CN115287332A - Poly (A) polymerase activity determination method - Google Patents

Poly (A) polymerase activity determination method Download PDF

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CN115287332A
CN115287332A CN202211025669.7A CN202211025669A CN115287332A CN 115287332 A CN115287332 A CN 115287332A CN 202211025669 A CN202211025669 A CN 202211025669A CN 115287332 A CN115287332 A CN 115287332A
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polymerase
rna
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邓汉卿
陈雅
陆青
燕东平
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Mona Wuhan Biotechnology Co ltd
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Abstract

The invention discloses a method for measuring activity of Poly (A) polymerase, which comprises the steps of carrying out tailing reaction on RNA by using the Poly (A) polymerase to generate a PPi product, hydrolyzing the PPi to obtain a product Pi, calculating the amount of the PPi product by measuring the amount of the Pi product, and calculating the activity of the Poly (A) polymerase according to enzyme activity definition. Compared with other schemes for measuring the consumption of the substrate or the tailing length of the product in the scheme of the Poly (A) polymerase, the scheme does not need to use a radioactive isotope or an expensive capillary electrophoresis instrument, has low detection cost, and is more visual, accurate, simple and efficient in result.

Description

Poly (A) polymerase activity determination method
Technical Field
The invention belongs to the technical field of enzyme activity detection, and particularly relates to a method for determining the activity of Poly (A) polymerase.
Background
Poly (A) Polymerase takes RNA as a template, and catalyzes ATP to be sequentially incorporated into the 3 'end of the RNA in the form of AMP, namely, poly A tail is added to the 3' end of the RNA. The reaction equation is as follows:
Figure BDA0003815441020000011
the Poly (A) tail structure at the 3' end is one of the most important structures for mRNA stability, and most mRNA degradation begins from the Poly (A) tail. The addition of a Poly (a) tail can improve the stability of RNA in eukaryotic cells and enhance its translation efficiency after transfection or microinjection. Poly (A) tail is added to synthesized RNA by using Poly (A) Polymerase in the production process of the mRNA vaccine to increase the stability of the RNA, play an important role in protecting the mRNA, and can enhance the translation efficiency of the mRNA vaccine after entering a human body, thereby improving the storage performance of the vaccine and the performance of the mRNA vaccine.
In addition, the Poly (A) tail can also provide a universal primer binding site for cDNA synthesis, and can be used for end labeling of RNA or quantification of microRNA. The miRNA is about 20-24 nt in length and participates in various regulation ways including development, virus defense, hematopoietic process, signal path, cell proliferation and apoptosis, cell metabolism and the like. But also indispensable for prediction of miRNA target genes, mRNA-miRNA interaction, detection of miRNA expression levels, and the like. Since miRNA is too short in length, a Poly (a) tail can be added to the 3' end of miRNA by Poly (a) polymerase, increasing its length. Then, the reverse transcription primer Oligo (dT) n is combined with the Poly (A) sequence, so that the reverse transcription product cDNA can be obtained, and then the subsequent quantitative detection is carried out. Poly (A) Polymerase is the main reagent component in miRNA detection kit (tailing method).
Accurately determining the activity of the Poly (A) enzyme, and having important significance for the performance of related products. The standard Poly (A) enzyme activity determination method adopts a radioactive substrate doping method which is easy to generate radioactive pollution, has multiple steps and long period, and the use of radioactive isotopes needs to obtain the relevant laboratory permission quality, so that the use threshold is high. The scheme of detecting the length of the tail added with A by adopting a capillary electrophoresis apparatus or a PAGE gel electrophoresis method has the defects of needing expensive instruments or having low precision. In addition, the scheme of detecting the consumption of ATP as a substrate has more experimental steps, complex experimental design and inconvenient operation.
Therefore, it is necessary to design a method for measuring Poly (A) polymerase activity, which is simple in operation, short in cycle time, and low in multiple measurement error.
Disclosure of Invention
In view of the above, the present invention provides a method for measuring Poly (A) polymerase activity, which solves the problems of inconvenient operation, long cycle and low precision of the prior art.
In order to solve the problems, the scheme of the invention is as follows:
a method for measuring activity of Poly (A) polymerase comprises the following steps: PAP enzyme catalyzed tailing reaction is firstly carried out by using RNA, PPi is hydrolyzed into Pi after the reaction is finished, the Pi content in the product is determined, and the activity of Poly (A) polymerase is calculated.
Further, the Pi was measured by a malachite green-phosphomolybdic acid spectrophotometry.
Preferably, the Poly (a) polymerase is pre-diluted so that the concentration of the hydrolysis product Pi is within the linear range of the measurement curve.
Preferably, the tailing reaction process is carried out according to enzyme activity definition; the calculation formula is as follows: enzyme activity = Pi mole number ÷ 2 × dilution factor ÷ enzyme input volume.
Further, the RNA is artificially synthesized RNA or RNA extracted from organisms.
Preferably, the length of the artificially synthesized RNA is 3-1000bp.
Preferably, the artificially synthesized RNA sequence comprises an arbitrary arrangement sequence of four bases of AUCG.
Preferably, the input amount of the artificially synthesized RNA is in the range of 1 to 100nmol.
Further, the hydrolysis of PPi to Pi is performed by sufficiently hydrolyzing the product PPi using pyrophosphorohydrolase.
The invention also provides the application of the determination method in quality control of Poly (A) polymerase.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the determination method, the pyrophosphoric acid product generated after the Poly (A) polymerase tailing reaction is hydrolyzed, the enzyme activity can be calculated by determining the content of phosphate radical, the operation is simple, the result determination can be completed within two hours, the error of multiple determination is low, and the product requirements are met.
2. The determination method of the invention uses conventional chemicals as chemical reagents, does not use radioactive substances or expensive instruments, has low cost and has strong popularization and use values.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.
FIG. 1 is a standard curve of phosphorus content versus OD value fitted in the determination of Poly (A) polymerase of the present invention.
Detailed Description
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
The nomenclature used in connection with, and the techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques for, analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well known and commonly employed in the art.
