Method for detecting mRNA capping rate

Abstract

The invention discloses a method for detecting an mRNA capping rate, and belongs to the technical field of biology. The detection method comprises the following steps: carrying out hybridization reaction on mRNA and a base-free modified probe, and carrying out enzyme digestion reaction on the product; and adding magnetic particles into the reaction product, incubating, cleaning and eluting to obtain the mRNA pretreatment product. According to the method, the unmodified probe is used for detecting the cap adding rate of the mRNA, the pretreatment process of the mRNA is mainly improved, and the reaction condition of hybridization combination of the probe and the mRNA is optimized, so that the higher detection fragment yield is obtained; by optimizing the elution mode, the proportion of the probe in a capping rate detection sample can be remarkably reduced, and the proportion of a target fragment is increased. Therefore, compared with a traditional detection method, the method has the advantages that the detection sensitivity can be improved, the detection lower limit is reduced, the resolution and accuracy are improved, the process is simplified, the cost can be reduced, and the application prospect is better.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for detecting mRNA capping rate. Background Technology

[0002] mRNA (messenger ribonucleic acid) is an important molecule in living organisms. Its main function is to transmit genetic information from DNA and guide protein synthesis. mRNA vaccines utilize this mechanism. They do not directly contain the virus, but rather mRNA encoding a specific part of the virus. When this mRNA enters human cells, the cells produce that part of the virus according to the mRNA's instructions, triggering an immune response and providing the body with resistance. Compared to traditional vaccines, mRNA vaccines offer greater design flexibility and a shorter production cycle. Because their design and development only require the viral genome, if the virus mutates, only the sequence needs to be adjusted to obtain a new vaccine. mRNA vaccines have brought mRNA technology into the public eye. mRNA technology not only has applications in vaccine production but also holds immense potential in treating genetic diseases, cancer, and gene editing.

[0003] In vitro transcription (IVT) allows for the synthesis of RNA in vitro using RNA polymerase. Most eukaryotic mRNAs possess a 5' cap structure, which protects the 5' end from damage by phosphatases and nucleases and promotes translation initiation. RNA synthesized through in vitro transcription lacks a 5' cap structure and requires an additional capping step to obtain it. Two types of capping methods are commonly used in mRNA production: enzymatic capping and co-transcriptional capping. Enzymatic capping utilizes the RNA triphosphatase, guanylate acyltransferase, and guanine methyltransferase activities of vaccinia virus capping enzymes to generate Cap0-mRNA, followed by Cap1-mRNA generation using 2'-O-methyltransferase. Under appropriate capping buffer concentrations, and in the presence of guanosine triphosphate (GTP) and S-adenosylmethionine (SAM), RNA can be capped within one hour while maintaining the correct orientation. Co-transcriptional capping involves directly adding a certain proportion of cap analogs to the in vitro transcription reaction system, directly yielding capped mRNA. Currently, third-generation cap analogs have been developed. Regardless of whether enzymatic capping or co-transcriptional capping is performed, the capping rate of the resulting mRNA must be measured after the reaction, as the capping rate is an important indicator of mRNA quality.

[0004] mRNA typically consists of hundreds or thousands of nucleotides. Capped and uncapped mRNAs may differ by a single nucleotide or methyl group, making them difficult to capture using conventional separation and analysis methods. Currently, common methods involve obtaining 5' mRNA fragments of tens of nucleotides through enzymatic degradation or cleavage, followed by differentiation and quantification of capped fragments using acrylamide gel electrophoresis, capillary gel electrophoresis, or LC-MS to determine the mRNA capping rate. The most ideal approach currently involves capturing the fragment using a fully oxygen-methylated probe, cleaving it with RNase H, and then detecting it using LC-MS. This approach improves capture efficiency through oxygen-methylation, achieving higher recovery rates compared to other methods, and combined with the low detection limit of LC-MS, it achieves good detection results. However, the recovery efficiency of this approach is still relatively low, and the fully oxygen-methylated probe is expensive, resulting in a high overall cost. Furthermore, the residual probe component in the mRNA fragment product often exceeds 70%, further reducing the amount of effectively detectable fragments.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting mRNA capping rate. The detection method of this invention can obtain more detection fragments, reduce the probe content in the sample, and reduce costs while improving resolution.

[0007] This invention is implemented as follows: This invention provides a method for detecting mRNA capping rate, comprising: hybridizing mRNA with a base-free probe and performing an enzymatic digestion reaction on the product; then adding magnetic microparticles to the reaction product, incubating, and then washing and eluting to obtain the mRNA pretreatment product; and finally performing LC-MS detection on the mRNA pretreatment product. The hybridization reaction conditions are as follows: heating at 68~72℃ for 2~10 min, ice bath for 0.5~1 h, and incubation at 35~38℃ for 1~4 h.

