Kit for enzyme digestion of mRNA-polyA and preparation method thereof
By using H-Rnase T1 enzyme and H-oligo dT25 magnetic beads, the problem of total polyA loss and long processing time in mRNA PolyA tail detection was solved, and efficient and stable enzyme cleavage treatment was achieved, improving recovery rate and enzyme cleavage accuracy.
Patent Information
- Application Number
- CN202510606302.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, mRNA PolyA tail detection has large total loss of polyA, low recovery rate, long processing time, cumbersome and unstable enzyme cutting steps, making it difficult to achieve standardized processing.
The H-Rnase T1 enzyme and H-oligo dT25 magnetic beads were used. The H-Rnase T1 enzyme was connected with d5mC at the N-terminal and C-terminal surface of the oligo 5' and 3' of the oligo surface of the H-oligo dT25 magnetic beads, and combined with a specific reaction solution and a cleaning solution to improve the enzyme cleavage efficiency and stability.
The polyA recovery rate is improved to 90%, the enzyme cutting time is shortened to 1 hour, the stability and accuracy of the enzyme cutting system are enhanced, and standardized processing is achieved.
Smart Images
Figure CN120366404A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and particularly relates to a kit for digesting mRNA-polyA and a preparation method thereof. Background Art
[0002] Detecting the length and integrity of the PolyA tail of mRNA is crucial for ensuring the quality and efficacy of mRNA drugs. The length and integrity of the PolyA tail directly affect the stability and translation efficiency of mRNA, and thus affect the expression level and function of proteins. Therefore, accurate PolyA tail detection is a key step in the development and production process of mRNA drugs.
[0003] In the prior art, the detection of the mRNA PolyA tail is carried out by digesting the PolyA tail, purifying with magnetic beads, recovering, and analyzing. However, shearing the polyA in mRNA requires multiple steps to complete, including annealing - digestion - purification - washing - terminating the reaction - eluting and collecting - evaporating and spinning dry - resuspending.
[0004] However, there are some problems in shearing the PolyA tail in the prior art: 1. The total amount of polyA is lost, and the recovery rate is low, and the polyA recovery rate is about 10%; 2. The processing time is long and the efficiency is low, which takes about 4 hours, and the excessive time is likely to cause the target RNA fragment to degrade in the reaction system; 3. Purification usually uses oligo magnetic beads, and its principle is to form superparamagnetic composite iron balls by chemically coupling oligos. This substance is easily decomposed by nucleases or the activity is reduced by chemical substances in the reaction system. The reason is that only one end of the 5' or 3' of the traditional magnetic bead is connected to the magnetic bead, and one end of it will inevitably be exposed, resulting in its instability; 4. The RNAse T1 enzyme used in the prior art belongs to a natural protein, and its enzyme properties are poor; 5. The digestion step is cumbersome and there are many variables, which is not conducive to standardized processing. Summary of the Invention
[0005] In view of this, the present invention provides a kit for digesting mRNA-polyA and a preparation method thereof, which can improve the polyA recovery rate, shorten the digestion time, enhance the stability of the digestion system, reduce the processing steps, and achieve standardized digestion processing.
[0006] To achieve the above object, the present invention provides a kit for digesting mRNA-polyA, comprising the following components: H-Rnase T1 enzyme, H-oligo dT25 magnetic beads, reaction solution, termination solution, washing solution; The H-Rnase T1 enzyme has 1 molecule of nucleic acid monomer d5mC at each of its N-terminus and C-terminus; the d5mC is a monomer deoxyribonucleotide dC base with methylation at the 5th position in the pyrimidine ring; The H-oligo dT25 magnetic beads have oligos densely distributed on the surface of each magnetic bead, with both the 5' and 3' ends of the oligos connected to the surface of the magnetic bead.
[0007] Optionally, the reaction solution includes Tris-HCl, ethylenediaminetetraacetic acid, and glycerol; the concentration of Tris-HCl is 45 - 55 mM, the pH of Tris-HCl is 7 - 8, the concentration of ethylenediaminetetraacetic acid is 1.8 - 2.2 mM, and the concentration of glycerol is 1.8 - 2.2 mM.
[0008] Optionally, the concentration of Tris-HCl is 50 mM, the pH of Tris-HCl is 7.5, the concentration of ethylenediaminetetraacetic acid is 2 mM, and the concentration of glycerol is 2 mM.
[0009] Optionally, the termination solution includes isopropanol and water; the mass fraction of isopropanol is 83 - 87%, the mass fraction of water is 13 - 17%, and the sum of the mass fractions of isopropanol and water is 100%.
[0010] Optionally, the mass fraction of isopropanol is 85%, and the mass fraction of water is 15%.
