Method for detecting mRNA fracture fragment by using CE and MPRT method

Abstract

The invention discloses a method for detecting mRNA (messenger Ribonucleic Acid) fragmentation fragments by using CE and MPRT methods, and belongs to the technical field of biology. Compared with a traditional capillary electrophoresis analysis method, the method can more conveniently, accurately and efficiently analyze the fracture degree on the mononucleotide level, the resolution ratio of a generated result is clearer than that of a traditional method, and the method has important development value and wide application prospects in the field of quality control of mRNA vaccine production.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically is a method for detecting mRNA breakage fragments by using CE and MPRT methods. Background Art

[0002] mRNA vaccines have emerged as a promising strategy for viral infections.

[0003] These vaccines can induce innate immune responses, prevent viral infection or further induce cancer. However, due to the instability of mRNA, measures must be taken to prevent its degradation during storage and transportation, which relies on RNA fragment analysis methods.

[0004] For existing RNA fragment analysis methods, the most commonly used are high-performance liquid chromatography-mass spectrometry / mass spectrometry (HPLC-MS / MS) and microfluidic electrophoresis. HPLC-MS / MS is known for its high sensitivity and specificity, and is suitable for high-precision detailed analysis. However, HPLC-MS / MS is expensive, time-consuming, and requires expertise and equipment. Microfluidic electrophoresis is characterized by rapidity, automation, and low sample consumption. However, it also requires specific equipment and incurs certain consumable costs. In addition to RNA fragment analysis, second-generation and third-generation sequencing can also be used for mRNA quality analysis. Second-generation sequencing provides short to medium read lengths with high throughput and high accuracy. Third-generation traditional sequencing, such as PacBio and Nanopore, can provide real-time sequencing with long or ultra-long read lengths. However, they are more expensive and have higher error rates. In addition, mRNA quality analysis using third-generation sequencing requires reverse transcription, followed by cDNAPCR or cDNA synthesis base pairing, which may introduce additional biases.

[0005] The technology currently used to ensure the integrity of mRNA is capillary electrophoresis. However, after research, it was found that capillary electrophoresis has a very limited resolution and cannot screen complete mRNA. In addition, if small fragments break from the mRNA, CE cannot detect any changes. These fragments are likely to contain truncated antigens and lack their 3'UTR and poly(A) sequences. Secondly, since CE lacks all sequence information, it cannot provide sequence degradation correlation. Therefore, it is meaningful to develop a method for efficiently detecting RNA broken fragments. Summary of the invention

[0006] 1. Technical problem to be solved by the invention

[0007] The purpose of the present invention is to solve the problem of low efficiency and high error rate in the existing RNA fragment detection.

[0008] 2. Technical solution

[0009] In order to achieve the above object, the technical solution provided by the present invention is:

[0010] The present invention uses CE and MPRT methods to detect mRNA fragmentation fragments, and the method comprises the following steps:

[0011] S100, preparing mRNA samples to be tested;

[0012] S200, stimulates mRNA to break and form fragments;

[0013] S300, capillary electrophoresis quality detection;

[0014] S400, select specific reverse transcription primers and perform MPRT-Seq;

[0015] S500, sequencing the generated cDNA using STR;

[0016] S600, data analysis through SHAPE;

[0017] The reverse transcription primer of step S400 includes at least one of the following:

[0018] RTPrimer1(pairingposition:554–573):5′-FAM-GGAGGTAGATGAGATGTGAC-3′;

[0019] RTPrimer2(pairingposition:1036–1053):5′-FAM-GGCAGATGGAACCTCTTG-3′;

[0020] RTPrimer3(pairingposition:1561–1578):5′-FAM-GCGGTTGTTACTTGACTG-3′;

[0021] RTPrimer4(pairingposition:2065–2082):5′-FAM-CCGTCGTCCTTGAAGAAG-3′;

[0022] RTPrimer5(pairingposition:2493–2510):5′-FAM-CATGCAGTACCAGCTCGA-3′;

[0023] RTPrimer6(pairingposition:2771–2788):5′-FAM-TTGCTAGCTCCAGGGTGT-3′.

[0024] Preferably, the mRNA sample in step S100 consists of and LUC.

[0025] Preferably, in step S400, mRNA breakpoints are detected by MQRT-Seq reactivity.

