CN114480570A - Method for detecting mRNA 2' -O-methyltransferase activity - Google Patents
Method for detecting mRNA 2' -O-methyltransferase activity Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention provides a method for detecting the activity of mRNA 2' -O-methyltransferase. Specifically, the method comprises the following steps: (S1) providing a single-stranded RNA molecule having a 5 '-triphosphate at its 5' end; (S2) performing the following test reactions on the RNA molecule in (S1), the test reactions including reactions in a negative test system, a first test system, a second test system, wherein: in the negative test system, no enzyme is contained; a first test system comprising Cap0 capping enzyme; a second test system comprising Cap0 and mRNA 2' -O-methyltransferase; (S3) detecting (S2) the formation of SAH in the negative test system, the first test system and the second test system, and comparing with the standard curve to obtain the quantitative result of SAH. The method has the advantages of high safety, no radioactive pollution, simple steps, low experimental environment requirement and low cost.
Description
Technical Field
The invention belongs to the field of biological engineering, and particularly relates to a method for detecting the activity of mRNA 2' -O-methyltransferase.
Background
The main method for detecting the activity of 2' -O-methyltransferase at present is substrate radiolabeling by using [ methyl-3H]SAM characterizes all families of methyltransferases that use SAM (S-adenosylmethionine). Reaction in [ methyl-3H]Transferred [ 2 ] between SAM and short GpppX-RNA3H]CH3 was quantitatively analyzed and then fractionated using TLC product separation and radioactive counting. By using radiolabeled RNA/nucleotides, changes occurring in the structure of the nucleotide substrate can be observed. The method has the advantages of low safety, radioactive pollution, complex steps, high experimental environment requirement and high cost.
Therefore, there is a need in the art to develop a method for detecting the activity of mRNA 2' -O-methyltransferase, which is safe, free from radioactive contamination, simple in procedure, low in experimental environment requirements, and low in cost.
Disclosure of Invention
The invention aims to provide a method for detecting the activity of mRNA 2' -O-methyltransferase.
In a first aspect of the invention, there is provided a method for in vitro detection of mRNA 2' -O-methyltransferase activity, comprising the steps of:
(S1) providing a single-stranded RNA molecule having a 5 '-triphosphate at its 5' end;
(S2) performing the following test reactions on the RNA molecules in (S1), respectively, the test reactions including reactions in a negative test system, a first test system, and a second test system, the reaction time being t, wherein:
in the negative test system, no enzyme is contained;
a first test system comprising Cap0 capping enzyme;
a second test system comprising Cap0 and mRNA 2' -O-methyltransferase;
(S3) detecting (S2) the formation of SAH in the negative test system, the first test system and the second test system, and comparing the formation with a standard curve or standard value to obtain a quantitative result of SAH: wherein the content of the first and second substances,
the amount of SAH produced in the negative test system was A0; the generation amount of SAH in the first test system is A1; the generation amount of SAH in the second test system is A2; wherein, ΔQuality of food=A1-A0；△1=A2-A1；
If ΔQuality of foodIf the value is lower than the preset value, the test is considered to fail;
if Δ1/△Quality of foodIf > 1, the 2' -O-methyltransferase to be detected is regarded as enzymatically active, and the enzymatic activity is Delta1/t；
If Δ1/△Quality of foodIf =1, the 2' -O-methyltransferase to be detected is considered inactive.
Wherein, ". DELTA")Quality of food"represents a quality control value, i.e., a difference between the amounts of SAH produced in the first test system and the negative test system,
“△1"indicates the amount of SAH production by mRNA 2' -O-methyltransferase, i.e., the difference between the amount of SAH production in the second test system and the first test system.
In another preferred example, the predetermined value is 0.
In another preferred embodiment, the length of the single-stranded RNA molecule is 500- & ltwbr & gt 2000 nt, preferably 1000 nt.
