CN116640833A - Method for automatically detecting mRNA capping rate in batches by utilizing capillary electrophoresis technology - Google Patents
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Abstract
The invention provides a method for automatically detecting mRNA capping rate in batches by utilizing capillary electrophoresis technology, belonging to the technical field of biological detection. Hybridizing a hybridization probe designed according to the 5' UTR region sequence of mRNA to be detected with the mRNA to be detected to obtain a heterozygous sequence, and carrying out enzymolysis, purification and high-temperature denaturation and dissociation on the heterozygous sequence to obtain probe, capped and uncapped mRNA sequences. The capillary electrophoresis equipment is used for analyzing the mRNA capping rate, and the automatic batch quantitative detection of the mRNA capping rate can be realized by setting a reverse electrophoresis program and a specific gel casting and gel storage program. The capillary electrophoresis spectrum obtained by the invention has smooth characteristic peak base line, high signal-to-noise ratio and high separation degree between signal peaks of the capped sample and the uncapped sample although the molecular weights of the capped sample and the uncapped sample are similar. The invention has simple operation, low consumable cost, accurate and repeatable experimental result, and is very suitable for quality verification in mRNA medicine industrialization.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for automatically detecting mRNA capping rate in batches by utilizing a capillary electrophoresis technology.
Background
mRNA is used as a transient carrier of genetic information and can be synthesized into proteins by cellular machinery. The method has excellent potential in drug development due to the characteristics of safety, strong, quick, flexible and programmable production. Thus, mRNA drugs are very attractive as a novel nucleic acid drug, and related needs are increasing.
The most critical step in mRNA drug preparation is the production of structurally intact and biologically active mRNA stocks. The in vitro synthesized mRNA needs to have a complete structure to perform normal biological functions. The smallest mRNA structure typically contains a5 'cap, a 5'/3 'untranslated region (Untranslated region, UTR), an open reading frame encoding a protein (Open reading frame, ORF) and a 3' poly a tail. The structure of mRNA transcribed by eukaryotic RNA polymerase II, which is co-transcribed and modified at its 5' end, is called a cap. The cap structure is critical for mRNA: (1) mRNA caps promote efficient translation of mRNA by recruiting translation initiation factors; (2) the cap structure can protect mRNA from rapid degradation of exonuclease, so that the relative stability of mRNA is effectively maintained; (3) for drug development, mRNA lacking the cap structure will be recognized in the body as "abnormal" by the innate immune sensor pattern recognition receptor (Pattern recognition receptor, PRR), thereby eliciting excessive activation of the innate immune response and degradation of mRNA.
In recent years, based on species diversity, with intensive research, more Cap structures have been discovered, and in addition to the most common m7GPPPN Cap structures in eukaryotes, 2,7-m3GPPPN, 5' -gamma-mPPPN, mGpppN, gpppN, gpppmNm, m G (5 ') ppp (5 ') m62 AmpapCmpcm 3Um (Cap 4) and the like, as well as Nicotinamide Adenine Dinucleotide (NAD) caps originally found in bacteria and yeast, have also been identified in mammalian cells. Furthermore, commercially available co-transcribed caps for mRNA have been developed with capping analogs such as m7G (5 ') ppp (5') (2 'OMeA) pG, m7 (3' OMeG) (5 ') ppp (5') (2 'OMeA) pG, m7G (5') ppp (5 ') (2' OMe, m 6A) pG, etc., and mRNA co-transcribed cap analogs based on sense strand RNA viruses
m7G (5 ') ppp (5 ') (2 ' OMeA) pU. Because of the increasing variety of existing cap structures and the increasing demand for mass detection of mRNA capping rate, there is a need to develop a technical method for mass detection of mRNA capping rate. The current method for determining mRNA cap type and capping efficiency mainly comprises the following steps: ELISA (for example, chinese patent application CN105209633A combines with cap structure by using anti-m 7G specific antibody, and uses ELISA to quantitatively detect RNA capping efficiency by using primary antibody, the method needs specific reagent to make standard curve, applicable cap structure is limited, because of difference of sample, there is easy difference of accuracy between batches), HPLC chromatography (for example, chinese patent application CN105051213A, the method needs to use proper chromatographic column, the requirement of cap and uncapped sample is high, cap structure needs to have larger size to separate cap and uncapped sample peak easily from chromatogram), imageJ software analysis (for example, chinese patent application CN112626177A uses