[0011] Optionally, the washing solution includes sodium citrate and water; the washing solution is an aqueous solution of sodium citrate with a concentration of 9 - 11 mM.
[0012] Optionally, the washing solution is an aqueous solution of sodium citrate with a concentration of 10 mM.
[0013] To achieve the above object, the present invention also provides a method for preparing a kit for digesting mRNA-polyA, including the following steps: A. Prepare H-Rnase T1 enzyme: Insert the target DNA fragment into a plasmid, introduce the prepared plasmid into Escherichia coli, culture, collect and purify the target protein Rnase T1 expressed in Escherichia coli; use a chemical reaction to make glycine (H2N-CH2-COOH) and monomer d5mC undergo a chemical reaction to form a compound H2N-CHd5mC-COOH; through a chemical reaction, make RnaseT1 react with H2N-CHd5mC-COOH to obtain the target H-Rnase T1 enzyme; B. Prepare H-oligo dT25 magnetic beads: Using uniformly dispersed magnetic polystyrene microspheres as the matrix, combine with Fe3O4 to form superparamagnetism, and through the phosphoramidite method and covalent coupling, make both the 5' and 3' ends of oligo dT 25 connected to the surface of the magnetic bead, and purify and wash to obtain H-oligo dT 25; C. Configure the reaction solution, termination solution, and washing solution.
[0014] Optionally, the conditions for the chemical reaction are to react for 30 - 60 minutes under metal catalysis and at 20 - 30 °C.
[0015] The above technical solutions of the present invention have at least the following beneficial effects: 1. The kit for cleaving mRNA - polyA provided by the present invention introduces H - Rnase T1 enzyme. The difference between it and the common Rnase T1 enzyme in the prior art is that the H - Rnase T1 enzyme has 1 molecule of nucleic acid monomer d5mC (deoxyribonucleotide dC base with methylation at the 5th position in the pyrimidine ring) at each of its N - terminal and C - terminal. Its function is to enable the H - Rnase T1 enzyme to target and locate G bases during the cleavage reaction, and can significantly improve enzyme activity, stability, cleavage ability, cleavage efficiency and accuracy.
[0016] 2. The kit for cleaving mRNA - polyA provided by the present invention introduces H - oligo dT25 magnetic beads. Compared with the common magnetic beads in the prior art, the difference is that for traditional magnetic beads, only one end of the oligo is connected to the magnetic bead at either the 5' or 3' end, and one end will inevitably be exposed, resulting in instability. However, for H - oligo dT25 magnetic beads, due to their structure that the surface of a single magnetic bead is covered with oligos, both the 5' and 3' ends of the oligos are connected to the magnetic bead surface, and the oligos are no longer in a free state at one end, making them more stable and not easily decomposed by nucleases.
[0017] 3. Compared with the reaction solution in the prior art, the reaction solution provided by the present invention reduces the inhibition of enzyme activity by metal ions in the prior art; compared with the termination solution in the prior art, the termination solution of the present invention reduces enzyme residue and can increase the separation effect of the cleaved target fragment and the magnetic bead; the washing solution of the present invention can remove non - target fragments.
[0018] 4. The kit for cleaving mRNA - polyA of the present invention increases the polyA recovery rate to 90%, reduces the time of the mRNA - polyA cleavage experiment to 1 hour, and increases the stability and accuracy of the cleavage system, reduces the processing steps, and completes the mRNA - polyA cleavage in one step, achieving a high degree of standardization. Description of the Drawings
[0019] Figure 1 It is the distribution ratio diagram of 100nt PolA in Example 1 of the present invention; Figure 2 It is the distribution ratio diagram of 100nt PolA in Comparative Example 1 of the present invention; Figure 3 It is the comparative diagram of the distribution ratios of 100nt PolA between Example 1 and Comparative Example 1 of the present invention. Detailed Embodiments
[0020] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention fall within the scope of protection of the present invention.
[0021] Example 1 A. Preparation of H-Rnase T1 enzyme: Insert the target DNA fragment into a plasmid, introduce the prepared plasmid into Escherichia coli, culture it, collect and purify the target protein Rnase T1 expressed in Escherichia coli; use a chemical reaction to react for 60 minutes under metal catalysis and at 20 °C to cause glycine (H2N-CH2-COOH) and monomer d5mC to undergo a chemical reaction to form the compound H2N-CHd5mC-COOH; through a chemical reaction, react for 60 minutes under metal catalysis and at 20 °C to cause Rnase T1 and H2N-CHd5mC-COOH to react to obtain the target H-Rnase T1 enzyme; B. Preparation of H-oligo dT25 magnetic beads: Use uniformly dispersed magnetic polystyrene microspheres as the matrix, combine with Fe3O4 to form superparamagnetism, and connect oligo dT 25 at both its 5' and 3' ends to the surface of the magnetic beads through the phosphoramidite method and covalent coupling, and purify and wash to obtain H-oligo dT 25; C. Prepare reaction solution, termination solution, and washing solution; The reaction solution contains Tris-HCl, ethylenediaminetetraacetic acid, and glycerol; the concentration of Tris-HCl is 50 mM, the pH of Tris-HCl is 7.5, the concentration of ethylenediaminetetraacetic acid is 2 mM, and the concentration of glycerol is 2 mM; The termination solution includes isopropanol and water; the concentration of isopropanol is 85%, and the concentration of water is 15%; The washing solution includes sodium citrate and water; the washing solution is an aqueous solution of sodium citrate with a concentration of 10 mM.