In another preferred embodiment, the final concentration of the RNA in the negative test system, the first test system or the second test system is 150. mu.g/500. mu.l to 350. mu.g/500. mu.l, preferably 250. mu.g/500. mu.l, respectively.
In another preferred embodiment, the negative test system, the first test system, or the second test system each independently comprises SAM, GTP, 10 x capping reaction buffer, and RNase inhibitor.
In another preferred embodiment, the final concentration of the 10 × capping reaction buffer is 1 × capping reaction buffer.
In another preferred embodiment, the final concentration of SAM is 0.25 mM-0.75 mM, preferably 0.5 mM.
In another preferred embodiment, the final GTP concentration is between 0.5mM and 2mM, preferably 1 mM.
In another preferred embodiment, the final concentration of the RNase inhibitor is 0.5U/. mu.l-2U/. mu.l, preferably 1U/. mu.l.
In another preferred embodiment, the Cap0 capping enzyme is a vaccinia virus capping enzyme.
In another preferred embodiment, the final concentration of Cap0 capping enzyme in the first test system is 0.3U/. mu.l to 0.8U/. mu.l, preferably 0.4U/. mu.l.
In another preferred embodiment, the final concentration of Cap0 capping enzyme in the second test system is 0.3U/. mu.l to 0.8U/. mu.l, preferably 0.4U/. mu.l.
In another preferred embodiment, the final concentration of the mRNA 2' -O-methyltransferase in the second test system is from 2U/. mu.l to 8U/. mu.l, preferably 4U/. mu.l.
In another preferred embodiment, the reaction time t is 0.8-1.4 h; preferably, the reaction time t is 1 h.
In another preferred embodiment, the test reaction is carried out at 35-38 ℃; preferably, it is carried out at 37 ℃.
In another preferred embodiment, in the first test system, the reaction conditions are: the reaction time t is 0.8 to 1.4 hours at 37 ℃; preferably, the reaction time t is 1h at 37 ℃.
In another preferred embodiment, in the second test system, the reaction conditions are: the reaction time t is 0.8 to 1.4 hours at 37 ℃; preferably, the reaction time t is 1h at 37 ℃.
In another preferred example, (S1), the single-stranded RNA molecule having a 5 '-triphosphate at the 5' -end is mRNA obtained by in vitro transcription of DNA.
In another preferred example, (S1) further includes:
(S1.1) providing a DNA;
(S1.2) transcribing the DNA in vitro (S1) to obtain mRNA transcribed from the DNA.
In another preferred example, (S1.2) further includes: the mRNA after transcription and purification is placed in a PCR instrument at 65 ℃ for 5min, and immediately placed on ice for 5min after the reaction is finished.
In another preferred example, (S1.1), the target gene is eGFP gene.
In another preferred example, (S1.2), DNA is transcribed using T7 RNA polymerase to obtain mRNA transcribed from the DNA.
In another preferred example, (S3), the detection is HPLC detection.
In another preferred example, (S3) includes:
(S3.1) setting chromatographic conditions suitable for SAH separation;
(S3.2) plotting a standard curve of SAH: diluting SAH standard substance into SAH standard solution with different molar concentrations in a gradient manner, and drawing a standard curve of SAH according to the SAH peak area in liquid chromatogram under the chromatographic condition of (S3.1) by taking the SAH molar quantity as a horizontal coordinate and the peak area as a vertical coordinate;
(S3.3) calculating the content of SAH: and (3) respectively detecting the first test system, the second test system and the negative control group under the chromatographic condition in the step (S3.1), obtaining corresponding SAH peak areas, and respectively substituting the corresponding SAH peak areas into the standard curve in the step (S3.2) for calculation to obtain corresponding SAH contents in the first test system, the second test system and the negative control group.
In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.
In another preferred embodiment, the method is an in vitro method.
In another preferred example, the "chromatographic conditions suitable for SAH separation" means that the peak position t1 of SAH is more than or equal to 2min (t1-t0) compared with the peak position t0 of SAM in the test system; preferably more than or equal to 4 min; more preferably more than or equal to 6 min.