ImageJ software to analyze gel pattern to rapidly and quantitatively detect RNA capping efficiency, the method can only quantitatively analyze mRNA capping and uncapped sample amount difference obvious sample, the detection effect of mRNA sample with higher capping rate is limited for in vitro transcription), HPLC tandem mass spectrometry (for example, the method of Chinese patent application CN112111558A uses-MS to measure the activity of vaccinia virus capping enzyme), the method needs to have high precision in PCR (for example, the method of detecting mRNA capping enzyme activity by using cap and qPCR is limited by using a high-quality PCR device, the method is limited by using the PCR method of detecting the peak of the cap and the PCR device is limited by using the PCR method is limited by using the PCR device and the required to provide high-quality PCR method, thereby determining the activity of vaccinia virus capping enzyme. But this method is applicable only to enzymatic capping detection) and the like. In addition, most of the existing detection methods are manually operated, and no automatic processing and batch processing methods of mRNA samples exist, so that large differences exist in manually operated samples among batches, and the standard requirements of quality controllability of the samples among batches are difficult to realize.
Capillary electrophoresis (Capillary Electrophoresis, CE) technology is a method of separating and analyzing biomolecules (nucleic acids, proteins, polypeptides, sugars, etc.) based on differences in charge and molecular weight. The charged biological molecules are moved in the charged capillary by an electric field, and molecules with different sizes, charges and shapes are separated. mRNA is a single-stranded biological macromolecule composed of nucleotides, with negatively charged properties. Since nucleic acid mRNA has a negative charge, under the action of an electric field, the differences based on mRNA size, morphology, and charge density can move and segregate differently in capillaries. The specific process of mRNA isolation involves a number of factors including pH of the buffer, ion concentration and type, type and size of capillary material, and the like. In practical applications, adjustments and optimizations are required for different samples and experimental purposes. By optimizing experimental conditions, efficient, high-resolution separation and quantification of nucleic acid samples can be achieved. In summary, capillary electrophoresis technology is widely used in biomedical, molecular biology, food science, and other fields.
Chinese patent application CN115678968A discloses a method for detecting mRNA capping efficiency using capillary electrophoresis. The patent adopts a manual operation method, is mainly used for detecting the cap structure of the m7GPPPN, and has a relatively single detection object; the selected site of the probe is nucleotide in the 10 th-85 th region of the 5 'end of the 5' UTR, and the probe is not designed from the beginning, so that the selectable sequence interval of the probe is limited; because the probe designed from the head can be more difficult to distinguish between uncapped sequences and electrophoresis peaks of the capping sequences in capillary electrophoresis, a detection method technology with better resolution is needed, however, the peak images of the results obtained by the method are not independent but are adhered together, so that the accuracy of calculation of the detection result of the capping rate can be affected; the DNA probe designed by the patent needs to be subjected to enzymolysis twice after being combined with the mRNA to be tested, namely DNase enzymolysis is needed after RNase enzymolysis.
Therefore, developing a complete set of simple, economical, efficient, batch and high-resolution method for detecting mRNA capping efficiency through automatic processing is one of the problems to be solved urgently in mRNA drug research and drug production quality inspection.
Disclosure of Invention
The invention aims to provide a method for automatically detecting mRNA capping rate in batches by utilizing a capillary electrophoresis technology.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for automatically detecting mRNA capping rate in batches by utilizing capillary electrophoresis technology, which comprises the following steps:
(1) Designing a hybridization probe according to the sequence of the 5' UTR region of mRNA to be detected;
(2) Denaturing and annealing the hybridization probe and mRNA to be detected to obtain a heterozygous sequence;
(3) Directionally cutting the heterozygous sequence by using RNA hydrolase, purifying and then carrying out high-temperature denaturation and dissociation to obtain a mixture containing the probe, capped mRNA and uncapped mRNA;
(4) And (3) performing capillary electrophoresis on the mixture extracted in the step (3), and calculating the capping rate of the mRNA to be detected according to the capillary electrophoresis result.
