthermore, optimized immobilization and washing procedures ensure the stability and activity of the enzyme on the carrier, as well as the purity of the reaction system. This method not only simplifies the enzyme preparation process but also significantly improves the efficiency and yield of RNA synthesis, enabling large-scale production and better aligning with the requirements of green chemistry and environmental protection.

[0051] In a preferred embodiment, S1 includes: performing two first washes on the immobilized carrier using a first buffer solution, the first buffer solution comprising 0.05-0.2M PB, 0.2-1M NaCl, 20-100mM imidazole, and pH 7.0-8.0; preferably, the first buffer solution comprises 0.1M PB, 0.5M NaCl, 50mM imidazole, and pH 7.0-8.0.

[0052] In a preferred embodiment, the immobilization incubation in S2 includes: mixing the cleaned immobilized carrier with an enzyme that catalyzes the RNA ligation reaction, stirring and incubating at 20°C for 4 to 48 hours, followed by filtration; preferably, the enzyme that catalyzes the RNA ligation reaction includes an enzyme solution, and the volume ratio of the enzyme solution to the cleaned immobilized carrier is 2-6:1, preferably 4:1; more preferably, the enzyme solution includes crude enzyme solution or pure enzyme solution; even more preferably, the protein concentration in the crude enzyme solution is 20 to 50 mg / mL, and the protein concentration in the pure enzyme solution is 1 to 5 mg / mL; preferably, the stirring time is 4 to 16 hours.

[0053] In a preferred embodiment, S3 includes: performing a second wash 3-4 times on the incubated immobilized enzyme using a second buffer solution, the second buffer solution comprising 0.05-0.2M PB, 10-50mM imidazole, and pH 7.0-8.0; preferably, the second buffer solution comprises 0.1M PB, 30mM imidazole, and pH 7.0-8.0. Preferably, the salt ion concentration in the second buffer solution is lower than the salt ion concentration in the first buffer solution, and the salt ions include, but are not limited to, sodium chloride and phosphate salt ions.

[0054] In the above-described method for preparing immobilized enzymes, the incubation enzyme solution can be either pure or crude. This method is a rapid and simple way to prepare high-purity immobilized enzymes that catalyze RNA ligation reactions. It can utilize crude enzyme solutions, greatly simplifying the immobilized enzyme preparation process and significantly reducing costs. Simultaneously, purified enzymes can be used to prepare immobilized enzymes, allowing for easy enzyme recovery and reuse, thus improving enzyme utilization. Washing with salts removes impurities such as host proteins, RNA, or DNA bound to the immobilization carrier, resulting in a contamination-free immobilized enzyme. Reactions catalyzed by this contamination-free immobilized enzyme have low impurity content in the reaction products, meeting the high-quality requirements of subsequent production processes such as injectable drug manufacturing. This reduces additional purification steps, thereby saving costs and improving yield, and facilitating industrial-scale production.

[0055] In a third typical embodiment of this application, an enzyme that catalyzes the RNA ligation reaction as described above, or an immobilized enzyme that catalyzes the RNA ligation reaction prepared using the above-described method for preparing immobilized enzymes, is provided for use in RNA synthesis.

[0056] In a preferred embodiment, the application includes setting an immobilized enzyme for catalyzing RNA ligation reactions in a continuous flow apparatus to achieve continuous RNA synthesis.

[0057] In a preferred embodiment, continuous RNA synthesis includes synthesizing RNA using a single-channel continuous reaction or a multi-channel continuous reaction.

[0058] In a preferred embodiment, the continuous flow device includes a pump, a reaction system, and a reaction column.

[0059] In a preferred embodiment, the continuous flow device includes a pump, a reaction system, a mixer, and a reaction column.

[0060] In a preferred embodiment, the pump described above includes one or more of a micro-peristaltic pump, a high-efficiency liquid phase pump, or a syringe pump.

[0061] In a preferred embodiment, the above reaction system includes one, two, three, or four RNA substrates.