In a second aspect of the present invention, there is provided a kit for detecting mRNA 2' -O-methyltransferase activity, the kit comprising:
(a) a first container, and a negative test system located in the first container;
(b) a second container, and a first test system located in the second container;
(c) a third container, and a second test system located in the third container; and
(d) a fourth container, SAH standard; wherein the content of the first and second substances,
in the negative test system, no enzyme is contained; a first test system comprising Cap0 capping enzyme; in a second test system, Cap0 capping enzyme and mRNA 2' -O-methyltransferase were included.
In another preferred embodiment, the negative test system, the first test system, or the second test system each independently comprises SAM, GTP, 10 x capping reaction buffer, and RNase inhibitor.
In another preferred embodiment, the kit further comprises instructions for use.
In another preferred example, the SAH standard is used to plot a standard curve of SAH.
In the third invention of the present invention, there is provided a device for detecting an mRNA 2' -O-methyltransferase activity, the device comprising:
(d1) a reaction module comprising a negative control chamber, a first reaction chamber, and a second reaction chamber, wherein,
the negative control chamber contains a negative test system;
the first reaction chamber comprises a first reaction module comprising a first test system;
the second reaction chamber comprises a second reaction module comprising a second test system;
wherein, the negative test system does not contain enzyme; a first test system comprising Cap0 capping enzyme; a second test system comprising Cap0 and mRNA 2' -O-methyltransferase;
(d2) a data acquisition module configured to perform data acquisition on the SAH peak areas in each reaction chamber in a reaction module;
(d3) an analysis module configured to analyze the SAH peak area from the data acquisition module through a "peak area-concentration" standard curve of SAH to obtain a 2' -O-methyltransferase activity analysis result to be screened;
(d4) and the output module outputs the analysis result of the screening analysis module.
In another preferred embodiment, the final concentration of Cap0 capping enzyme in the first test system is 0.3U/. mu.l to 0.8U/. mu.l, preferably 0.4U/. mu.l.
In another preferred embodiment, the final concentration of Cap0 capping enzyme in the second test system is 0.3U/. mu.l to 0.8U/. mu.l, preferably 0.4U/. mu.l.
In another preferred embodiment, the final concentration of the mRNA 2' -O-methyltransferase in the second test system is from 2U/. mu.l to 8U/. mu.l, preferably 4U/. mu.l.
In another preferred embodiment, in the first test system, the reaction conditions are: reacting for 0.8-1.4h at 37 ℃; preferably, the reaction is carried out at 37 ℃ for 1 h.
In another preferred embodiment, in the second test system, the reaction conditions are: reacting for 0.8-1.4h at 37 ℃; preferably, the reaction is carried out at 37 ℃ for 1 h.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is an agarose gel electrophoresis image of the DNA template obtained by PCR amplification. The fragment size of the DNA template is shown, and M is the molecular weight marker.
FIG. 2 is an agarose gel electrophoresis of transcribed RNA. The fragment size of the DNA template is shown, and M is the molecular weight marker.
FIG. 3 is a liquid chromatogram (Waters) of the SAH standard, with the peak position of the SAH standard being around 10 min.
FIG. 4 is a liquid chromatogram (Waters) of SAM, and the peak position of SAM as a component in the reaction system is about 3 min.
Fig. 5 is a SAH standard graph. The linear relationship of SAH molar quantity X and peak area Y is shown.
FIG. 6 is a liquid chromatogram (Waters) of a sample, where the position marked by an arrow is the peak position of SAH.