[0022] Example 2 A. Preparation of H-Rnase T1 enzyme: Insert the target DNA fragment into a plasmid, introduce the prepared plasmid into Escherichia coli, culture it, collect and purify the target protein Rnase T1 expressed in Escherichia coli; use a chemical reaction, react for 30 minutes under metal catalysis and at 30 °C to cause glycine (H2N-CH2-COOH) and monomer d5mC to undergo a chemical reaction to form the compound H2N-CHd5mC-COOH; through a chemical reaction, react for 30 minutes under metal catalysis and at 30 °C to react Rnase T1 and H2N-CHd5mC-COOH to obtain the target H-Rnase T1 enzyme; B. Preparation of H-oligo dT25 magnetic beads: Using uniformly dispersed magnetic polystyrene microspheres as the matrix, combine with Fe3O4 to form superparamagnetism, and through the phosphoramidite method and covalent coupling, connect both the 5' and 3' of oligo dT 25 to the surface of the magnetic beads, and purify and wash to obtain H-oligo dT 25; C. Prepare reaction solution, termination solution, and washing solution; The reaction solution is Tris-HCl, ethylenediaminetetraacetic acid, and glycerol; the concentration of Tris-HCl is 45 mM, the pH of Tris-HCl is 7, the concentration of ethylenediaminetetraacetic acid is 2.2 mM, and the concentration of glycerol is 1.8 mM; The termination solution includes isopropanol and water; the concentration of isopropanol is 83% and the concentration of water is 17%; The washing solution includes sodium citrate and water; the washing solution is an aqueous solution of sodium citrate with a concentration of 11 mM.
[0023] Example 3 A. Preparation of H-Rnase T1 enzyme: Insert the target DNA fragment into a plasmid, introduce the prepared plasmid into Escherichia coli, culture it, collect and purify the target protein Rnase T1 expressed in Escherichia coli; use a chemical reaction, react for 45 minutes under metal catalysis and at 25 °C to cause glycine (H2N-CH2-COOH) and monomer d5mC to undergo a chemical reaction to form the compound H2N-CHd5mC-COOH; through a chemical reaction, react for 45 minutes under metal catalysis and at 25 °C to react Rnase T1 and H2N-CHd5mC-COOH to obtain the target H-Rnase T1 enzyme; B. Preparation of H-oligo dT25 magnetic beads: Using uniformly dispersed magnetic polystyrene microspheres as the matrix, combine with Fe3O4 to form superparamagnetism, and through the phosphoramidite method and covalent coupling, connect both the 5' and 3' of oligo dT 25 to the surface of the magnetic beads, and purify and wash to obtain H-oligo dT 25; C. Prepare reaction solution, termination solution, and washing solution; Reaction solution: Tris-HCl, ethylenediaminetetraacetic acid, glycerol; the concentration of Tris-HCl is 55 mM, the pH of Tris-HCl is 8, the concentration of ethylenediaminetetraacetic acid is 1.8 mM, and the concentration of glycerol is 2.2 mM; The termination solution includes isopropanol and water; the concentration of isopropanol is 87% and the concentration of water is 13%; The cleaning solution includes sodium citrate and water; the cleaning solution is an aqueous solution with a concentration of 9 mM.
[0024] Comparative Example 1 The conventional method for shearing polyA in mRNA in the prior art: annealing - enzymatic digestion - purification - cleaning - termination of reaction - elution and collection - evaporation and rotary drying - resuspension; Rnase T1 enzyme is used for enzymatic digestion, and conventional oligo dT 25 magnetic beads are used for purification.
[0025] Table 1 is obtained by comparing the enzymes in Example 1 and Comparative Example 1; Table 2 is obtained by comparing the magnetic beads in Example 1 and Comparative Example 1.