[0026] Preferably, the method of simulating mRNA degradation is treatment at 37°C.

[0027] Preferably, the means of simulating mRNA degradation is freeze-thaw cycling.

[0028] 3. Beneficial effects

[0029] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0030] The present invention utilizes CE and MPRT methods to detect mRNA breakage fragments. Compared with the traditional capillary electrophoresis analysis method, the method of the present invention can more conveniently, accurately and efficiently analyze the degree of breakage at the single nucleotide level, and the resolution of the results produced is also clearer than that of the traditional method. It has important development value and broad application prospects in the field of quality control of mRNA vaccine production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The schematic diagram of the location of GFP-LUCmRNA and six reverse transcription primers and the research process;

[0032] Figure 2 Schematic diagram of capillary electrophoresis (CE) to assess GFP-Luc mRNA integrity under accelerated degradation;

[0033] Figure 3 Multi-primer reverse transcription sequencing (MPRT-seq) revealed the schematic diagram of the reactivity changes of GFP-Luc mRNA samples under accelerated degradation;

[0034] Figure 4 Schematic diagram of GFP-Luc mRNA secondary structure prediction and high degradation reactive sequence evaluation. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present application.

[0036] It should be noted that the terms "first", "second", etc. in the specification and claims of the present application and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequential order. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present application described here. In addition, the terms "including" and "having" and any of their variations are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device comprising a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0037] In the present application, the terms "upper", "lower", "left", "right", "front", "back", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings. These terms are mainly used to better describe the present application and its embodiments, and are not used to limit the indicated devices, elements or components to have a specific orientation, or to be constructed and operated in a specific orientation.

[0038] In addition, some of the above terms may be used to express other meanings in addition to indicating orientation or positional relationship. For example, the term "on" may also be used to express a certain dependency or connection relationship in some cases. For those of ordinary skill in the art, the specific meanings of these terms in this application can be understood according to specific circumstances.

[0039] In addition, the terms "installed", "set", "provided with", "connected", "connected", and "socketed" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral structure; it can be a mechanical connection, or an electrical connection; it can be a direct connection, or an indirect connection through an intermediate medium, or it can be an internal connection between two devices, elements, or components. For those of ordinary skill in the art, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0040] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0041] Example 1

[0042] Refer to the attached Figure 1-4 In this embodiment, a method of detecting mRNA fragmentation using CE and MPRT method comprises the following steps:

[0043] S100, preparing mRNA samples to be tested;

[0044] S200, stimulates mRNA to break and form fragments;

[0045] S300, capillary electrophoresis quality detection;

[0046] S400, select specific reverse transcription primers and perform MPRT-Seq;

[0047] S500, sequencing the generated cDNA using STR;

[0048] S600, data analysis through SHAPE;

[0049] The reverse transcription primer of step S400 includes at least one of the following:

[0050] RTPrimer1(pairingposition:554–573):5′-FAM-GGAGGTAGATGAGATGTGAC-3′;

[0051] RTPrimer2(pairingposition:1036–1053):5′-FAM-GGCAGATGGAACCTCTTG-3′;

[0052] RTPrimer3(pairingposition:1561–1578):5′-FAM-GCGGTTGTTACTTGACTG-3′;

[0053] RTPrimer4(pairingposition:2065–2082):5′-FAM-CCGTCGTCCTTGAAGAAG-3′;

[0054] RTPrimer5(pairingposition:2493–2510):5′-FAM-CATGCAGTACCAGCTCGA-3′;

[0055] RTPrimer6(pairingposition:2771–2788):5′-FA

[0056] The relevant test process using the method of this embodiment is as follows:

[0057] 1. RNA synthesis and secondary structure prediction

[0058] Design and synthesize an mRNA chain containing GFP protein and luciferase, and dissolve it in double distilled water without RNase to a concentration of 2 mg / ml. Use the RNAfold program to predict the secondary structure of RNA, and mRNA 3'poly (A) will not form base complementarity with other mRNAs.

[0059] 2. Simulate mRNA degradation

[0060] Two methods were used to simulate mRNA degradation: repeated freezing and thawing and high temperature at 37 degrees.