Detailed Description
The present inventors have conducted extensive and intensive studies and, for the first time, have developed a method for efficiently and safely detecting the activity of mRNA 2' -O-methyltransferase. Specifically, the activity of mRNA 2 '-O-methyltransferase was determined by performing Cap0 reaction on RNA transcribed from DNA with vaccinia virus capping enzyme and setting a negative control, further performing Cap1 reaction on target gene RNA with vaccinia virus capping enzyme and mRNA 2' -O-methyltransferase, and detecting the amount of SAH (S-adenosylhomocysteine) produced as a by-product in the above reaction by HPLC method. In particular, in the Cap1 capped two-enzyme reaction system, the activities of vaccinia virus capping enzyme and 2' -O-methyltransferase were not affected by each other, so that the enzyme activity assay could be performed simply and rapidly (within 2 hours). The present invention has been completed based on this finding.
Cap Structure and 2' -O-methyltransferase
The rapid development of mRNA vaccine technology has led to an increasing demand for RNA biology. Cap Cap structure biosynthesis is an important step in mRNA maturation. It has become a hotspot for research because of its impact on the development and treatment of many viral infections.
Eukaryotic mRNA is a complex molecule, consisting of various nucleotide regions. The central part of the mRNA sequence is the protein coding segment located between the 5 'and 3' untranslated region (UTR). The 3' end of the mRNA is protected by a poly (A) tail, which consists of multiple adenosines monophosphates, and is stably transcribed and involved in translation. A unique nucleotide structure called a "Cap" is located at the 5' mRNA end, i.e., a "Cap". It has a 7-methylguanosine component linked to the first nucleotide of the nascent transcript via a 5'-5' -triphosphate bridge.
Cap Cap structure biosynthesis is an important step in mRNA maturation. Cap Cap biosynthesis involves three consecutive enzymatic reactions mediated by RNA triphosphatase (TPase), guanylyltransferase (GTase) and N7-guanine methyltransferase (N7-MTase), ultimately forming the Cap0 structure, which is the predominant form of simple structures, such as yeast in eukaryotes, structures in plants, etc. However, in higher organisms, including humans, mRNA exists primarily in the form of Cap1 in vivo.
For the Cap structure of Cap0, the Cap structure of Cap1 has some more protection function for RNA on the basis of its function. First, it protects the mRNA from rapid degradation by exonucleases. Secondly, compared with the Cap structure of Cap0, the Cap structure of 5' Cap1 can significantly improve the translation efficiency of RNA during translation and improve the expression of mRNA after microinjection and transfection. In addition, the 5' Cap1 Cap structure also plays a further role in preventing the innate immune sensor from recognizing mRNA, and it has been speculated that it protects mRNA from translational shutdown triggered by innate immune response through the type I interferon signaling pathway.
Cap0 RNA can be methylated to Cap1 RNA. mRNA 2 '-O-methyltransferase (2' -O-MTase) can add a methyl group to the 2'-O of the first nucleotide of the Cap structure at the 5' -end of the RNA, and methylate capped Cap0 RNA into Cap1 RNA using S-adenosylmethionine (SAM) as a methyl donor. This enzyme can only use RNA with a 7-methylguanosine Cap structure (m 7GpppN, Cap 0) as a substrate, and does not act on RNA whose 5' -end is pN, ppN, pppN, or GpppN.
As used herein, "Cap" or "Cap" structures can be used interchangeably to refer to a unique nucleotide structure located at the end of a 5' mRNA. It has a 7-methylguanosine component linked to the first nucleotide of the nascent transcript by a 5'-5' -triphosphate bridge.
As used herein, "SAM (S-Adenosyl-L-methionine)", "S-adenosylmethionine" are used interchangeably and refer to a compound having C15H22N6O5The molecular formula of S is shown as the following.
As used herein, "SAH (S-adenosyl homocysteine)", "S-adenosyl homocysteine" are used interchangeably and refer to molecules having the formula shown below: c14H20N6O5And S. SAH is formed from S-adenosyl-L-methionine (SAM) via demethylation.
In the present invention, SAH is a byproduct of Cap0 capping reactions as well as Cap1 capping reactions.