[0008] In some embodiments, the elution solution in the elution step is nuclease-free water.

[0009] In some embodiments, the elution method is as follows: add nuclease-free water to the washed magnetic microparticles, heat at 60-80°C for 2-5 min, use a magnetic rack to attract the magnetic microparticles, and obtain the mRNA pretreatment product after treatment.

[0010] In some embodiments, the hybridization reaction system contains mRNA, RNase H probe and incubation buffer; the incubation buffer consists of 10 mM Tris-HCl (pH 7.5) and 0.1 M LiCl.

[0011] In some embodiments, the molar ratio of mRNA to RNase H probe in the reaction system is 0.8 to 1:1.

[0012] In some embodiments, the composition of the base-free probe is: 5'-4~6nt RNA-4~6nt DNA-10~20nt RNA-Biotin-3'.

[0013] In some embodiments, the mRNA includes ψU-modified mRNA and mRNA without base modifications.

[0014] In some embodiments, the reaction system of the enzyme digestion reaction contains hybridization reaction products, reaction buffer and RNsaeH enzyme; the conditions for the enzyme digestion reaction are: reaction at 37°C for 0.5~1.5 h.

[0015] In some embodiments, the incubation method after adding magnetic microparticles is as follows: incubate at 20-50 rpm and room temperature for 20-40 min on a rotary mixer.

[0016] In some embodiments, the washing method is as follows: the incubated magnetic microparticles are treated with a magnetic rack to remove the supernatant, and then washed sequentially with magnetic bead washing buffer and nuclease-free water.

[0017] The present invention has the following beneficial effects: This invention utilizes unmodified probes to detect mRNA capping rate. The main improvements focus on the mRNA pretreatment process. By optimizing the reaction conditions for probe-mRNA hybridization, a higher fragment yield is achieved. Furthermore, by optimizing the elution method, the proportion of probes in the capping rate detection sample is significantly reduced, increasing the proportion of target fragments. Therefore, compared to traditional detection methods, this invention improves detection sensitivity, lowers the detection limit, enhances resolution and accuracy, simplifies the process, and reduces costs, demonstrating promising application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1This is the deconvoluted mass spectrum of Example 1. Figure 2 The Extract Masses Table of Example 1; Figure 3 This is the deconvolution mass spectrum of Example 2; Figure 4 The Extract Masses Table of Example 2; Figure 5 This is the deconvolution mass spectrum of Comparative Example 1; Figure 6 This is the Xtract Masses Table for Comparative Example 1. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] The common method for detecting mRNA capping rate is as follows: A DNA probe complementary to the 5' end sequence of the mRNA to be tested is designed and specifically hybridized with the mRNA, allowing the DNA probe to precisely bind to the 5' end region of the mRNA, facilitating the next step of enzyme digestion. RNase H enzyme is added; this enzyme specifically recognizes and cleaves the RNA strand in the DNA-RNA hybrid double strand. Guided by the DNA probe, it precisely cleaves the 5' end of the mRNA (including the cap structure and the first few nucleotides), generating a separate, short 5' oligonucleotide fragment. The digestion product is then analyzed by liquid chromatography-mass spectrometry (LC-MS). LC separates the different oligonucleotide fragments generated by digestion, and MS accurately measures the molecular weight of the separated fragments. By calculating the matching degree between the actual measured mass and the theoretical mass (capped / uncapped), the proportion of capped molecules (capping rate) is directly and absolutely quantified.

[0022] Probes are typically chemically modified, such as by 2'-O-methylation. Compared to unmodified probes, these modifications enhance their binding affinity to mRNA, increase the recovery efficiency of the short ends of the mRNA being detected, and thus improve the accuracy of LC-MS detection results. However, in practical applications, this modification increases detection costs. Therefore, the inventors attempted to detect mRNA capping rate using unmodified probes. Since mRNA pretreatment is a crucial step affecting detection results, this invention primarily optimizes and improves the mRNA pretreatment method, enabling it to achieve or even surpass the recovery efficiency of the comparative method when using unmodified probes.

[0023] Therefore, this invention provides a method for detecting mRNA capping rate, which involves sequentially performing mRNA synthesis, mRNA pretreatment, and LC-MS detection. The pretreatment method includes: hybridizing mRNA with a base-free probe and performing enzymatic digestion on the product; then adding magnetic microparticles to the reaction product, incubating, and finally washing and eluting to obtain the pretreated mRNA product.

