CN116590382B - Probe for detecting capping rate of mRNA sample, kit and use method thereof - Google Patents
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Abstract
The application relates to the field of biotechnology, in particular to a probe for detecting the capping rate of an mRNA sample, a kit and a use method thereof; the kit comprises probes, wherein the specific probe design form is that RNA fragments 0-50nt+DNA fragments 1-10nt+RNA fragments 0-50nt, and selenium substitution is carried out on one or more bases on the DNA fragments; the non-seleno part of the DNA fragment of the probe is specifically cut by RNase H fast cutting enzyme; the kit can detect capping rates of mRNA with different lengths, and has accurate and stable result, retention time and capping rate result RSD <1%.
Description
Technical Field
The application relates to the technical field of biology, in particular to a probe and a kit for detecting capping rate of an mRNA sample and a use method thereof.
Background
The cap structure is an essential element for mRNA to perform biological functions in vivo. Briefly, the mRNA cap structure has 2 primary functions: first, protein translation machinery is recruited in cells such that mRNA translation expresses proteins that perform specific biological functions; second, mRNA stability is improved, avoiding degradation by intracellular endonucleases. Since cap structure has such important functions, detecting the rate of mRNA capping is undoubtedly one of the most critical indicators in quality analysis of mRNA production processes.
mRNA molecular weight is generally hundreds to thousands of kDa, and the cap structure is only about 500 and Da. The extremely small cap structure appears slightly indistinguishable in the presence of large mRNA molecules, which results in extremely fine differences between capped and uncapped mRNA, i.e., one nucleotide or methyl, which is difficult to capture by conventional separation analysis methods. Researchers in the detection of mRNA capping rates have become aware of this problem initially, and therefore they enzymatically degrade or cleave mRNA molecules of enormous molecular mass into analyzable isotopically labeled mononucleotides or 5' terminal short sequences, such as RNase T2, RNase H, ribozyme, etc. Detection of cap orientation can also be achieved by matching with pyrophosphatase (TAP or RppH). Researchers use polyacrylamide gel electrophoresis, capillary gel electrophoresis or LC-MS systems to distinguish and quantify short fragments carrying caps, and ultimately determine mRNA capping rates.
RNase H is an endonuclease that removes RNA primers from fragments of DNA replication or processes R-Loops to modulate R-Loops mediated biological processes such as gene expression, DNA replication and DNA or histone modification. In vitro, studies have reported that E.coli RNase H can be cleaved at a specific site in single stranded RNA using a DNA-RNA chimeric structure. However, the RNase H cleavage site is 1 or more, resulting in the generation of 5' -terminal short cleavage fragments which are heterogeneous, differing from each other by 1 or several nucleotides.
The liquid chromatography-mass spectrometry can convert the composition and content changes of different sample effluents into mass spectrum signals, and the mass spectrum signals are measured by a mass spectrum instrument, so that qualitative and quantitative analysis is realized, and the product purity and the integrity of mRNA are characterized. However, the method has high requirement on sample size, is easy to generate ion addition peaks, and has the phenomenon of unstable result on mRNA detection by liquid chromatography mass spectrometry, which is reflected by the fact that the retention time of chromatographic peaks is not repeated and the change of capping rate results is large.
Disclosure of Invention
In order to solve the defects of the prior art, solve the problems that the existing liquid chromatography mass spectrometry is easy to generate ion addition peaks, and the capping rate detection result of mRNA fragments obtained by cutting RNase H fast cutting enzyme is unstable, and the sample consumption is large, the application discloses the following scheme:
the application discloses a customized probe in a first aspect, wherein the specific customized probe is designed in a form that one or more bases on a DNA fragment are subjected to selenium substitution, wherein the RNA fragment is 0-50nt+DNA fragment 1-10nt+RNA fragment 0-50 nt; selenium substitution specifically refers to the substitution of an oxygen atom in the phosphate group on the deoxynucleic acid with a selenium atom. The customized probe is reversely complementary with a specific section at the 5' end of the mRNA to be detected; the portion of the DNA fragment of the customized probe that is not seleno is specifically cleaved by RNase H rapid-cutting enzyme.
Preferably, the probe design is RNA fragment 0-50nt+DNA fragment 2-10nt+RNA fragment 0-50nt.