Vaccinia virus capping enzyme
Vaccinia virus capping system a 7-methylguanosine Cap structure (m 7Gppp, Cap 0) was added to the 5' end of the RNA using vaccinia virus capping enzymes and related components. In eukaryotes, this structure is closely related to the stabilization, transport and translation of mRNA. Capping RNA using enzymatic reactions is a simple and efficient method, and the Cap structure is completely identical to the native Cap0 structure, significantly improving the stability and translation ability of RNA for in vitro transcription, transfection and microinjection. This enzyme is composed of two subunits (D1 and D12), the D1 subunit performing the functions of RNA triphosphatase and guanosine transferase and the D12 subunit performing the function of guanine methyltransferase, which are all necessary for the addition of one complete Cap0 structure, m7Gppp 5' N.
The capped RNA products constructed from vaccinia virus capping enzymes have the "cap 0" structure. Cap 0-RNA can be converted to a "Cap 1" structure by using both mRNA Cap 2' -O-methyltransferase and vaccinia virus capping enzyme in a capping reaction. mRNA Cap 2' -O-methyltransferase enzymes Cap 1-RNA is prepared from Cap 0-RNA by transferring a methyl group from the donor molecule SAM to the 2' -O position at the 5' end of Cap 0-RNA, immediately adjacent to the first nucleotide of the Cap structure.
Detection method of the invention
The application discloses a method for detecting mRNA 2' -O-methyltransferase activity. The method comprises the following steps:
step A, transcribing target gene RNA by utilizing T7 RNA polymerase;
step B, performing Cap0 capping reaction on the RNA in the step A by utilizing vaccinia virus capping enzyme and setting a negative control group; performing Cap1 capping reaction on the RNA in step A by using vaccinia virus capping enzyme and mRNA 2' -O-methyltransferase; the amount of production of SAH (S-adenosylhomocysteine) as a by-product in step A, B, C was measured by HPLC and it was observed whether the amount produced in step C was significantly higher than in step B, to determine the activity of mRNA 2' -O-methyltransferase in step C.
Later data analysis showed that: the method has good repeatability, the difference between the SAH production amount of Cap1 and Cap0 is obvious, and the activity of mRNA 2' -O-methyltransferase can be successfully detected.
In a specific example, Vaccinia Virus Capping Enzyme (VCE) used in the Cap0 Capping reaction adds a 7-methylguanosine Cap structure (Cap 0) to the 5' end of the RNA.
Wherein the vaccinia virus capping enzyme has functions of RNA triphosphatase (TPase), guanylyltransferase (GTase) and N7-guanine methyltransferase (N7-MTase).
The invention establishes a method for detecting the activity of mRNA 2' -O-methyltransferase through research, and the specific process is as follows:
firstly, a pair of primers T7-F/R is designed and synthesized according to the existing target gene, and the primers are used for carrying out PCR amplification on a plasmid containing the target gene to obtain a transcribed DNA template; then, RNA is transcribed and synthesized under the action of T7 RNA polymerase and 4 NTPs;
subsequently, Cap0 reaction was performed on the transcription-synthesized RNA, and a negative control group without enzyme was set; then, Cap reaction of Cap1 is carried out on the transcription synthesized RNA; finally, the transcribed RNA, Cap RNA of Cap0 and Cap RNA of Cap1 were subjected to HPLC analysis to detect the level of SAH, a reaction by-product, to determine the activity of mRNA 2' -O-methyltransferase.
If the mRNA 2' -O-methyltransferase is inactive, the SAH production of Cap RNA of Cap0 and Cap RNA of Cap1 has no significant difference; if mRNA 2' -O-methyltransferase is active, there is a significant difference in SAH production between Cap0 and Cap1 RNA.
The main advantages of the invention include
1) No radioactive label is needed, no radioactive pollution is caused, and a severe-grade laboratory is not needed;
2) the operation is simple, and the activity detection of the enzyme can be completed within 2 hours.
3) The enzyme activity is determined by the amount of by-products generated in the reaction without using a mass spectrometer.