Preferably, the hybridization probe is reverse-complementary to the mRNA to be detected.
Preferably, the hybridization probe is designed according to the base sequence of the 5'UTR region of the mRNA to be detected from the 1 st to 100 th region of the 5' end.
Preferably, the length of the hybridization probe is more than or equal to 15nt.
Preferably, the cap structure of the mRNA to be tested comprises: cap structure of the 5 'end of natural mRNA, synthetic cap structure analogs with modified or unmodified mRNA5' end.
Preferably, the addition ratio of the mRNA to be detected and the hybridization probe in the step (2) is 1:1-1:5.
Preferably, the denaturing conditions of step (2) are: denaturation at 90-100 ℃ for 1-10 min, and annealing: annealing at 70 deg.C, and cooling to room temperature at-10 deg.C/2 min.
Preferably, the RNA hydrolase in the step (3) is one of RNase H, RNase HI and RNase II.
Preferably, the temperature of the high-temperature denaturation dissociation in the step (3) is 70-100 ℃ and the time is 2-10 minutes.
Preferably, the length of the capillary tube in the step (4) is 10-60 cm.
Preferably, the capillary electrophoresis in the step (4) further comprises a reverse electrophoresis program, wherein the reverse electrophoresis time is 10-60 minutes, and the gel preservation temperature is 2-8 ℃ during electrophoresis.
The invention designs a hybridization probe according to the 5' UTR region sequence of mRNA to be detected, then hybridizes the hybridization probe with the mRNA to be detected to obtain a heterozygous sequence, and carries out enzymolysis, purification and high-temperature denaturation and dissociation on the heterozygous sequence to obtain probe, capped and uncapped mRNA sequences. The mRNA capping rate is analyzed by an automatic nucleic acid sample processing system and capillary electrophoresis equipment with good sensitivity, high resolution and accurate precision, and the automatic batch quantitative detection of the mRNA capping rate can be realized by setting a reverse electrophoresis program and a specific gel casting and gel storage program, and once the program is set, the mRNA capping rate can be stably operated without complex operation. And the capillary gel is stored at 2-8 ℃, so that the stability of the gel property can be maintained within 24 hours, the repeated balance and gel filling of the editing program are carried out at intervals, and a stable mRNA capping rate batch detection result can be obtained.
The capillary electrophoresis spectrum obtained by the method has smooth characteristic peak base line, high signal-to-noise ratio and high separation degree between signal peaks although the molecular weights of the capped sample and the uncapped sample are similar. For a plurality of mRNA capping samples, reverse electrophoresis procedures are added through single glue filling, and electrophoresis can be performed through multiple sample injections, so that capping rate detection results of the plurality of samples are obtained, and batch detection is realized. The invention has simple operation, low consumable cost, accurate and repeatable experimental result, and is very suitable for quality verification in mRNA medicine industrialization.
Drawings
FIG. 1 is a capillary electrophoresis diagram of an enzyme-cleaved capped (G) probe of example 3;
FIG. 2 is a capillary electrophoresis of the capped (G) sample of example 3;
FIG. 3 is a capillary electrophoresis of an uncapped (G) sample of example 3;
FIG. 4 is a capillary electrophoresis of a sample mixture of capped (G) and uncapped (G) of example 3;
FIG. 5 is a capillary electrophoresis of a capped (U) sample of example 3;
FIG. 6 is a capillary electrophoresis chart of internal reference markers (20 nt, 50nt, 70nt and 100 nt) in example 3.