Wherein the DNA fragment may be further selected from any one of 3, 4, 5, 6, 7, 8, 9nt in length.
Preferably, the customized probe is designed in the form of an RNA fragment 10nt+a DNA fragment 6nt+an RNA fragment 10nt, and selenium substitution is performed on one or more bases on the DNA fragment.
The reduced likelihood of cleavage after selenium substitution results in a greater propensity for cleavage at the fourth unsubstituted DNA only, i.e., increased cleavage specificity. Such designs can raise the enzyme cleavage specificity to more than 90% with less than 50% before.
In a second aspect, the application discloses the use of a combination of a tailored nucleic acid analysis-specific mobile phase, a nucleic acid analysis-specific system wash, and the above-described tailored probes in a kit for detecting mRNA capping rate.
Wherein the nucleic acid mobile phase and the flushing liquid are prepared by taking perfluoro triethylamine and benzene as main raw materials; the special formula of the mobile phase for nucleic acid and the preparation process are as follows: phase a 1% hexafluoroisopropanol +0.1% perfluorotriethylamine +0.1% benzene +98.8% aqueous solution; phase B0.5% hexafluoroisopropanol +0.05% perfluorotriethylamine +0.05% benzene +65% ethanol +34.4% aqueous solution; the whole plastic PET material container is prepared, uniformly mixed, subjected to ice water bath ultrasonic treatment for 10-30 hours, bottled and then stored by nitrogen filling.
The liquid phase system flushing fluid has the following unique formula and manufacturing process: 25% methanol, 25% acetonitrile, 25% isopropanol, 24.8% water, 0.1% perfluorotriethylamine, 0.1% benzene, 10 mM EDTA; and (3) preparing a plastic PET material container in the whole process, bottling, and filling nitrogen for preservation.
The mobile phase special for nucleic acid analysis and the flushing liquid special for nucleic acid analysis have the functions that firstly, the signal of the ion addition peak can be obviously reduced, and the ion addition peak from more than ten ion addition peaks to only the ion addition peak of remaining Na is reduced; and secondly, the peak type and the substance separation degree are obviously improved.
Through the special mobile phase for nucleic acid, the high-efficiency system flushing liquid and the customized probe, the accurate detection of the mRNA capping rate by the liquid chromatography-mass spectrometry can be realized.
The kit further comprises the following components in actual use: RNase H fast cutting enzyme, a non-ribozyme buffer set, high-load SA magnetic beads, a non-ribozyme low-adsorption consumable set and a chromatographic column special for ultra-efficient oligonucleotide separation.
Preferably, the coreless enzyme buffer set comprises solution 1: RNase H reaction buffer; solution 2:0.1 M NaCl; solution 3:5 mM Tris-HCl,0.5 mM EDTA,60 mM NaCl,pH7.5; solution 4:1% methanol/water solution, 100. Mu.m EDTA.
Preferably, the non-ribozyme low adsorption consumable set comprises a non-ribozyme low adsorption EP tube, a non-ribozyme low adsorption gun head and a non-ribozyme low adsorption sample bottle.
The third aspect of the application discloses a method for detecting mRNA capping rate by the kit, which comprises the following steps:
s1: mixing mRNA to be detected, a customized probe and the solution 1 in a non-ribozyme low adsorption EP tube by using a non-ribozyme low adsorption gun head, and annealing according to an annealing program;
s2: transferring the high-load SA magnetic beads into a non-ribozyme low-adsorption EP tube by using a non-ribozyme low-adsorption gun head, and cleaning the magnetic beads by using a solution 2;
s3: mixing the annealed solution with the cleaned magnetic beads by using a non-ribozyme low adsorption gun head, and incubating for 30 min at 37 ℃;
s4: transferring RNase H fast-cutting enzyme into an S3 solution by using a non-ribozyme low-adsorption gun head, incubating for 3H at 37 ℃, and carrying out enzymolysis on the hybridization double chains connected with the magnetic beads;
s5: washing the magnetic beads in S4 by using the solution 3;
s6: and (3) mixing the solution 4 with the magnetic beads in the step (S5) by using a non-ribozyme low adsorption gun head, incubating at 80 ℃ for 3 min, transferring to a magnetic rack for adsorption, and rapidly sucking the supernatant and transferring to a non-ribozyme low adsorption sample bottle.