The scheme provides a method for measuring the activity of Poly (A) polymerase, which comprises the following steps: PAP enzyme catalyzed tailing reaction is firstly carried out by using RNA, PPi is hydrolyzed into Pi after the reaction is finished, the Pi content in the product is determined, and the activity of Poly (A) polymerase is calculated.
In a preferred embodiment, the method for hydrolyzing PPi to Pi is not limited, and any method capable of hydrolyzing PPi to Pi may be used in the prior art, for example, pyrophosphohydrolase. Similarly, a common measurement method for the amount of Pi production may be selected, and any method capable of accurately measuring the amount of Pi production may be used in the prior art.
In a preferred embodiment, the amount of Pi produced is determined spectrophotometrically using malachite green-phosphomolybdic acid. Poly (A) polymerase needs to be diluted before enzyme activity is measured, so that the range of products Pi can fall within the range of the linear interval of the phosphorus standard curve of the scheme adopted at this time.
In a preferred embodiment, artificially synthesized RNA or RNA extracted from organisms or RNA of other sources can be adopted, the artificially synthesized RNA substrate sequence can be any arrangement of AUCG, the length is 3-1000bp, and the input amount needs to be in a range of 1-100nmol to ensure that the tailing rate is not limited by the molecular weight of the substrate. The RNA selected in the scheme is mainly considered as a substrate for a reaction with Poly (A) polymerase and A tail, and is not limited to the genetic information and the function of the RNA as nucleic acid material.
The basic principle of the scheme for measuring the activity of the Poly (A) polymerase is as follows:
1. reaction by adding A tail
Poly (A) polymerase (PAP) catalyzes the sequential incorporation of ATP into the 3' end of RNA in the form of AMP, according to the equation:
Figure BDA0003815441020000051
2. hydrolysis reaction of pyrophosphoric acid
Pyrophosphate can be decomposed into phosphate by an excess of pyrophosphate hydrolase (IPP):
Figure BDA0003815441020000052
3. malachite green-phosphomolybdic acid spectrophotometry
Phosphate radical and molybdate radical form phosphomolybdic heteropoly acid association complex under the acid condition, then form weak bond complex with malachite green as the chromogenic complex, under the acid condition, the chromogenic complex keeps stable, the shade of the color is in direct proportion to the content of phosphate within a certain concentration range. The malachite green phosphorus determination method is a very sensitive method for analyzing inorganic phosphorus, and can be used for determining the content of trace phosphorus in solutions such as water, soil, blood and the like. The Poly (A) polymerase tailing activity was calculated from the concentration of phosphorus.
In a preferred embodiment, the tailing reaction is the same as the enzyme activity defining reaction, so the tailing reaction is preferably carried out in a reaction system and under a condition defined by the enzyme activity, the standard enzyme activity is obtained, and the calculation of the enzyme activity result is facilitated.
For manufacturers, the determination method can be used for controlling the enzyme activity of the product and preparing products with different enzyme activities; for users, the enzyme activity can be measured in real time, and a basis is provided for experiments.
Examples
Poly (A) polymerase tailing reaction
The desired reaction system was set up as in table 1.
Table 1:
Figure BDA0003815441020000053
Figure BDA0003815441020000061
reaction conditions are as follows: 10min at 37 ℃; 15min at 85 ℃.
2. Hydrolysis reaction of pyrophosphoric acid
And adding excessive inorganic pyrophosphatase into the product after the tailing reaction is finished, and centrifuging after flicking and uniformly mixing. 25 ℃ and 1h.
3. Pi content measurement by malachite green-phosphomolybdic acid spectrophotometry
1) Phosphorus standard curve sample preparation
An inorganic phosphorus standard solution was prepared as shown in Table 2.
Table 2:
Figure BDA0003815441020000062
2) Malachite green color development reaction
And (3) taking 20 mul of hydrolysis reaction completion solution and phosphorus standard curve solution, adding 180 mul of malachite green dye solution into each tube, and standing at room temperature for 20min.
3) Absorbance reading
And (3) sucking 180 mu l of solution with complete color reaction into a transparent cuvette, reading an absorbance OD value at the wavelength of 660nm, and judging and selecting a dilution gradient falling in the linear range of the phosphorus standard curve as an effective reading value according to the OD value. The results of reading the OD values of the inorganic phosphorus standard solutions are shown in Table 3, and the standard curve fitting is shown in FIG. 1.
Table 3:
Figure BDA0003815441020000063
Figure BDA0003815441020000071
4. the enzyme activity was calculated (1 unit of enzyme required for incorporation of 1nmol ATP into RNA oligo in a 20. Mu.l reaction system at 37 ℃ for 10 minutes).
The calculation formula is as follows: enzyme activity = phosphorus concentration ÷ 2 × 20 × 10 ÷ 1000 × dilution factor ÷ enzyme input volume.
In the formula, 20 × 10 means: 20 μ l is the volume of the hydrolysis reaction completion solution for malachite green color development reaction, and 10 is the dilution multiple of 20 μ l hydrolysate added with color development liquid and the volume is increased to 200 μ l, which can also be directly understood as: phosphorus concentration 2X 200. Mu.l. μ M is converted to nM and is therefore divided by 1000.
The phosphorus concentration is obtained by reading the OD value through repeated experiments on the same sample, and the phosphorus concentration is kept stable among independent experiments. The results of the experiment are shown in table 4.
Table 4:
repetition of Enzyme activity data U/mul
1 5.4931
2 4.8005
3 5.1544
4 5.3738
5 4.4317
6 4.7171
The average value of the data of enzyme activity measured in multiple replicates is shown in Table 5.
Table 5:
Figure BDA0003815441020000072
as can be seen from Table 5, the error of repetition of the enzyme activity measurement using this protocol was small. The whole enzyme activity determination process can be completed within 2 hours, reagents of all reaction systems are available in the market, expensive equipment or instruments are not needed, and the method is simple, convenient and fast and has strong reliability of determination results. And the enzyme activity data of the Poly (A) polymerase manufacturer used in the embodiment of the scheme is 5U/mul, and the accuracy and reliability of the method can be verified by adopting the scheme that the determination method is consistent with the enzyme activity data obtained by the traditional method.
In addition, the experimental means of the embodiment is adopted to detect that the activity limit value of the Poly (A) polymerase is 0.2U, and the solution has higher sensitivity.
Particularly, the determination method of the scheme overcomes the technical bias of the prior art or the traditional method for determining from the substrate, determines the activity of the Poly (A) polymerase from the corresponding molar ratio product generation amount in the molecular, is simple, convenient and low in cost, and has remarkable popularization significance compared with the traditional determination method.
The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.