[0061] Dissolve 1 mL of RNA sample in a 2 mL tube, immerse the bottom of the tube in liquid nitrogen, ensure that the RNA sample is at the liquid nitrogen level, freeze for 20 seconds, and then thaw in room temperature water. Perform 5, 10, and 20 freeze-thaw cycles for three samples respectively. After reaching the corresponding cycle, take out 300 μL of RNA solution and transfer it to a new EP tube.

[0062] Heat treatment at 37°C, place 600 μL RNA sample deposited in EP tube into a constant temperature metal heating block, and after 24 hours and 48 hours, take out 300 μL RNA solution each time and transfer it to a new EP tube. The two treated samples use mRNA samples from the same batch without any treatment as controls. All samples are stored at -80°C for the next step of testing.

[0063] 3. Hair cell electrophoresis detection

[0064] The above mRNA samples were detected using the Bioptic Qsep1 capillary electrophoresis analyzer. 20 μL of the processed mRNA sample was taken out and analyzed using the Bioptic Qsep1 CE fragment analyzer (BiOptic Inc., Taiwan, China) and the cartridge kit with the R1 cartridge (RNA cartridge number R1-O-240222-58). The alignment marker was RNA-LM (20nt). The detection was performed using the full RNA QC program according to the manufacturer's protocol. The CE program analyzed the sample smear and the main peak cutoff, and calculated the distribution of the peak area and the average size of the fragment. The length range of the analyzed mRNA was approximately 2100 to 3300 nt, covering the entire main mRNA fragment peak. The result file was generated by Bioptic Software (version 3.4.3) report. The reported data include capillary electropherogram, relative fluorescence intensity migration time graph and RNA quality (RNA fragment range, average fragment length and its distribution).

[0065] The results are as follows Figure 2 As shown in the figure, first, we use standard CE to obtain a measurement baseline. Figure 2The electropherogram in A shows that the mRNA peaks elute in the length range of approximately 2100 nt to 3300 nt, with an average size of approximately 2500 nt. The integrity of the control mRNA was 91.1%, while the mRNAs treated with 5 and 10 freeze-thaw cycles both showed similar integrity of 91.8%. After 20 cycles, the loss of integrity of the mRNA samples increased to 88.2% compared to the control ( Figure 2 B, left panel, blue bars). On the other hand, incubation at 37°C could more effectively degrade our target mRNA. CE results showed that mRNA integrity dropped to 85.5% after 24 h and to 78.0% after 48 h ( Figure 2 A, B). Using CE analysis, we could determine a general loss of integrity of the target mRNA after incubation at 37°C, but not as high integrity for mRNA subjected to less severe freeze-thaw treatments.

[0066] 4. MPRT-Seq and Data Analysis

[0067] SHAPE is a widely used technique for RNA structural analysis. mRNA strand breaks due to degradation can also be detected using similar chemistry. However, a single reverse transcriptase read of 300-650 nt often limits the depth of a single reaction. To further analyze mRNA degradation, an optimized SHAPE method was used along with MPRT-seq. The reverse transcription (RT) primer pairing positions are as follows Figure 1 As shown in A. GFP-Luc mRNA RNA was annealed with 6 RT DNA primers respectively. 10 μL of annealed RNA solution was mixed with RT assay reagents, including 4 μL 1× HiScript III buffer, 1 μL 10 mM dNTPs and 10 units of HiScript III reverse transcription. The mRNA solution was incubated at 50°C for 30 minutes, then quenched with 0.8 μL 5M NaOH at 95°C for 5 minutes and neutralized by adding 29 μL of acid stop mixture. The resulting cDNA fragments were detected using STR.

[0068] The raw data were analyzed using ShapeFinder version 1.0 program. Reactivity was processed in 6 parts with 5′-FAM labeled RT primers; the reactivity quantification covered a range of approximately 500 nt between two adjacent primer pairing positions, and all quantitative data were assembled, normalized, and subtracted from the control reactivity using Excel. The average degradation line was generated according to the following rescaling equation:

[0069] IA(N)=A*(1+q)^N(1)

[0070] where IA(N) is the rescaled average intensity at position N, A is the overall average degradation response value obtained directly from a comprehensive gradient test with freeze-thaw cycles or heating at 37°C, and q is the rescaling factor.