S7: flushing the fluid system with a nucleic acid analysis specific system;
s8: carrying out liquid chromatography on the mRNA fragment obtained in the step S6 by utilizing a special mobile phase for nucleic acid analysis and a special chromatographic column for ultra-high-efficiency oligonucleotide separation;
s9: and analyzing the obtained chromatographic mass spectrogram, and calculating the capping rate.
Preferably, the ratio of mRNA to the customized probe is in the range of 1:10-10:1.
Preferably, the high-load SA magnetic beads are ferroferric oxide magnetic beads with the particle size of 30-3000 nm, and are coated with streptavidin.
Compared with the prior art, the application has the following remarkable effects:
(1) The application is not provided with a complete mRNA detection kit of each module in the market at present; by using the kit, the sample demand is reduced by one tenth compared with the literature method, and the result is accurate and stable. Retention time and capping rate results RSD <1%. And (3) injection: because the production cost of mRNA per unit mass is high, the cost of the detected sample is high, and the sample requirement is reduced to one tenth, so that the cost of the detected sample can be remarkably reduced. Specifically, the cost of the mRNA preparation is about 20 yuan per 100. Mu.g. The existing mRNA capping rate detection method requires 500pmol of mRNA and has a mass of 500 mug calculated according to the molecular weight of 1000000 Da. Thus, the sample cost required for detection is up to 100 yuan. By using the method described in this patent, the sample size can be reduced to 50pmol mRNA and the sample cost can be reduced to 10 yuan due to the increase of the signal intensity of the unit sample.
(2) By utilizing the technical scheme of the application for obtaining the customized probe by selenium substitution, the possibility that other sites are cut by endonuclease is reduced, the enzyme cutting is more prone to be carried out at the position of the fourth unsubstituted DNA, the enzyme cutting specificity is improved, and the enzyme cutting specificity which is less than 50% before is improved to more than 90%; so that the fragments after enzyme digestion are consistent, and the retention time consistency of chromatographic peaks in the chromatographic mass spectrometry is high; the capping rate measurement and calculation results are stable and accurate.
(3) The mobile phase special for nucleic acid analysis and the flushing liquid special for nucleic acid analysis have the functions that firstly, the signal of the ion addition peak can be obviously reduced, and the ion addition peak from more than ten ion addition peaks to only the ion addition peak of remaining Na is reduced; and secondly, the peak type and the substance separation degree are obviously improved.
(4) mRNA capping rates of different lengths were measured using the proprietary method, with 7 runs of each length sample in parallel. The results were accurate and stable with retention times and cap rate results RSD <1%.
Drawings
FIG. 1 is a sample pattern of LC-MS detection in example 1.
FIG. 2 is a graph of a sample obtained after pretreatment of the sample using a conventional probe in example 2.
FIG. 3 is a graph of a sample obtained after pretreatment of the sample with a selenium substitution probe in example 2.
Detailed Description
The application is further illustrated by means of the following examples, which are not intended to limit the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1 actual sample detection
Through continuous experiments and verification, the inventor of the application establishes a novel kit detection method of capping rate.
The application range is as follows: can be used for the production of mRNA by different capping processes, the detection of different types of cap structures and samples of different LC-MS systems.
Technical difficulties: the production cost of mRNA unit mass is high, and the sample detection cost is high; detection of mRNA capping rate using LC-MS is prone to ion addition peaks; the phenomenon of unstable result exists in the detection of the capping rate of mRNA, which is reflected in that the retention time of chromatographic peaks is not repeated and the result of the capping rate is changed greatly.
The solution method comprises the following steps: the combined kit of the special mobile phase for the special nucleic acid, the high-efficiency system flushing liquid and the customized probe can remarkably reduce the signal of the ion addition peak, and the signal is reduced from more than ten ion addition peaks to only the Na ion addition peak; and secondly, the peak type and the substance separation degree are obviously improved.
The kit assembly includes: (1) a mobile phase dedicated to nucleic acid analysis; (2) a system wash solution dedicated to nucleic acid analysis; (3) customizing the probe; (4) RNase H fast-cutting enzyme; (5) a no-ribozyme buffer set; (6) high-load SA magnetic beads; (7) a non-ribozyme low adsorption consumable set; (8) ultra-efficient oligonucleotide separation special chromatographic column.