Claims (10)

1. A method for measuring the activity of Poly (A) polymerase, comprising the steps of: PAP enzyme catalyzed tailing reaction is firstly carried out by using RNA, PPi is hydrolyzed into Pi after the reaction is finished, the Pi content in the product is determined, and the activity of Poly (A) polymerase is calculated.
2. The method of claim 1, wherein Pi is measured using malachite green-phosphomolybdic acid spectrophotometry.
3. The assay method according to claim 2, wherein the Poly (a) polymerase is pre-diluted so that the concentration of the hydrolysis product Pi is within the linear range of the measurement curve.
4. An assay method according to claim 3, wherein the tailing reaction process is carried out according to enzyme activity definition; the calculation formula is as follows: enzyme activity = Pi mole number ÷ 2 × dilution factor ÷ input enzyme volume.
5. The method for measuring according to claim 1, wherein the RNA is artificially synthesized RNA or organism-extracted RNA.
6. The assay method according to claim 5, wherein the length of the artificially synthesized RNA is 3 to 1000bp.
7. The method of claim 5, wherein the synthetic RNA sequence has an arbitrary sequence of four bases including AUCG.
8. The method according to claim 5, wherein the amount of the artificially synthesized RNA to be added is in the range of 1 to 100nmol.
9. The method of claim 1, wherein the hydrolysis of PPi to Pi is performed by hydrolyzing the product PPi sufficiently with pyrophosphorohydrolase.
10. Use of the assay according to any one of claims 1 to 9 for the quality control of Poly (a) polymerase.
CN202211025669.7A 2022-08-25 2022-08-25 Poly (A) polymerase activity determination method Pending CN115287332A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117778523A (en) * 2024-02-26 2024-03-29 苏州近岸蛋白质科技股份有限公司 Poly (A) polymerase activity determination method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117778523A (en) * 2024-02-26 2024-03-29 苏州近岸蛋白质科技股份有限公司 Poly (A) polymerase activity determination method
CN117778523B (en) * 2024-02-26 2024-05-28 苏州近岸蛋白质科技股份有限公司 Poly (A) polymerase activity determination method

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