Detailed Description
The invention provides a method for automatically detecting mRNA capping rate in batches by utilizing capillary electrophoresis technology, which comprises the following steps:
(1) Designing a hybridization probe according to the sequence of the 5' UTR region of mRNA to be detected;
(2) Denaturing and annealing the hybridization probe and mRNA to be detected to obtain a heterozygous sequence;
(3) Directionally cutting the heterozygous sequence by using RNA hydrolase, purifying and then carrying out high-temperature denaturation and dissociation to obtain a mixture containing the probe, capped mRNA and uncapped mRNA;
(4) And (3) performing capillary electrophoresis on the mixture obtained in the step (3), and calculating the capping rate of the mRNA to be detected according to the capillary electrophoresis result.
In the present invention, the hybridization probe is reverse-complementary to the mRNA to be measured.
In the invention, the hybridization probe is designed according to the base sequence in the 1 st to 100 th regions of the 5 'end of the 5' UTR region sequence of mRNA to be detected, and the length of the hybridization probe is more than or equal to 15nt.
In the present invention, the hybridization probe is preferably further coupled with biotin for purification by coupling with streptavidin magnetic beads.
In the present invention, the cap structure of the mRNA to be measured comprises: cap structure of the 5 'end of natural mRNA, synthetic cap structure analogues with modified or unmodified mRNA5' end, preferably G, U, A, C caps and N nucleotide analogues thereof (n.gtoreq.1).
In the invention, the addition ratio of the mRNA to be detected and the hybridization probe in the step (2) is 1:1-1:5.
In the present invention, the conditions for the denaturation in the step (2) are as follows: denaturation at 90-100℃for 1-10 min, preferably 95℃for 5 min.
In the present invention, the annealing in step (2) is: annealing at 70 deg.C, and cooling to room temperature at-10 deg.C/2 min.
In the present invention, the RNA hydrolase in step (3) is one of RNase H, RNase H I and RNase H II, preferably RNase H.
In the present invention, the temperature of the RNA hydrolase cleavage reaction is 35 to 40℃for 1 to 4 hours, preferably 37℃for 3 hours.
In the present invention, the temperature of the high temperature denaturation dissociation in the step (3) is 70 to 100℃for 2 to 10 minutes, preferably 80℃for 3 minutes.
In the present invention, the processes of steps (2) and (3) are performed on an automatic nucleic acid extractor, which is: thermo Scientific KingFisherApex nucleic acid purification system or Roche MagNAPure LC fully automated nucleic acid purification and sample addition system, preferably Thermo Scientific KingFisherApex nucleic acid purification system.
In the invention, the length of the capillary tube in the step (4) is 10-60 cm.
In the present invention, the time for the capillary electrophoresis in the step (4) is 50 to 70 minutes, preferably 60 minutes.
In the present invention, the capillary electrophoresis of step (4) further comprises a step of adding reverse electrophoresis for 10 to 60 minutes, preferably 20 minutes, after completion of electrophoresis of each needle sample.
In the present invention, it is also necessary to add an internal nucleic acid standard for performing the capillary electrophoresis described in step (4).
In the present invention, the process of step (4) is performed on a capillary electrophoresis apparatus, which is: BECKMAN PA800 or SCIEX PA800plus.
In the present invention, the capillary electrophoresis in the step (4) further includes a step of enzyme-cutting probe electrophoresis, which can be used to detect whether the designed probe has self-cutting, and the accuracy of the result is affected by the self-cutting of the probe.
In the invention, the storage temperature of the gel used in the capillary electrophoresis in the step (4) is 2-8 ℃, the stability of the gel property can be maintained within 24 hours, the repeated balance and gel filling are performed at intervals of the editing program, and the stable mRNA capping rate batch detection result can be obtained.
In the invention, the detection method of the mRNA capping rate can be widely applied to quality control application of mRNA in basic biological research, clinical diagnosis, drug development and drug production.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
In this example, uncapped firefly luciferase (Firefly luciferase, fluc) mRNA and capped Fluc mRNA were used as detection targets. Uncapped and capped Fluc mRNA samples were prepared by in vitro transcription using the co-transcription method.
The preparation of in vitro transcription reactions and mRNA capping test samples must be performed in a completely DNase/RNase free environment and unless specified, all consumables, reagents, buffers, etc. involved require no nuclease.