[0062] In a preferred embodiment, the reaction column is packed with the immobilized enzyme described above for catalyzing the RNA ligation reaction.

[0063] In a preferred embodiment, the mixer includes a dynamic mixer or a static mixer; preferably, the static mixer includes a static baffle mixer, an orifice plate mixer, or a three-way mixer.

[0064] In a preferred embodiment, the synthesized RNA product includes natural RNA and non-natural RNA. Preferably, the non-natural RNA includes one or more ribonucleotides having a 2'-ribose modification, a ribose backbone modification, or a base modification. Preferably, the 2'-ribose modification includes 2'-methoxy modification, 2'-fluorine modification, 2'-hydrogen modification, 2'-methoxyethyl modification, 2'-FANA modification, locked nucleic acid modification, or hexitol nucleic acid modification.

[0065] Among them, locked nucleic acid modification is Hexitol nucleic acid modification 2'-methoxyethyl modification 2' methoxy group modified to 2' Fluorine modification 2'-FANA is In the above structures, Base represents a base.

[0066] Non-natural RNA is an important tool in biomedical and life science research. Currently, most non-natural RNA is synthesized using automated equipment via a solid-phase phosphoramide chemical process, which consists of four cyclic steps: deprotection, coupling, oxidation, and capping. However, solid-phase synthesis has significant drawbacks. It can only be used for relatively short RNA fragments, and the yield drops drastically with increasing chain length. Furthermore, the yield of solid-phase synthesis is low, the cost of synthesized RNA fragments remains high, and large-scale production is not yet feasible. Additionally, the excessive use of toxic chemicals in the process creates significant waste disposal challenges. Synthesizing long-chain RNA products using enzymes that catalyze RNA ligation requires the preparation of purified enzymes, which is time-consuming and labor-intensive, incurring substantial material and labor costs. Moreover, the purified liquid enzyme cannot be recovered from the reaction system, impacting post-processing enzyme removal. Therefore, developing a more environmentally friendly and efficient method for synthesizing non-natural RNA using immobilized enzymes is essential.

[0067] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0068] Example 1: Construction and expression of RNA ligase

[0069] This embodiment uses the enzyme used in Chinese patent CN117070512B as an example. The RNA ligase protein was codon-optimized to create a host bacterium for recombinant expression in *E. coli*. The optimized codons were synthesized to obtain the target gene, which was then constructed into the pET28a expression vector with NcoI and XhoI restriction sites and a His tag at the N-terminus. The recombinant plasmid containing the ligase gene was transformed into the cloning strain DH5α and cultured overnight on LB + 1.5% agar medium containing 50 μg / mL kanamycin to obtain single clones. The sequence of the single clones was confirmed by sequencing.

[0070] Single colonies were isolated and cultured in LB liquid medium containing 50 μg / mL kanamycin at 37°C for 16 h. The cells were centrifuged, and the supernatant was discarded to obtain bacterial sludge. Plasmids were extracted from the bacterial sludge using a plasmid extraction kit. The obtained plasmids were transformed into the BL21(DE3) expression strain and cultured overnight in LB medium containing 50 μg / mL kanamycin and 1.5% agar to obtain single colonies. Each single colony was isolated and cultured in 5 mL of LB medium containing 50 μg / mL kanamycin at 37°C for 16 h to obtain a seed culture. The seed culture was inoculated at a rate of 0.1% into 500 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C until OD (dryness). 600=0.6, add 0.1M IPTG for induction, adjust the culture temperature to 20℃, and incubate at 220rpm for 16h to obtain the induced expression of Escherichia coli ligase.

[0071] The bacterial culture was centrifuged at 8000 rpm, and the supernatant was discarded to obtain induced expression *E. coli* bacterial sludge. The obtained ligase-containing *E. coli* bacterial sludge was resuspended in 100 mM Tris-CL buffer to prepare a 10% bacterial concentration. The sludge was then sonicated at 20% power for 2 seconds followed by a 6-second pause. The resulting enzyme solution was centrifuged at 12000 rpm for 30 minutes at 4°C. The supernatant was the crude RNA ligase solution. 10 mM imidazole was added to the crude enzyme solution, and the solution was filtered through a 0.22 μm filter. The resulting enzyme solution was purified using a nickel column. After desalting and medium exchange, it was purified a second time using a strong anion exchange column. The resulting enzyme solution was then desalted and medium exchanged before being stored in 30% glycerol to obtain the pure RNA ligase solution.