[0024] In this invention, the hybridization reaction of mRNA with a base-free probe refers to the formation of a complex (double strand or hybrid) between nucleotide sequences that are complementary to Watson-Crick bases. The term "complementary" as used herein refers to a nucleic acid molecule that forms a stable double strand with its complementary sequence under specific conditions. If all nucleic acid bases match, the complementarity between two nucleic acid molecules is referred to as "complete" or "full," otherwise as "partial."

[0025] The mRNA samples detected by this invention can be in vitro transcribed mRNA, or isolated eukaryotic mRNA and viral RNA; at the same time, the mRNA can be ψU modified mRNA or mRNA without base modification, and this invention does not limit this.

[0026] The base-free probe structure of the present invention is: 5'-4~6nt RNA-4~6nt DNA-10~20nt RNA-biotin-3'. In a specific embodiment, the sequence of the probe can be designed as: 5'-AGAA / dG / / dA / / dA / / dT / ACUAGUUUAUUCCU-biotin-3' (SEQ ID NO.2).

[0027] The ability to detect mRNA capping rate using the above-mentioned base-free probes is mainly achieved by optimizing the conditions and incubation buffer during the hybridization reaction between the probe and mRNA. Compared with the traditional processing method - annealing, the method of this invention allows the probe to achieve higher binding efficiency than the traditional system without modification, thereby obtaining a higher detection fragment yield.

[0028] In some embodiments, the reaction system for the hybridization reaction contains mRNA, RNase H probe, and incubation buffer.

[0029] In some embodiments, the incubation buffer consists of 10 mM Tris-HCl (pH 7.5) and 0.1 M LiCl.

[0030] In some embodiments, the hybridization reaction conditions are: heating at 68-72°C for 2-10 min, ice bath for 0.5-1 h, and incubation at 35-38°C for 1-4 h. More preferably, the reaction conditions are: heating at 70°C for 10 min, ice bath for 1 h, and incubation at 37°C for 3 h.

[0031] In some embodiments, the molar ratio of mRNA to RNase H probe in the reaction system is 0.8 to 1:1.

[0032] In some embodiments, the reaction system for the enzymatic digestion reaction contains hybridization reaction products, reaction buffer, and RNsaeH enzyme; the conditions for the enzymatic digestion reaction are: reaction at 37°C for 0.5–1.5 h. More preferably, the conditions for the enzymatic digestion reaction are: reaction at 37°C for 1 h.

[0033] In some embodiments, the incubation method after adding magnetic microparticles is as follows: incubation at 20-50 rpm and room temperature for 20-40 min on a rotary mixer. More preferably, the conditions are incubation at 30 rpm and room temperature for 30 min.

[0034] In some embodiments, the cleaning method is as follows: the incubated magnetic microparticles are treated with a magnetic rack to remove the supernatant, and then washed sequentially with magnetic bead washing buffer and nuclease-free water (NF H2O).

[0035] On the other hand, based on the above-mentioned scheme, the present invention further optimizes the elution step. The elution method of the present invention involves adding nuclease-free water to the washed magnetic microparticles, heating at 60-80°C for 2-5 min, using a magnetic rack to attract the magnetic microparticles, and obtaining the mRNA pretreatment product after treatment. More preferably, the reaction conditions are heating at 65°C for 3 min.

[0036] Compared to the traditional elution method using methanol at 80°C, the elution method of this invention can reduce the proportion of probe peaks. This is mainly achieved by adjusting the reagents and temperature to find a condition that can break most of the hydrogen bonds (the fragment to be detected and the probe) while minimizing damage to the probe and the surface modification of the magnetic beads. To achieve the above effect, the elution conditions of these two combinations need to be as different as possible, and the optimal conditions need to be optimized and screened.

[0037] The above elution method can significantly reduce the proportion of probes in the capped sample, thereby increasing the proportion of the detection fragment in the sample. At the same time, the eluted sample can be directly detected by LC-MS without evaporation and reconstitution, simplifying the operation.

[0038] The advantages of the detection method of the present invention are mainly reflected in four dimensions: (1) High fragment yield, which can generate a sufficiently strong LC-MS signal with less mRNA raw material in the detection, thereby improving sensitivity; it can also lower the detection limit and is suitable for micro sample analysis; (2) High signal-to-noise ratio, which significantly reduces the proportion of probes in the sample with capping rate and increases the proportion of target fragments. Under this environment, the LC-MS spectrum is cleaner and the target peak is more prominent, which is convenient for integration and accurate quantification, reduces background interference, and is conducive to improving resolution and accuracy; (3) Simplified operation process, the obtained eluted sample can be directly used for subsequent detection, eliminating the need for steps such as reconstitution, shortening the pretreatment time, reducing sample loss or variation caused by multi-step operation, and thus improving detection efficiency and robustness; (4) Reduced cost, the detection probe of the present invention does not require the use of expensive base-modified probes. In addition, the detection method of the present invention is also universal, and is applicable to mRNAs of different sequences and lengths in addition to the sequences verified by experimental examples.