The main method flow of the application is as follows: the probe is annealed, then the magnetic beads are combined, then dissociated and eluted, and finally LC-MS detection is carried out.
Sample processing
1. Probe annealing
a. The annealing system is prepared according to the following proportion
b. Annealing is performed at the following temperature
2. Magnetic bead bonding
a. Pretreatment magnetic beads
The beaver 2.8 mu m SA magnetic beads are balanced to room temperature, and are vibrated and mixed uniformly, 100 mu L/reaction is taken into a centrifuge tube, and the centrifuge tube is placed on a magnetic rack, and the storage liquid is removed. The mixture was then resuspended in equal amount of buffer (0.1M NaOH,0.05M NaCl). Placed on a magnetic rack and the supernatant removed. Add 1 mL of 0.1 m naci solution, reverse upside down, centrifuge on palm, continue washing until the supernatant is neutral.
b. Incubation
The resuspended beads were separated into centrifuge tubes at 100. Mu.L/reaction, placed on a magnetic rack and the supernatant removed. Then, 100. Mu.L of an annealing system was added, and the mixture was homogenized by pipetting, and incubated at 1000 rpm and 37℃for 30 minutes on a spin mixer.
RNase H cleavage
RNase H (50 units) was added to the incubated mixture, and the mixture was homogenized by pipetting and incubated at 37℃for 3H.
d. Cleaning
The incubated mixture was placed on a magnetic rack, washed 3 times with 100. Mu.L of wash buffer (5 mM Tris-HCl,0.5 mM EDTA,60 mM NaCl,pH7.5), and then washed 3 times again with nuclease-free water.
3. Dissociation elution
50. Mu.L of dissociation solution (1% methanol/water solution, 100. Mu.M EDTA) which had been preheated to 80℃was added, then incubated for 3 min at 80℃at 1000 rpm on a spin mixer, immediately transferred to a magnetic rack for adsorption for about 1 min, quickly aspirated supernatant transferred to a new centrifuge tube, centrifuged, and 40. Mu.L of supernatant was aspirated into a sample bottle.
4. LC-MS analysis
Mobile phase a: 494 mL ultrapure water (plastic graduated cylinder), 5mL hexafluoroisopropanol, 0.5mL perfluorotriethylamine, 0.5mL benzene were added; mobile phase B:2.5 mL hexafluoroisopropanol, 0.25 mL perfluorotriethylamine, 0.25 mL benzene, 325 mL ethanol (glass cylinder), 172 mL ultrapure water (plastic cylinder). After shaking and mixing, ice bath ultrasound 6 h.
Instrument parameters:
notice that: (1) The gun head, the centrifuge tube and the sample bottle used for pretreatment are all low-adsorption and Rnase free. The reagent used was sigma. (2) mobile phase bottles Agilent-specific PET mobile phase bottles were used. (3) System rinse LC-MS system 12 h was rinsed with rinse (25% methanol, 25% acetonitrile, 25% isopropyl alcohol, 24.8% water, 0.1% perfluorotriethylamine, 0.1% benzene, 10 mM EDTA). After the mobile phase was changed over, purge was added and the system was flushed 0.5. 0.5 h before the column was accessed.
mRNA capping rates of different lengths were measured using the kit method of the application, with samples of each length being run in parallel 7 times. The results were accurate and stable, retention times and cap rate results RSD <1%, see tables 1 and 2.
TABLE 1
TABLE 2
Example 2 (Effect verification of cleavage specificity after hybridization of seleno Probe)
mRNA capping rate detection was performed using mRNA samples and probes of the following sequences.
The sequence mRNA is 5'GGAAAUAAACUAGUACUUUUCUGGU3';
Probe1：5'dAdCdCdAdGdAAAAGUACUAGUUUAUUUCC3'；
Probe2：5'dAdCdCdAdGdAAAAGUACUAGUUUAUUUCC3'；
the 6 DNAs of Probe1 are DNA having no specific substitution. The first 4 DNA of Probe2 is selenium substitution, the 5 th DNA has no special modification, and the 6 th DNA is selenium substitution.
In addition to probes, other specific capping rate detection methods employ the methods of the kits of the application.