[0072] It should be noted that in subsequent experiments, the N-terminal His tag of the RNA ligase used for immobilization with the glutathione ligand affinity vector was replaced with a GST tag; the N-terminal His tag of the RNA ligase used for immobilization with the Halo Tag ligand affinity vector was replaced with a Halo Tag tag; the N-terminal His tag of the RNA ligase used for immobilization with the dextrin ligand affinity vector was replaced with an MBP tag; and the N-terminal His tag of the RNA ligase used for immobilization with the Strep-Tactin ligand affinity vector was replaced with a Strep-II Tag tag.

[0073] Example 2 Preparation of Immobilized RNA Ligase

[0074] a. Wash the vector 1-2 times with 0.1M PB 7.0-8.0 buffer (containing 0.2-1M NaCl + 20-100mM imidazole); b. Mix the vector with 4 times (4V) of RNA ligation pure enzyme solution or crude enzyme solution, stir at 20℃ for 4-48 hours, filter, and wash 3-4 times with 0.1M PB 7.0-8.0 buffer (containing 0.5M NaCl + 50mM imidazole) to obtain the immobilized RNA ligase of the affinity vector.

[0075] The above-mentioned method for preparing immobilized RNA ligase is not limited to a specific RNA ligase. For RNA ligases of different sources and sequences, the above method can be used to prepare the immobilized RNA ligase described in this application.

[0076] Example 3 utilizes an immobilized RNA ligase prepared from purified enzyme to catalyze the synthesis of natural tRNA.

[0077] Substrate 1 and Substrate 2 were added to a clean reagent bottle (substrate 1 and substrate 2 are shown in Table 3) to prepare a reaction system with a final volume of 100 μL. The concentrations of substrate 1 and substrate 2 were both 500 μM, ATP was 1 mM, Tris-HCl (pH 7.5 @ 25℃) was 50 mM, MgCl2 was 10 mM, DTT was 1 mM, and 30 mg of immobilized ligase prepared in Example 2 using pure RNA ligase solution was added. The reaction system was reacted at 16℃ for 16 h. The resulting reaction system was centrifuged at 3000 rpm for 2 min to remove the immobilized ligase solid. The supernatant was analyzed by SDS-PAGE and MS. The results showed that the immobilized ligase could catalyze the synthesis of the final product, and the UPLC results showed that the substrate had been almost completely converted. The molecular weight was identified by mass spectrometry as 23560.3, and the theoretical molecular weight was 23564 ± 5, which is the target product. This indicates that the method of catalyzing the synthesis of natural tRNA using immobilized ligase was successful.

[0078] Table 3

[0079] P represents the phosphate group at position 5 of the C atom of the 5' end nucleotide.

[0080] The carriers used and the specific results are shown in Table 4:

[0081] Table 4

[0082] Example 4: Synthesis of non-natural tRNA catalyzed by immobilized RNA ligase prepared from crude enzyme.

[0083] The reaction system was as shown in Example 3 (substrate 3 and substrate 4 are shown in Table 5), except that 30 mg of the immobilized ligase prepared in Example 2 using crude RNA ligase solution was added. Results showed that the immobilized ligase catalyzed the synthesis of the final product, and UPLC results indicated that the substrates were essentially completely converted. Mass spectrometry analysis revealed a molecular weight of 24601.6, with a theoretical molecular weight of 24606.2 ± 5, confirming it as the target product. This demonstrates the success of the immobilized ligase-catalyzed synthesis of non-natural tRNA.

[0084] Table 5

[0085] In this context, m before A, C, G, or U indicates a 2' methoxy modification of the ribonucleotide, and P indicates a phosphate group on the C atom at position 5 of the 5' end of the nucleotide.