[0039] It should be noted that in the method for detecting mRNA capping rate of the present invention, both mRNA synthesis and LC-MS detection are performed using conventional methods in the art. The present invention does not limit these methods, and those skilled in the art can select and set them according to actual needs and conditions.

[0040] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0041] The nucleotide sequences of the mRNAs used in the following experiments are shown in SEQ ID NO.1. The IVT buffer used consisted of: 400mM Tris-HCl pH 8.0, 350mM MgCl2, 50mM DTT, and 20mM spermidine. Capanalogs were purchased from Shenji (catalog number CAP3011). T7 RNA Polymerase was from Sangon Biotech (B110083-0005). RNase inhibitor was from Sangon Biotech (B600478-0005). Ppase was from Sangon Biotech (B110084-0010). RNase H and 10×reaction buffer were purchased from Shenji (10115). SAbeads were purchased from Rebecca (NBS205). The SAbeads binding buffer consisted of: 10mM Tris-HCl pH 7.5, 1mM EDTA, and 2M NaCl.

[0042] Example 1 This embodiment describes a method for detecting the capping rate of base-free mRNA. The specific steps are as follows: 1. Base-modified mRNA of the synthesized product (1) Prepare the IVT reaction system and incubate it at 37°C for 3 h. The reaction system is shown in Table 1: Table 1 IVT Reaction System

[0043] (2) Add 1 μL of DNase I and incubate at 37°C for 30 min.

[0044] (3) Add 30 μL of NF H2O and 30 μL of 7.5 M LiCl precipitation solution (Invitrogen# AM9480) to the IVT reaction product. Transfer to a 1.5 mL centrifuge tube, mix well, and incubate at -20℃ for 30 min.

[0045] (4) Centrifuge at 4℃ and the highest speed for 15 min.

[0046] (5) Remove the supernatant and wash the precipitate twice with 70% ethanol.

[0047] (6) Remove the supernatant, dry for 5-10 min, and dissolve the precipitate with NF H2O.

[0048] (7) RNA concentration was determined using nanodrop.

[0049] 2. mRNA pretreatment (1) 100 pmol mRNA and 100 pmol probe were heated at 70℃ for 10 min, placed on ice for 1 h, and incubated at 37℃ for 3 h. The sequence of the probe is as follows: AGAA / dG / / dA / / dA / / dT / ACUAGUUUAUUCCU-biotin.

[0050] (2) Prepare the RNsae H enzyme digestion reaction system and incubate it at 37℃ for 1 h. The reaction system is shown in Table 2: Table 2 RNsae H enzyme digestion reaction system

[0051] (3) 100 μL of SAbeads were washed 3 times with NF H2O and 3 times with SA beads washing buffer, and then set aside. Note: SAbeads washing buffer is obtained by diluting 2×SA beads binding buffer by one-fold.

[0052] (4) Add 100 μL of 2×SAbeads binding buffer to the reaction product and mix it with SAbeads. Place it on a rotary mixer and incubate at 30 rpm for 30 min at room temperature.

[0053] (5) Place on a magnetic rack for 5 min, remove the supernatant, wash 3 times with SA beads washing buffer, and wash 3 times with N-FH2O.

[0054] (6) Add 30 μL NF H2O, heat at 65℃ for 3 min, place on a magnetic rack for 5 min, and aspirate the supernatant.

[0055] 3. LC-MS detection The supernatant obtained in the above steps was added to LC-MS for analysis, with the relevant parameters as follows: (1) Chromatographic column: XBridge Oligonuleotide BEH C18 Column, 130 Å, 2.5 μm, 4.6 mm × 50 mm; (2) Mobile phase A: 0.1 mol / L TEAA; Mobile phase B: Acetonitrile; (3) Column temperature: 60℃; (4) Flow rate: 1.0 mL / min; (5) Gradient elution: mobile phase B from 5% to 30%, 15 min; (6) Detection wavelength: 260 nm.

[0056] Example 2 This embodiment is a method for detecting the capping rate of ψUTP-modified mRNA. The difference between this method and Example 1 is that ψUTP-modified mRNA is synthesized in step 1.