4) In the invention, the vaccinia virus capping enzyme is used for replacing the functions of RNA triphosphatase (TPase), guanylate transferase (GTase) and N7-guanine methyltransferase (N7-MTase), thereby simplifying the reaction system.
The invention is further illustrated by the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
EXAMPLE 1 establishment of RNA reaction System
1. Sample and material preparation
a. Materials, reagents and instruments: 2x Fast Pfu Master Mix (2 x Fast Pfu polymerase Mix), Novoprotein (near shore protein science, Inc.), cat #: e035; t7 High Yield RNA Transcription kit (T7 High-Yield RNA Transcription kit), Novoprotein (near shore protein science, ltd.), cat # n: e131; cap1 Capping System (Cap1 Capping System), Novoprotein (near shore protein science ltd.), cat #: m082; DEPC-H2O, sterilizing water after the treatment of diethyl cokenate; freshly prepared 80% ethanol, purified magnetic beads; 0.2ml EP tube, magnetic frame, PCR instrument, and Qubit nucleic acid detector. The Cap1 capping system comprises SAM, GTP, 10 Xcap reaction buffer, RNase inhibitor, vaccinia virus capping enzyme, mRNA 2' -O-methyltransferase.
b. Sample preparation: a plasmid containing a gene of interest.
2. Design of primers T7-F and T7-R
In the experiment, an eGFP gene is selected as a target gene, a primer is designed, wherein T7-F has a T7 promoter sequence, and the specific sequence is as follows:
T7-F：TAATACGACTCACTATAGGGagatcgcctggagacgcc（SEQ ID NO:1）
T7-R：gctatggcagggcctgc（SEQ ID NO:2）
3. amplifying a DNA fragment containing a target gene from the plasmid, and adding the following reaction systems:
TABLE 1 reaction System for amplifying DNA fragment containing target Gene on plasmid
And (4) lightly beating the mixture by using a pipettor, fully and uniformly mixing the mixture, and centrifuging the reaction solution to the bottom of the tube for a short time. The samples were placed in a PCR instrument and the reaction program set up as follows:
TABLE 2 PCR reaction procedure
After the reaction is finished, the product is recovered through isopropanol precipitation, as shown in figure 1, the band is clear and single, and has no impurity band, and a Qubit nucleic acid detector detects the concentration.
4. In vitro transcription RNA reaction, the following configuration d was added:
TABLE 3 reaction System for in vitro RNA transcription reaction
And 3h at 37 ℃, adding 2U DNase I after the reaction is finished, purifying by magnetic beads at 37 ℃ for 30min, wherein the band is clear, single and has no impurity band as shown in figure 2, and the concentration is detected by a Qubit nucleic acid detector.
5. Cap0 reaction, placing the RNA after transcription and purification in a PCR instrument at 65 ℃ for 5min, immediately placing on ice for 5min after the reaction is finished, and adding the following reaction system:
TABLE 4 reaction System for Cap0 capping reaction
The temperature is 37 ℃ for 1h, and the mixture is preserved at the temperature of minus 20 ℃ for standby after the reaction is finished.
Wherein the enzyme unit (U) is the amount of enzyme capable of converting 1. mu. mol of substrate in 1 min.
6. Cap1 reaction, placing the RNA after transcription and purification in a PCR instrument at 65 ℃ for 5min, immediately placing on ice for 5min after the reaction is finished, and adding the following reaction system:
TABLE 5 reaction System for Cap1 capping reaction
After the reaction is finished, the reaction solution is placed at the temperature of minus 20 ℃ for storage for later use.
Example 2: method for detecting mRNA 2' -O-methyltransferase activity
1. Chromatographic conditions and system applicability test:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column comprises the following components: sepax Bio-C18 analytical column, 4.6X 250mm, mobile phase: 5% of methanol and 1 per mill of glacial acetic acid. Flow rate: 1.0ml/min, detection wavelength 254nm, column temperature: 25 ℃, sample introduction: 10 μ l.