[0086] All ribonucleotides of substrates 3 and 4 have a 2' methoxy group modified on them.

[0087] The carriers used and the specific results are shown in Table 6:

[0088] Table 6

[0089] Example 5 utilizes an immobilized RNA ligase prepared from crude enzyme to catalyze the synthesis of double-stranded ligations of non-natural RNA.

[0090] Substrate 5 to substrate 8 were mixed in equimolar proportions (substrate 5 to substrate 8 are shown in Table 7), 10 μL of 1 mM ATP was added, 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) was added, and water was added to make the substrate concentration 50 μM. 30 mg of the immobilized ligase prepared in Example 2 was added, and the reaction was carried out at 25 °C for 3 h. The resulting reaction system was centrifuged at 3000 rpm for 2 min, the immobilized ligase solid was removed, and the supernatant was analyzed by denaturing polyacrylamide gel electrophoresis. The results showed that new bands were generated and the substrate bands disappeared. UPLC analysis of the sample revealed a target peak with increased molecular weight, identified as the product peak, with virtually no substrate remaining. MS analysis detected peaks of 4613.12 and 6633.07, with a theoretical molecular weight of 4609.91±5 for the sense strand and 6634.99±6 for the antisense strand. This indicates that the immobilized ligase catalyzed the product formation, generating ligated double-stranded RNA.

[0091] Table 7

[0092] Wherein, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, and f indicates 2' fluorine modification of the ribonucleotide.

[0093] Substrate 5 has 2' methoxy modifications at positions 1, 2, 5, and 6 of the ribonucleotides, and 2' fluorine modifications at positions 3 and 4 of the ribonucleotides.

[0094] Substrate 6 has 2' methoxy modifications at positions 3, 4, 7, and 8, and 2' fluorine modifications at positions 1, 2, 5, and 6.

[0095] Substrate 7 has 2' methoxy modification at positions 2, 3, 6, 7, 10, and 11 of the ribonucleotides, and 2' fluorine modification at positions 1, 4, 5, 8, and 9 of the ribonucleotides.

[0096] Substrate 8 has 2' methoxy modification at positions 1, 4, 5, 8 and 9 of the ribonucleotides, and 2' fluorine modification at positions 2, 3, 6 and 7 of the ribonucleotides.

[0097] The carriers used and the specific results are shown in Table 8:

[0098] Table 8

[0099] Example 6 utilizes an immobilized ligase prepared from crude enzyme to catalyze the gap ligation of non-natural RNA.

[0100] Substrate 9–12 was mixed in equimolar proportions (substrate 9–12 are shown in Table 9) and added to a reaction flask. 10 μL of 1 mM ATP was added, along with 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT). 50 mg of the immobilized ligase prepared in Example 2 was added, and water was added to bring the substrate concentration to 400 μM. The reaction was carried out at 25 °C for 16 h. The resulting reaction system was centrifuged at 3000 rpm for 2 min, and the immobilized ligase solid was removed to obtain the supernatant. Analysis was performed using denaturing polyacrylamide gel electrophoresis. High-brightness product bands were generated in the catalytic systems of immobilized enzymes using different types of carriers. UPLC analysis revealed a target peak with increased molecular weight, identified as the product peak. MS analysis confirmed consistency with the standard, confirming product formation. This demonstrates that the immobilized ligase can synthesize non-natural RNA products using nicked double-stranded non-natural RNA as a substrate.

[0101] Table 9

[0102] Wherein, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, and f indicates 2' fluorine modification of the ribonucleotide.

[0103] Substrate 9 has 2' methoxy modification on the 1st and 3rd ribonucleotides and 2' fluorine modification on the 2nd and 4th ribonucleotides.

[0104] Substrate 10 has 2' methoxy modifications at positions 1, 3, and 5 of the ribonucleotide, and 2' fluorine modifications at positions 2 and 4 of the ribonucleotide.