[0026] Table 1 Comparison of Enzymes in Example 1 and Comparative Example 1
[0027] Table 2 Comparison of Magnetic Beads in Example 1 and Comparative Example 1
[0028] Test Example Use the enzymatic digestion methods of Example 1 and Comparative Example 1 to digest mRNA-polyA respectively, select mRNA lengths (2000 nt, 3000 nt, 4000 nt, 5000 nt, 6000 nt), where the polyA length is 100 nt, and use Thermo OE 240 high-resolution mass spectrometry to analyze and detect the enzymatic digestion polyA products to obtain a polyA distribution map, see Figure 1 、 Figure 2 、 Figure 3 , and the data of the total polyA recovery rate, processing time, and enzymatic digestion accuracy rate are shown in Table 3; the average values of the ion abundances of the main component peaks of the mass spectrometry and the accuracy of the mass spectrometry fragment sizes are repeated for detection, as shown in Table 4.
[0029] Table 3 Related Data Table after Enzymatic Digestion of mRNA-polyA by the Methods of Example 1 and Comparative Example 1 (mRNA Concentration 2 mg / ml)
[0030] Table 4 Related Data Table after Enzymatic Digestion of mRNA-polyA by the Methods of Example 1 and Comparative Example 1
[0031] Combined with TableFigure 1 , 2 As can be seen from 3 and Tables 3 and 4, the technical solution provided by the present invention has significantly better cleavage effect on the target polyA than the comparative example.
[0032] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A kit for cleaving mRNA-polyA, characterized in that, It includes the following components: H-Rnase T1 enzyme, H-oligodT25 magnetic beads, reaction solution, termination solution, and washing solution; The H-Rnase T1 enzyme has 1 molecule of nucleic acid monomer d5mC at each of its N-terminus and C-terminus; the d5mC is a monomer deoxyribonucleotide dC base with methylation at the 5th position in the pyrimidine ring; The H-oligo dT25 magnetic beads have oligo densely distributed on the surface of a single magnetic bead, and both the 5' and 3' of the oligo are connected to the surface of the magnetic bead.
2. The kit for cleaving mRNA-polyA according to claim 1, wherein, The reaction solution includes Tris-HCl, ethylenediaminetetraacetic acid, and glycerol; the concentration of Tris-HCl is 45 - 55 mM, the pH of Tris-HCl is 7 - 8, the concentration of ethylenediaminetetraacetic acid is 1.8 - 2.2 mM, and the concentration of glycerol is 1.8 - 2.2 mM.
3. The kit for cleaving mRNA-polyA according to claim 2, characterized in that, The concentration of Tris-HCl is 50 mM, the pH of Tris-HCl is 7.5, the concentration of ethylenediaminetetraacetic acid is 2 mM, and the concentration of glycerol is 2 mM.
4. The kit for cleaving mRNA-polyA according to claim 1, wherein The termination solution includes isopropanol and water; the mass fraction of isopropanol is 83 - 87%, the mass fraction of water is 13 - 17%, and the sum of the mass fractions of isopropanol and water is 100%.
5. The kit for cleaving mRNA-polyA according to claim 4, wherein The mass fraction of isopropanol is 85%, and the mass fraction of water is 15%.
6. The kit for cleaving mRNA-polyA according to claim 1, characterized in that, The washing solution includes sodium citrate and water; the washing solution is an aqueous solution of sodium citrate with a concentration of 9 - 11 mM.
7. The kit for cleaving mRNA-polyA according to claim 1, wherein The washing solution is an aqueous solution of sodium citrate with a concentration of 10 mM.
8. A method for preparing a kit for cleaving mRNA-polyA as described in any one of claims 1 to 4, characterized in that, It includes the following steps: A. Preparation of H-Rnase T1 enzyme: Insert the target DNA fragment into a plasmid, introduce the prepared plasmid into Escherichia coli, culture it, collect and purify the target protein Rnase T1 expressed in Escherichia coli; use a chemical reaction to make glycine (H2N-CH2-COOH) and monomer d5mC undergo a chemical reaction to form a compound H2N-CHd5mC-COOH; through a chemical reaction, make Rnase T1 and H2N-CHd5mC-COOH react to obtain the target H-Rnase T1 enzyme; B. Preparation of H-oligo dT25 magnetic beads: Use uniformly dispersed magnetic polystyrene microspheres as a matrix, combine with Fe3O4 to form superparamagnetism, and through the phosphoramidite method and covalent coupling, make both the 5' and 3' of oligo dT 25 connected to the surface of the magnetic bead, and purify and wash to obtain H-oligo dT 25; C. Prepare the reaction solution, termination solution, and washing solution.
9. The preparation method of the kit for cleaving mRNA-polyA according to claim 8, wherein, The conditions of the chemical reaction are to react for 30 - 60 minutes under metal catalysis and at 20 - 30 °C.