(1) Preparation of conventional codon optimized uncapped Fluc mRNA samples by in vitro transcription
The linearized DNA plasmid containing the Fluc gene sequence was used as transcription template after enzyme removal purification, and was transcribed using a commercially available T7 in vitro transcription kit. The reaction system is shown in Table 1.
TABLE 1 uncapped mRNA in vitro transcription System
| Component (A) | 20 mu L of reaction system (mu L) |
| DNase/RNase-Free Water | 6 |
| GTP(100mM) | 2 |
| ATP(100mM) | 2 |
| UTP1(100mM) | 2 |
| CTP1(100mM) | 2 |
| 10X Transcription Buffer | 2 |
| DNA template(2μg)1000ng/μL | 2 |
| T7RNA Polymerase Mix | 2 |
| Total volume of | 20 |
The components were added to a 200. Mu.L PCR tube without enzyme according to the above system, mixed and centrifuged, and then placed on a PCR instrument for reaction at 37℃for 2 hours. After completion of the transcription reaction, 2U of DNase I was added to each system and the reaction was continued at 37℃for 15 minutes to remove the template DNA. And detecting the integrity of the synthesized mRNA by using denaturing agarose gel electrophoresis, purifying to obtain an uncapped mRNA sample after the detection is qualified, and detecting the concentration of the mRNA by using a micro ultraviolet spectrophotometer. The uncapped Fluc mRNA sequence of the conventional codon optimized firefly luciferase is shown in SEQ ID No. 1.
(2) Preparation of conventional and replicative codon optimized capped Fluc mRNA samples by in vitro transcription
Using linearized DNA plasmids containing the Fluc gene sequence, after enzyme removal purification, as transcription templates, transcription synthesis was performed using a commercially available T7 in vitro transcription kit, the reaction system is shown with reference to Table 2, and cap analogs were prepared according to conventional and replicative templates using commercially available m7 (3 ' OMeG) (5 ') ppp (5 ') (2 ' OMeA) pG (G caps, then G-caps) or m7G (5 ') ppp (5 ') (2 ' OMeA) pU (U caps, then U-caps), respectively. The replication codon optimized uncapped Fluc mRNA sequence is shown in SEQ ID No. 2.
TABLE 2 capping mRNA in vitro transcription System
| Component (A) | 20 mu L of reaction system (mu L) |
| Dnase/RNase-Free Water | 4.4 |
| GTP(100mM) | 2 |
| ATP(100mM) | 2 |
| UTP1(100mM) | 2 |
| CTP1(100mM) | 2 |
| G hat/U hat (100 mM) | 1.6 |
| 10X Transcription Buffer | 2 |
| DNA template(2μg)1000ng/μL | 2 |
| T7RNA Polymerase Mix | 2 |
| Total volume of | 20 |
The components were added to a 200. Mu.L PCR tube without enzyme according to the above system, mixed and centrifuged, and then placed on a PCR instrument for reaction at 37℃for 2 hours. After completion of the transcription reaction, 2U of DNase I was added to each system and the reaction was continued at 37℃for 15 minutes to remove the template DNA. And detecting the integrity of the synthesized mRNA by using denaturing agarose gel electrophoresis, purifying to obtain a capped mRNA sample after the detection is qualified, and detecting the concentration of the mRNA by using a micro ultraviolet spectrophotometer.
Example 2
An on-press sample for mRNA capping rate detection was prepared.
The mRNA used for the preparation of the sample on the capillary electrophoresis was an uncapped mRNA sample prepared in example 1 and two capped mRNA samples. Two hybridization probes were commissioned and biotin was coupled to were synthesized by Shanghai Biotechnology Inc.:
Probe G(SEQ ID NO：3)：AATACTAGUUUAUUCUCCCU/3BioTEG/
Probe U(SEQ ID NO：4)：AAAATCUUUUGGCGUCGUGU/3BioTEG/
mRNA synthesized in example 1 was denatured and annealed with each probe according to the system of Table 3. The reaction procedure for automated sample processing is shown in table 4.
TABLE 3 denaturation annealing of mRNA to probes
After the addition of the components is completed, the components are evenly mixed and centrifuged.