When probe1 was used for cap rate detection, the results are shown in FIG. 2, where there were 4 cleavage sites, each of which was 0 site,
1-position enzyme digestion, 2-position enzyme digestion and 3-position enzyme digestion. The 2-position enzyme digestion accounts for about 43 percent.
When probe2 was used for cap rate detection, the results are shown in FIG. 3, where there were only 2 cleavage sites, 1-position cleavage and 2-position cleavage, respectively. The 2-site enzyme digestion accounts for about 91 percent.
From the above results, it was found that the possibility of cleavage was reduced by using the selenium-substituted probe, resulting in a tendency of cleavage more at the fourth unsubstituted DNA alone, i.e., an improvement in cleavage specificity. Such designs can raise the enzyme cleavage specificity to more than 90% with less than 50% before.
The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.
Claims (8)
1. A probe for determining that the capping rate of mRNA is specifically cut by RNase H enzyme, which is characterized in that the probe is designed in the form of 10-50nt+2-10nt+10-50 nt of RNA fragment and selenium substitution is carried out on one or more bases on the DNA fragment; the selenium substitution refers to that the oxygen atom in the phosphate radical on the deoxidized nucleic acid is replaced by a selenium atom;
the probe is reversely complementary with a specific section at the 5' end of mRNA to be detected; the non-seleno portion of the DNA fragment of the probe is specifically cleaved by RNase H rapid-cutting enzyme.
2. A kit comprising a mobile phase dedicated for nucleic acid analysis, a system wash dedicated for nucleic acid analysis, and the probe of claim 1, said kit for detecting mRNA capping rate;
the mobile phase special for nucleic acid analysis and the flushing liquid special for the nucleic acid analysis are prepared from perfluoro triethylamine and benzene as raw materials.
3. The kit of claim 2, further comprising the following components: RNase H fast cutting enzyme, a non-ribozyme buffer set, high-load SA magnetic beads, a non-ribozyme low-adsorption consumable set and a chromatographic column special for ultra-efficient oligonucleotide separation.
4. The kit of claim 3, wherein the coreless buffer kit comprises solution 1: RNase H reaction buffer; solution 2:0.1 M NaCl; solution 3:5 mM Tris-HCl,0.5 mM EDTA,60 mM NaCl,pH7.5; solution 4: 100. mu m EDTA.
5. The kit of claim 4, wherein the kit of parts comprises a coreless enzyme low adsorption EP tube, a coreless enzyme low adsorption gun head, and a coreless enzyme low adsorption sample bottle.
6. The kit of claim 5, wherein the method for detecting mRNA capping rate of the kit comprises the steps of:
s1: mixing mRNA to be detected, a probe and the solution 1 in a non-ribozyme low adsorption EP tube by using a non-ribozyme low adsorption gun head, and annealing according to an annealing program;
s2: transferring the high-load SA magnetic beads into a non-ribozyme low-adsorption EP tube by using a non-ribozyme low-adsorption gun head, and cleaning the magnetic beads by using a solution 2;
s3: mixing the annealed solution with the cleaned magnetic beads by using a non-ribozyme low adsorption gun head, and incubating for 30 min at 37 ℃;
s4: transferring RNase H fast-cutting enzyme into an S3 solution by using a non-ribozyme low-adsorption gun head, incubating for 3H at 37 ℃, and carrying out enzymolysis on the hybridization double chains connected with the magnetic beads;
s5: washing the magnetic beads in S4 by using the solution 3;
s6: mixing the solution 4 with the magnetic beads in the step S5 by using a non-ribozyme low adsorption gun head, incubating at 80 ℃ for 3 min, transferring to a magnetic rack for adsorption, and rapidly sucking the supernatant and transferring to a non-ribozyme low adsorption sample bottle;
s7: flushing the fluid system with a nucleic acid analysis specific system;
s8: carrying out liquid chromatography on the mRNA fragment obtained in the step S6 by utilizing a special mobile phase for nucleic acid analysis and a special chromatographic column for ultra-high-efficiency oligonucleotide separation;
s9: and analyzing the obtained chromatographic mass spectrogram, and calculating the capping rate.
7. The kit of claim 6, wherein the ratio of mRNA to probe is in the range of 1:10 to 10:1.
8. The kit of claim 7, wherein the high-load SA magnetic beads are ferroferric oxide magnetic beads with the particle size of 30-3000 nm, and are coated with streptavidin.
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