[0071] For freeze-thaw cycles, the overall average degradation reactivity A was 3.5 with a rescaling factor q of 0.000001. For 37°C heating, A was 8.8 with a q of 0.00035. Degradation intensities above the relative average were defined as high. Based on this criterion, we identified four groups of sequences, each containing 5 to 10 consecutive nucleotides that showed high reactivity in both freeze-thaw cycles and 37°C heating incubation.

[0072] The results are as follows Figure 3 As the freeze-thaw cycles increased from 0 (control) to 20 cycles, we observed a linear increase in the average reactivity ( Figure 3 A, left panel). The average MPRT-seq reactivity increased from 0.71 (control) to 1.95, 4.14, and 6.54 ( Figure 3 A, right panel). While the control shows only a few small peaks, after freeze-thaw cycles, individual peaks corresponding to RNA lengths of 1000, 1500, 2000, and 2500 nt appear. The peak around 2000-2500 nt shows the strongest reactivity ( Figure 3 A, left panel). Similar to the CE results, MPRT-seq showed that heating at 37°C resulted in more degradation ( Figure 3 B, left panel), the average reactivity increased to 6.93 and 16.35 ( Figure 3 B, right panel). Figure 3 B shows that the peak of the 48-hour incubation sample is more intense than that of the 24-hour sample. Coincidentally, the results of the 24-hour sample are very similar to the 20-cycle freeze-thaw sample (CE: 88.2% vs. 85.5%, MPRT reactivity: 6.93 vs. 6.54). The two samples also share very similar SHAPE reactivity characteristics ( Figure 3 A, B, blue and orange traces).

[0073] The first aspect of the present invention utilizes MPRT-seq to detect mRNA samples, and the results not only agree well with CE analysis, but also provide the sequence identity of the truncation position ( Figure 3 and 4). Although second- and third-generation sequencing can also achieve sequence specificity, MPRT-seq is able to conveniently, accurately, and efficiently analyze the extent of 0 breaks at the single nucleotide level. Due to their reliability and wide acceptance in the field, the industry can adopt these mature methods without much trouble. If any obvious degradation sites are found, designers can quickly modify the mRNA sequence and optimize codons to eliminate vulnerable sites, thereby obtaining higher mRNA purity and longer shelf life without affecting antigen expression.

[0074] The second aspect of the present invention uses the SHAPE analysis method to provide high-precision quality control for mRNA production, and MPRT-seq can screen degradation-sensitive sequences and their structural features. Once developed, this information can provide an mRNA degradation model specific to each mRNA sequence and guide the improvement of mRNA stability, providing a more efficient detection method for mRNA drugs.

[0075] The above-described embodiments only express a certain implementation mode of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for detecting mRNA fragmentation using CE and MPRT, characterized in that: The method comprises the following steps: S100, preparing mRNA samples to be tested; S200, stimulates mRNA to break and form fragments; S300, capillary electrophoresis quality detection; S400, select specific reverse transcription primers and perform MPRT-Seq; S500, sequencing the generated cDNA using STR; S600, data analysis through SHAPE; The reverse transcription primer of step S400 includes at least one of the following: RT Primer 1(pairing position:554–573):5′-FAM-GGAGGTAGATGAGATGTGAC-3′; RT Primer 2(pairing position:1036–1053):5′-FAM-GGCAGATGGAACCTCTTG-3′; RT Primer 3(pairing position:1561–1578):5′-FAM-GCGGTTGTTACTTGACTG-3′; RT Primer 4(pairing position:2065–2082):5′-FAM-CCGTCGTCCTTGAAGAAG-3′; RT Primer 5(pairing position:2493–2510):5′-FAM-CATGCAGTACCAGCTCGA-3′; RT Primer 6(pairing position:2771–2788):5′-FAM-TTGCTAGCTCCAGGGTGT-3′.

2. The method for detecting mRNA fragmentation using CE and MPRT according to claim 1, characterized in that: The mRNA sample in step S100 is composed of and LUC.

3. The method for detecting GFP mRNA breakage fragments according to claim 1, characterized in that: In step S400, mRNA breakpoints are detected by MQRT-Seq reactivity.

4. The method of claim 1 for detecting mRNA fragmentation using CE and MPRT, characterized in that: The method of simulating mRNA degradation was treatment at 37°C.

5. The method of detecting mRNA fragmentation using CE and MPRT according to claim 1, characterized in that: A way to simulate mRNA degradation is freeze-thaw cycling.

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