The mixed samples of Table 3 were removed to 96-well deep well plates, placed on the control panel of a Thermo Scientific KingFisherApex nucleic acid purification system, and an automated mRNA sample processing procedure (magnetic beads were added during processing to purify the hybrid fragments of probe and mRNA) was set up according to Table 4.
TABLE 4 automated mRNA sample processing procedure
Through the procedure, purification of the mRNA capping rate detection sample is completed, and the sample can be detected by using capillary electrophoresis after cooling to room temperature.
Example 3
This example uses a capillary electrophoresis device to detect the capping rate of mRNA. The upper sample used was the upper sample obtained by the treatment in example 2: uncapped (G) on-machine samples, capped (G) on-machine samples, on-machine samples where uncapped (G) and capped (G) are mixed by equal mass, capped (U) on-machine samples. And (5) adjusting the sample loading volume to 20 mu L by using enzyme-free water, and performing on-machine detection.
The conditions used for capillary electrophoresis are: the capillary length is 60cm, the capillary electrophoresis temperature is 30 ℃, the electrophoresis voltage is 30kV, the ultraviolet light collection wavelength is 254nm, and the sample injection time is 15 seconds. The storage temperature of the gel was 8 ℃. The program sequence is set forth in table 5.
TABLE 5 automated mRNA (G) capping Rate detection procedure
In order to detect the presence or absence of self-cleaving sites in the probe, which affects the reaction efficiency of the probe, a step of adding a separate cleavage probe and running the capillary electrophoresis is required in the first test. In this example, the results of the capillary electrophoresis of the enzyme-cleaved probe G are shown in FIG. 1 (Table 5, no. 1). As shown in FIG. 1, the capillary electrophoresis diagram of the enzyme-cleaved probe G shows that the probe is a single peak, which indicates that the probe screened by computer assistance has no self-cleavage site, has good hybridization performance in theory, and is suitable for detecting the capping rate of mRNA.
In the in vitro co-transcription capping reaction, capping mRNA and unsuccessfully capped mRNA exist in the reaction system due to the competitive relationship between the cap structure and the nucleotide G and the difference of capping efficiency. After RNase H directional enzyme cutting, magnetic beads can be utilized to specifically capture mRNA fragments complementary to a hybridization region of the probe through biotin labels on the probe, after high-temperature denaturation, the mRNA fragments (containing capped mRNA and unsuccessfully capped mRNA fragments) of the complementary region of the probe are dissociated in a solution, and differences of electrophoresis speed of each fragment can be generated through capillary electrophoresis, so that each fragment is distinguished on a capillary electrophoresis chart, and the capping rate can be calculated through the peak area ratio of capped mRNA to total mRNA (containing capped mRNA and uncapped mRNA).
The ratio of peak areas of the individual peaks was obtained by software calculations (fig. 2, table 7), and the capping rate of the mRNA capping (G) samples was 4.71/(0.93+4.71) ×100% =83.51% according to the values of table 7.
TABLE 7 peak area ratio of individual peaks for mRNA capped (G) and uncapped samples
| PK# | Name | Time | Corrected Area | Corrected Area Percent |
| 1 | Probe with a probe tip | 42.48 | 5432.78 | 94.36 |
| 2 | Uncapped peaks in samples | 44.65 | 53.73 | 0.93 |
| 3 | Capping peaks in samples | 44.92 | 271.10 | 4.71 |
FIG. 3 is an uncapped (G) sample containing only probe peaks and uncapped (G) sample peaks using capillary electrophoresis.
FIG. 4 is a capillary electrophoresis plot of a mixture of capped (G) and uncapped (G) samples, labeled with the peak exit positions of the probe, uncapped (G) and capped (G), where the peak pattern of the probe, uncapped (G) and capped (G) samples in the mixture of FIG. 4 is consistent with the peak pattern of the capped (G) sample alone of FIG. 2.
Fig. 5 is a capillary electrophoresis chart of a capped (U) sample, and the ratio of peak areas of the peaks (table 8) was obtained by software calculation, so that the capping ratio of the U cap (fig. 5) was 27.46/(7.49+27.46) ×100% =78.57%.