[0105] Substrate 11 has 2' methoxy modification at positions 2, 4, and 6, and 2' fluorine modification at positions 1, 3, and 5.

[0106] Substrate 12 has 2' methoxy modification at positions 1, 3, 5, 7, 9, 11, 13, and 15, and 2' fluorine modification at positions 2, 4, 6, 8, 10, 12, and 14.

[0107] Example 7: Ligation of thio-modified double-stranded RNA using immobilized RNA ligase

[0108] A mixture of substrates 13-16 in equimolar proportions of 50 μM was added to a reaction flask to prepare a ligation reaction system consisting of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, and 20 mg of the immobilized RNA ligation crude enzyme prepared in Example 2. Water was added to bring the substrate concentration to 50 μM, and the reaction was carried out at 16 °C for 16 h. After the reaction was completed, the reaction was terminated by incubation at 80 °C for 5 min. UPLC analysis of the samples revealed that the target peak with increased molecular weight was obtained in the catalytic systems of the immobilized enzyme using different types of carriers, which was identified as the product peak. MS analysis confirmed that the results were consistent with the standard, confirming the formation of the product. This indicates that the immobilized ligase can synthesize non-natural RNA products using nicked double-stranded non-natural RNA as a substrate.

[0109] Table 10

[0110] Wherein, m after A, C, G or U indicates a 2' methoxy modification of the ribonucleotide, f indicates a 2' fluorine modification of the ribonucleotide, and s indicates a thiomodification of the 5' phosphate of the ribonucleotide.

[0111] Substrate 13 has 2' methoxy modification at positions 1, 2, 3, 4, 5, 6, 8, and 12, 2' fluorine modification at positions 7, 9, 10, and 11, and 5' phosphate thio modification at positions 2 and 3.

[0112] Substrate 14 has 2' methoxy modification on ribonucleotides 1-9 and 5' phosphate thio modification on ribonucleotides 1, 8 and 9.

[0113] Substrate 15 has 2' methoxy modification at positions 1, 3, 4, 5, 6, 7, and 8, 2' fluorine modification at position 2, and 5' phosphate thio modification at positions 1, 8, and 9.

[0114] Substrate 16 has 2' methoxy modification on ribonucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, and 12, 2' fluorine modification on ribonucleotides at positions 2, 6, and 14, and 5' phosphate thio modification on ribonucleotides at positions 2 and 3.

[0115] Example 8: Using immobilized ligase to catalyze the double-strand ligation of non-natural RNA

[0116] Substrate 17–20 were mixed in equimolar proportions (substrate 17–20 are shown in Table 11) and added to a reaction flask. 10 μL of 1 mM ATP was added, along with 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT), 50 mg of the immobilized RNA ligation crude enzyme prepared in Example 2, and water was added to bring the substrate concentration to 800 μM. The reaction was carried out at 25°C for 16 h. The resulting reaction system was centrifuged at 3000 rpm for 2 min, and the immobilized ligase solid was removed to obtain the supernatant. Analysis was performed using denaturing polyacrylamide gel electrophoresis, which showed a very bright product band. UPLC analysis of the samples revealed that the target peak with increased molecular weight was obtained in the catalytic systems of immobilized enzymes using different types of carriers, which was identified as the product peak. The UPLC results are shown in Figure 1. MS analysis confirmed the results were consistent with the standard, confirming the formation of the product and demonstrating that the immobilized ligase can synthesize double-stranded non-natural RNA.

[0117] Table 11

[0118] Wherein, m after A, C, G or U indicates 2' methoxy modification of the ribonucleotide, f indicates 2' fluorine modification of the ribonucleotide, s indicates thiomodification of the 5' phosphate of the ribonucleotide, and dT represents thymine.

[0119] Substrate 17 has 2' methoxy modification at positions 1, 2, 3, 4, 5, 6, 8, 10, 12, 13, and 14, 2' fluorine modification at positions 7 and 9, 5' phosphate thio modification at positions 2 and 3, and thymine deoxyribonucleotide at position 11.