[0057] Comparative Example 1 The difference from Example 1 lies in the mRNA pretreatment method, as detailed below: (1) Design an RNase H probe. The probe consists of 4nt DNA + 18nt RNA + biotin. The RNA is modified with 2'-O-Me nucleotides, and its sequence is: mAmGmAmA / dG / / dA / / dA / / dT / mAmCmUmAmGmUmUmUmAmUmCmCmU-biotin.

[0058] (2) Prepare the reaction system as shown in Table 3: Table 3 Reaction System

[0059] The mRNA in this case is ψUTP-modified mRNA.

[0060] (3) The reaction system was annealed and incubated according to the following procedure: 95℃ for 5 min, 65℃ for 2 min, 55℃ for 2 min, 40℃ for 2 min, and 22℃ for 2 min.

[0061] (4) Mix with 100 μL of clean SA beads and incubate at room temperature for 30 min.

[0062] (5) Add 25U RNase H, mix well, and react at 37℃ and 1200rpm for 2h.

[0063] (6) Place the entire system on a magnetic rack, clean the magnetic beads, add 100 μL of 75% methanol and mix well. Incubate at 80°C for 3 min, then place on a magnetic rack to remove the supernatant.

[0064] (7) After evaporating the supernatant to dryness, redissolve it with 25 μL RF H2O.

[0065] The subsequent LC-MS detection method is the same as in Example 1.

[0066] Table 4 shows the theoretical enzyme digestion fragments, probe sequences, and molecular weights involved in Examples 1-2 and Comparative Example 1, as follows: Table 4 Theoretical enzyme digestion fragments, probe sequences, and molecular weights

[0067] The detection results of Examples 1-2 and Comparative Example 1 are as follows: Figure 1-6 As shown in Table 5. Figure 1 This is the deconvolution mass spectrum of Example 1. Figure 2 The Extract Masses Table of Example 1; Figure 3 This is the deconvolution mass spectrum of Example 2. Figure 4 The Extract Masses Table of Example 2; Figure 5 This is the deconvolution mass spectrum of Comparative Example 1. Figure 6 This is the Xtract MassesTable for Comparative Example 1.

[0068] Table 5 Summary of detection results for Examples 1-2 and Comparative Example 1

[0069] The capping rate of each experimental group in this invention is above 99%, and compared with Comparative Example 1, the hybridization reaction method used in Example 1 has a higher total intensity of enzyme digestion fragment peak signal; at the same time, the elution method of Example 1 can reduce probe residue and reduce the proportion of probe in the capping rate detection sample.

[0070] 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. A method for detecting mRNA capping rate, characterized in that, The method includes: The mRNA was hybridized with a base-free probe, and the product was digested with enzymes. Magnetic microparticles were then added to the reaction product, and after incubation, washing and elution were performed to obtain the mRNA pretreatment product. The mRNA pretreatment product was then detected by LC-MS. The conditions for the hybridization reaction were: heating at 68-72℃ for 2-10 min, ice bath for 0.5-1 h, and incubation at 35-38℃ for 1-4 h.

2. The method according to claim 1, characterized in that, The elution solution in the elution step is nuclease-free water.

3. The method according to claim 2, characterized in that, The elution method is as follows: add nuclease-free water to the washed magnetic microparticles, heat at 60~80℃ for 2~5 min, use a magnetic rack to attract the magnetic microparticles, and obtain the mRNA pretreatment product after treatment.

4. The method according to claim 1, characterized in that, The reaction system for the hybridization reaction contains mRNA, a base-free probe, and an incubation buffer. The incubation buffer consists of: 10 mM Tris-HCl at pH 7.5 and 0.1 M LiCl.

5. The method according to claim 4, characterized in that, The molar ratio of mRNA to unmodified probe in the reaction system is 0.8 to 1:

1.

6. The method according to claim 1, characterized in that, The composition of the base-free probe is: 5'-4~6nt RNA-4~6nt DNA-10~20nt RNA-Biotin-3'.

7. The method according to claim 1, characterized in that, The mRNA includes ψU-modified mRNA and mRNA without base modification.

8. The method according to claim 1, characterized in that, The reaction system for the enzyme digestion reaction contains the hybridization reaction product, reaction buffer, and RNsae H enzyme; the conditions for the enzyme digestion reaction are: 0.5~1.5 h at 37℃.

9. The method according to claim 1, characterized in that, The incubation method after adding magnetic microparticles is as follows: incubate at 20-50 rpm and room temperature for 20-40 min on a rotary mixer.

10. The method according to claim 1, characterized in that, The cleaning method is as follows: after incubation, the magnetic microparticles are treated with a magnetic rack to remove the supernatant, and then washed sequentially with magnetic bead washing buffer and nuclease-free water.

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