2. Solution preparation:
preparing a test solution: mu.l of each of the transcribed RNA, the Cap0 reaction system, and the Cap1 reaction system was filtered through a 0.22. mu.M filter at 12000rpm for 5 min. Taking 10 μ l of the supernatant to obtain the test solution.
Preparation of a reference solution: 10mg of SAH powder was dissolved in 3.25ml of DEPC-H2O to obtain 8mM SAH standard solution. 8mM SAH was diluted to 4mM, 2mM, 1mM, 0.5mM, 0.25mM, respectively, according to a 2-fold gradient, to obtain a control solution.
3. HPLC determination
The sample solution and the reference solution were measured 10. mu.l each, and the measured solution was injected into a liquid chromatograph under the above-mentioned chromatographic conditions. And acquiring mass spectrum peak intensity data of corresponding quantitative signals of each molar mass standard. As shown in FIG. 3, the peak position of SAH standard is about 10 min. The SAH molar mass is taken as the abscissa and the peak area is taken as the ordinate, and a standard curve of the SAH is drawn, as shown in FIG. 5. Substituting the detected SAH peak areas of all samples into a standard curve linear equation to calculate, so as to obtain the molar content of the SAH in the actual sample, as shown in FIG. 6. The peak position of the component SAM in the reaction system is about 3min, as shown in FIG. 4. The results of calculating the actual production amount of SAH sample are shown in Table 6.
TABLE 6 actual SAH production calculation for test samples
The result shows that the negative control is not added with enzyme for reaction, so that the methyl transfer process does not occur, and no SAH is generated; the Cap0 RNA takes methyl transfer reaction by taking SAM as a methyl donor under the action of vaccinia virus capping enzyme to generate SAH with the concentration of 0.015 mM;
in the reaction of Cap1 RNA, firstly, the vaccinia virus capping enzyme reacts with SAM as a donor to generate Cap0 RNA, then under the action of mRNA 2 '-O-methyltransferase, SAM still reacts with the donor to generate Cap1 RNA, the final SAH generation concentration is 0.034mM, which is much higher than the generation amount of Cap0 reaction system, and the mRNA 2' -O-methyltransferase is active.
The capping efficiency of the Cap1 reaction system is detected by LC-MS, and the result shows that the capping rate of Cap0 in the Cap1 system can reach 99.6% (as shown in Table 7), which indicates that the addition of mRNA 2' -O-methyltransferase does not affect the capping efficiency of Cap0 capping enzyme.
TABLE 7 LC-MS detection of Cap0 capping Rate in Cap1 capping System
Comparative example the conventional method for measuring the mRNA 2' -O methyltransferase Activity
Step one, in a 20. mu.l reaction System (containing 50mM Tris-HCl [ pH 8.0 ]], 2mM DTT, 2mM MgCl240U RNase inhibitor, 0.01mM SAM, 0.5. mu. Ci 23H]-SAM[67.3Ci/mmol; 0.5μCi/μl]Mu.g mRNA 2' -O methyltransferase and 3. mu.g7MeGpppprna substrate) at 37 ℃ for 1 hour.
Step two, transfer tubes to ice, add an equal volume of 0.2% SDS, 20 mM EDTA.
Step three, placing the test tube on ice, and adding 1ml of 10mM NH4HCO3(pH 8.5)。
Step four, using 1ml of DEAE-Sephadex column separation and elution3The H-labeled product is quantified from the measurement result of a liquid scintillation counter3H]-SAM converted to an RNA substrate3H]The count signal of (A) represents the activity of the mRNA 2' -O methyltransferase tested.
The expected results are: mRNA 2' -O methyltransferase was not added to the negative control and cpm had almost no signal, indicating methyl-3H is not incorporated onto the substrate RNA; signals are generated in the cpm of the positive control and the experimental group, which indicates that methyl-3H is incorporated into the substrate RNA and mRNA 2' -O methyltransferase is active.