[0120] The ribonucleotides at positions 1-7 of substrate 18 are all modified with a 2' methoxy group.

[0121] Substrate 19 has 2' methoxy modification at positions 2, 4, 6, 7, 8, 9, and 10, 2' fluorine modification at positions 1, 3, and 5, and 5' phosphate thio modification at positions 9 and 10.

[0122] Substrate 20 has 2' methoxy modification at positions 1, 3, 7, 9, 11, and 13, 2' fluorine modification at positions 2, 4, 5, 6, 8, 10, and 12, and 5' phosphate thio modification at positions 2 and 3.

[0123] The structure of L96 is as follows:

[0124] Example 9

[0125] The preparation method of the immobilized ligase was the same as in Example 2, using Asymchem's AEC-9008 vector. The difference was that the vector was washed with solutions of different concentrations for the first wash, resulting in the affinity resin-immobilized ligase. The synthesis of natural tRNA using this immobilized ligase was performed using the same method as in Example 3. The conversion rate was measured, and the results are shown in Table 12.

[0126] Table 12

[0127] Example 10

[0128] The preparation method of the immobilized ligase was the same as in Example 2, using Asymchem's AEC-9008 carrier. The difference was that different concentrations of crude RNA ligase solutions were immobilized to prepare affinity resin-immobilized ligases. The method for synthesizing natural tRNA using this immobilized ligase was the same as in Example 3. The conversion rate was detected, and the results are shown in Table 13.

[0129] Table 13

[0130] Example 11

[0131] The preparation method of the immobilized ligase was the same as in Example 2, using Asymchem's AEC-9008 vector. The difference was that different concentrations of imidazole were used to wash the immobilized enzyme, followed by a second wash, to obtain the affinity resin-immobilized ligase. The method for synthesizing natural tRNA using this immobilized ligase was the same as in Example 3. The conversion rate was measured, and the results are shown in Table 14.

[0132] Table 14

[0133] Example 12

[0134] Add 0.5 g each of substrates 17-20 from Example 8 to a clean reaction flask, add reaction buffer to a final concentration of Tris-Cl 50 mM, MgCl2 10 mM, DTT 1 mM, add ATP to a final concentration of 1 mM, add the immobilized enzyme prepared in Example 2 (using Asymchem's AEC-9008 carrier) to make the protein amount 0.4 mg / mL and the final substrate concentration 800 μM, mix the system well, shake on a shaker, react at 16°C for 16 h, heat at 85°C for 15 min to inactivate the protein, centrifuge at 12000 rpm for 10 min, and take the supernatant for UPLC detection.

[0135] Add 0.5 g each of substrates 17-20 from Example 8 to another clean reaction flask, add reaction buffer to the final concentration of Tris-Cl 50 mM, MgCl2 10 mM, DTT 1 mM, add ATP to the final concentration of 1 mM, add purified enzyme to make the protein amount 0.4 mg / mL, and the final substrate concentration 800 μM, mix the system, shake on a shaker, react at 16°C for 16 h, heat at 85°C for 15 min to inactivate the protein, centrifuge at 12000 rpm for 10 min, and take the supernatant for UPLC detection.

[0136] After UPLC analysis, both reactions were found to be complete. The reaction system was then purified by ion column chromatography, followed by ultrafiltration concentration and lyophilization to obtain the final product. The purity, water content, and endotoxin content of the product were measured, and the results are shown in Table 15 below.

[0137] Table 15

[0138] The results showed that immobilized RNA ligase had higher activity, leading to a higher conversion rate. Furthermore, the post-processed product exhibited better purity and a higher yield. Although the reaction was conducted in a non-clean laboratory, endotoxin residues remained. Endotoxin removal in a clean area achieved a level of 0.25 EU / mg, comparable to injectable pharmaceutical grade. In contrast, the product prepared from liquid pure enzymes had a high initial endotoxin content, making it difficult to control endotoxin levels to injectable pharmaceutical grade standards even in a clean area.