TABLE 8 peak area ratio of individual peaks for mRNA capped (U) and uncapped samples
| PK# | Name | Time | Corrected Area | Corrected Area Percent |
| 1 | Probe with a probe tip | 41.88 | 3311.46 | 65.05 |
| 2 | Uncapped peaks in samples | 44.55 | 381.27 | 7.49 |
| 3 | Capping peaks in samples | 44.70 | 1398.15 | 27.46 |
In addition, the steady state of instruments, accessories and procedures can be monitored by adding internal reference markers (20 nt, 50nt, 70nt and 100 nt) (fig. 6), while the molecular weight of the sample can be accurately analyzed using the internal standard and the functions of the software itself. Thus, the invention is a method that is well suited for batch detection of mRNA capping rates.
In the embodiment 3, the characteristic peak baseline of each map is smooth, the signal to noise ratio is high, the molecular weight of a capped sample is similar to that of an uncapped sample, but the separation degree between the two signal peaks is high, so that the invention can design a synthetic probe according to the sequence of mRNA to be detected, protect mRNA from degradation of RNA hydrolase through hybridization of the mRNA to be detected and the probe, and obtain the sequence of the mRNA5' end containing the cap structure region after purification and high-temperature denaturation. The mRNA capping rate is analyzed by an automatic nucleic acid sample processing system and capillary electrophoresis equipment with good sensitivity, high resolution and accurate precision, and the automatic batch quantitative detection of the mRNA capping rate can be realized by setting a reverse electrophoresis program and a specific gel casting and gel storage program, and once the program is set, the mRNA capping rate can be stably operated without complex operation. The method disclosed by the invention is simple to operate, low in consumable cost, accurate and repeatable in experimental result, and very suitable for quality verification in the industrialization of mRNA medicaments.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A method for automatically detecting mRNA capping rate in batches by utilizing capillary electrophoresis technology, which is characterized by comprising the following steps:
(1) Designing a hybridization probe according to the sequence of the 5' UTR region of mRNA to be detected;
(2) Denaturing and annealing the hybridization probe and mRNA to be detected to obtain a heterozygous sequence;
(3) Directionally cutting the heterozygous sequence by using RNA hydrolase, purifying and then carrying out high-temperature denaturation and dissociation to obtain a mixture containing the probe, capped mRNA and uncapped mRNA;
(4) And (3) performing capillary electrophoresis on the mixture extracted in the step (3), and calculating the capping rate of the mRNA to be detected according to the capillary electrophoresis result.
2. The method of claim 1, wherein the hybridization probe is reverse-complementary to the mRNA to be detected; the hybridization probe is designed according to the base sequence in the 1 st to 100 th regions of the 5 'end of the 5' UTR region sequence of mRNA to be detected; the length of the hybridization probe is more than or equal to 15nt.
3. The method of claim 1, wherein the cap structure of the mRNA to be tested comprises: cap structure of the 5 'end of natural mRNA, synthetic cap structure analogs with modified or unmodified mRNA5' end.
4. The method of claim 1, wherein the ratio of the mRNA to be detected to the hybridization probe in step (2) is 1:1 to 1:5.
5. The method of claim 1, wherein the denaturing conditions of step (2) are: denaturation at 90-100 ℃ for 1-10 min, and annealing: annealing at 70 deg.C, and cooling to room temperature at-10 deg.C/2 min.
6. The method of claim 1, wherein the RNA hydrolase of step (3) is one of RNaseH, RNaseH i and RNaseH ii.
7. The method of claim 1, wherein the high temperature denaturing dissociation in step (3) is carried out at a temperature of 70 to 100 ℃ for a period of 2 to 10 minutes.
8. The method of claim 1, wherein the capillary tube of step (4) has a length of 10 to 60cm.
9. The method of claim 1, wherein the capillary electrophoresis of step (4) further comprises a reverse electrophoresis program, the reverse electrophoresis time being 10 to 60 minutes, and the temperature at which the gel is stored during electrophoresis being 2 to 8 ℃.
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