Compared with the method in the invention, the traditional method has complex detection step operation, long period and radioactive pollution.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Suzhou near shore protein science and technology GmbH
<120> a method for detecting mRNA 2' -O-methyltransferase activity
<130> P2021-2971
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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Claims (10)
1. An in vitro method for detecting mRNA 2' -O-methyltransferase activity, comprising the steps of:
(S1) providing a single-stranded RNA molecule having a 5 '-triphosphate at its 5' terminus;
(S2) performing the following test reactions on the RNA molecules in (S1), respectively, the test reactions including reactions in a negative test system, a first test system, and a second test system, the reaction time being t, wherein:
in the negative test system, no enzyme is contained;
a first test system comprising Cap0 capping enzyme;
a second test system comprising Cap0 and mRNA 2' -O-methyltransferase;
(S3) detecting (S2) the formation of SAH in the negative test system, the first test system and the second test system, and comparing the formation with a standard curve or standard value to obtain a quantitative result of SAH: wherein the content of the first and second substances,
the generation amount of SAH in a negative test system is A0; the generation amount of SAH in the first test system is A1; the generation amount of SAH in the second test system is A2; wherein, ΔQuality of food=A1-A0；△1=A2-A1；
If ΔQuality of foodIf the value is lower than the preset value, the test is considered to fail;
if Δ1/△Quality of foodIf > 1, the 2' -O-methyltransferase to be detected is regarded as enzymatically active, and the enzymatic activity is Delta1/t；
If Δ1/△Quality of foodIf =1, the 2' -O-methyltransferase to be detected is considered inactive.
2. The method of claim 1, wherein the single stranded RNA molecule is 500-2000 nt in length.
3. The method of claim 1, wherein said Cap0 capping enzyme is a vaccinia virus capping enzyme.
4. The method of claim 1, wherein the Cap0 capping enzyme is present in the first test system at a final concentration of 0.3U/μ l to 0.8U/μ l.
5. The method of claim 1, wherein the Cap0 capping enzyme is present in the second test system at a final concentration of 0.3U/μ l to 0.8U/μ l.
6. The method of claim 1, wherein the mRNA 2' -O-methyltransferase in the second test system is at a final concentration of 2U/μ l to 8U/μ l.
7. The process according to claim 1, wherein the reaction time t is from 0.8 to 1.4 h.
8. A kit for detecting mRNA 2' -O-methyltransferase activity, the kit comprising:
(a) a first container, and a negative test system located in the first container;
(b) a second container, and a first test system positioned in the second container;
(c) a third container, and a second test system located in the third container; and
(d) a fourth container, SAH standard; wherein the content of the first and second substances,
in the negative test system, no enzyme is contained; a first test system comprising Cap0 capping enzyme; in a second test system, Cap0 capping enzyme and mRNA 2' -O-methyltransferase were included.
9. The kit of claim 8, wherein the negative test system, the first test system, or the second test system each independently comprises SAM, GTP, 10 x capping reaction buffer, and RNase inhibitor.
10. An apparatus for detecting mRNA 2' -O-methyltransferase activity, the apparatus comprising:
(d1) a reaction module comprising a negative control chamber, a first reaction chamber, and a second reaction chamber, wherein,
the negative control chamber contains a negative test system;
the first reaction chamber comprises a first reaction module comprising a first test system;
the second reaction chamber comprises a second reaction module comprising a second test system;
wherein, the negative test system does not contain enzyme; a first test system comprising Cap0 capping enzyme; a second test system comprising Cap0 and mRNA 2' -O-methyltransferase;
(d2) a data acquisition module configured to perform data acquisition on SAH peak areas in respective reaction chambers in a reaction module;
(d3) an analysis module configured to analyze the SAH peak area from the data acquisition module through a "peak area-concentration" standard curve of SAH to obtain a 2' -O-methyltransferase activity analysis result to be screened;
(d4) and the output module outputs the analysis result of the screening analysis module.
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