[0139] Example 13: Utilizing immobilized ligase to continuously catalyze the double-strand ligation of non-natural RNA

[0140] Single-feed continuous reaction: Substrate 17-20 from Example 8 were mixed in equimolar proportions (substrate 17-20 are shown in Table 11) and added to a reaction flask. 1 mM ATP and 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) were added, and water was added to bring the substrate concentration to 600 μM. This was the reaction system. The affinity vector-immobilized ligase prepared in Example 2 (using Asymchem's AEC-9008 vector) was added to a 5 mL reaction column, approximately 3.5 g of immobilized enzyme. Using a peristaltic pump, the reaction system was continuously passed through the reaction column at 25°C (reaction apparatus shown in Figure 2). The retention time was set to 5-20 min, and samples were collected every 30 min for analysis. UPLC analysis of the samples showed that the product conversion rate reached over 92%, and the system could run continuously for over 100 hours without decreasing conversion rate.

[0141] Continuous reaction with split feed: Substrate 17–20 were prepared to a concentration of 600 μM, and 1 mM ATP and 10× ligation buffer (containing 50 mM Tris-Cl, 10 mM MgCl2, and 1 mM DTT) were added to each, resulting in four substrate systems. Approximately 3.5 g of immobilized ligase from the affinity vector prepared in step 2 was added to a 5 mL reaction column. Each of the four substrate systems was connected to a peristaltic pump, and the four reaction systems were simultaneously and continuously passed through the reaction column (reaction apparatus shown in Figure 3) at 25°C. Retention times were set from 5 to 20 min, and samples were collected every 30 min for analysis. When a significant amount of substrate remained in a particular strip, the flow rate of that substrate was immediately reduced to maximize substrate utilization. UPLC analysis showed that the product conversion rate reached over 94%, and the system could operate continuously for over 100 hours without decreasing conversion.

[0142] Both of the above continuous flow reaction modes can achieve a production capacity of >1 kg of product per liter of column reactor per day. Compared with immobilized enzyme batch reaction, the conversion rate is basically the same, but the production capacity is increased by about 20 times, and the step of separating enzyme from product is reduced.

[0143] The specific results are shown in Table 16:

[0144] Table 16

[0145] Example 14

[0146] The preparation method of the immobilized ligase was the same as in Example 2, except that the linker arms of the selected vectors were different. Asymchem vectors AEC-9002, AEC-9008, AEC-9050, AEC-9051, and AEC-9052 were used to prepare affinity resin immobilized ligases. The method for synthesizing double-stranded non-natural RNA using this immobilized ligase was the same as in Example 8. The conversion rate was measured, and the results are shown in Table 17.

[0147] Table 17

[0148] Example 15

[0149] The preparation method of the immobilized ligase was the same as in Example 2, except that the chelating metal ions of the selected carriers were different. Asymchem carriers AEC-9008, AEC-9060, AEC-9061, AEC-9062, and AEC-9063 were used to prepare affinity resin immobilized ligases. The method for synthesizing non-natural RNA double-stranded ligation using this immobilized ligase was the same as in Example 8. The conversion rate was detected, and the results are shown in Table 18.

[0150] Table 18

[0151] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: The above-mentioned immobilized RNA ligase was prepared by immobilizing the enzyme carrier using the above preparation method. The above-mentioned RNA ligase can improve the activity of RNA ligase and can be reused, reducing the purification pressure of RNA ligase before reaction, reducing the steps of separating reaction products from enzymes, reducing production costs, improving production efficiency and capacity, and the product obtained has a low endotoxin content. It can prepare reaction products with impurity content that meet the requirements and can be directly used to prepare injectable drugs, reducing the steps and costs required for product post-processing.

[0152] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An immobilized enzyme for catalyzing RNA ligation reactions, characterized in that, The immobilized enzyme includes an immobilization carrier and an enzyme immobilized on the immobilization carrier; The immobilization carrier is an affinity carrier; The enzyme is an enzyme that catalyzes RNA ligation reactions; The ligand of the immobilized carrier is one or more of the following: hypozinotriacetic acid, iminodiacetic acid, glutathione, Halo Tag, dextrin, or Strep-Tactin.

2. The immobilized enzyme for catalyzing RNA ligation reaction according to claim 1, characterized in that, The matrix of the immobilization carrier is one or more of styrene, polystyrene, acrylic acid, polyacrylic acid, methacrylate, butyl methacrylate, silica, dextran, or agarose.

3. The immobilized enzyme for catalyzing RNA ligation reactions according to claim 1, characterized in that, The connecting arms of the immobilized carrier are C6 to C14.

4. The immobilized enzyme for catalyzing RNA ligation reactions according to any one of claims 1-3, characterized in that, The immobilization vector includes one or more of the following affinity vectors: EziG Amber, EziG Coral, EziG Opal, IB-HIS-1, IB-HIS-2, IMAC Chromstar FF, Ni Chromstar FF, Ni-IDA Purose 6 FF, Ni-NTA Purose 6 FF, AEC-9001, AEC-9002, AEC-9007, AEC-9008, AEC-9050, AEC-9051, AEC-9052, AEC-9060, AEC-9061, AEC-9062, AEC-9063, Glutathione Beads 4FF, Halo Link™ Resin, MBPseq Dextrin Agarose Resin 6FF, or Streptactin Agarose Resin 4FF.

5. The immobilized enzyme for catalyzing RNA ligation reaction according to claim 1, characterized in that, The enzyme includes any one or more of RNA ligase families 1, 2, and 3.

6. A method for preparing an immobilized enzyme for catalyzing RNA ligation reaction according to any one of claims 1-5, characterized in that, The preparation method includes: The immobilization vector is mixed with the enzyme that catalyzes the RNA ligation reaction and then immobilized and incubated to obtain the immobilized enzyme.

7. The preparation method according to claim 6, characterized in that, The preparation method includes: S1, the immobilized carrier is first cleaned; S2, the cleaned immobilized carrier is mixed with the enzyme that catalyzes the RNA ligation reaction and then immobilized and incubated. S3, the incubated immobilized enzyme is washed a second time to obtain the immobilized enzyme used to catalyze the RNA ligation reaction.

8. The preparation method according to claim 7, characterized in that, S1 includes: performing the first wash twice on the immobilized carrier using a first buffer solution, the first buffer solution comprising 0.05-0.2M PB, 0.2-1M NaCl, 20-100mM imidazole, and pH 7.0-8.

0.

9. The preparation method according to claim 7, characterized in that, The immobilization incubation in S2 includes: The cleaned immobilized vector was mixed with the enzyme that catalyzes the RNA ligation reaction, stirred and incubated at 20°C for 4-48 hours, and then filtered.

10. The preparation method according to claim 7, characterized in that, The enzyme comprises an enzyme solution, and the volume ratio of the enzyme solution to the washed immobilized carrier is 2-6:

1.

11. The preparation method according to claim 10, characterized in that, The enzyme solution includes crude enzyme solution or pure enzyme solution.

12. The preparation method according to claim 11, characterized in that, The protein concentration in the crude enzyme solution is 20–50 mg / mL, and the protein concentration in the pure enzyme solution is 1–5 mg / mL.

13. The preparation method according to claim 9, characterized in that, The stirring time is 4h to 16h.

14. The preparation method according to claim 8, characterized in that, S3 includes: performing the second washing on the incubated immobilized enzyme 3 to 4 times using a second buffer solution, the second buffer solution comprising 0.05-0.2M PB, 10-50mM imidazole, pH 7.0-8.

0.

15. The immobilized enzyme for catalyzing RNA ligation reaction according to any one of claims 1-5, or the immobilized enzyme for catalyzing RNA ligation reaction prepared by any one of the preparation methods according to claims 6-14, is used in RNA synthesis.

16. The application according to claim 15, characterized in that, The application includes setting the immobilized enzyme used to catalyze the RNA ligation reaction in a continuous flow device to achieve continuous RNA synthesis.

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