CN117737210A - Method for detecting proportion of mRNA with poly A tail in mRNA product - Google Patents

Abstract

The present invention provides a method for detecting the proportion of mRNA having a polyA tail in an mRNA product. The method of the invention comprises the following steps: 1) Obtaining RNA with a connector at the 3' end; 2) Performing reverse transcription to obtain cDNA; 3) Designing synthetic primers and probes for the 5 'end of the cDNA, designing synthetic primers and probes for the 3' end of the cDNA, and preparing a digital PCR reaction system for determination. The invention designs a corresponding primer and a probe according to the requirement of mRNA product quality control, and provides a method for effectively, stably and accurately detecting the proportion of mRNA with poly A tail in an mRNA product by matching with a digital PCR method.

Description

Method for detecting proportion of mRNA with poly A tail in mRNA product

Technical Field

The invention relates to a method for detecting the proportion of mRNA with poly A tail in mRNA product, which can be applied in the fields of quality control of mRNA product and the like.

Background

The market of two mRNA vaccines in 2020 announces that mRNA technology formally enters the commercial era, and mRNA also enters the rapid development stage. mRNA is used as the technical basis of medicines and vaccines, and has great flexibility in production and application. Any protein can be encoded and expressed by mRNA and in principle prophylactic and therapeutic vaccines can be developed against various diseases, such as infections and cancers, as well as protein replacement therapies.

The preparation process of the mRNA product can be roughly divided into three stages: 1) Preparing linear DNA, wherein the stage mainly comprises constructing a vector containing a target gene, carrying out plasmid amplification by engineering bacteria and linearizing the DNA; 2) Preparing mRNA stock solution, wherein the mRNA is prepared mainly by taking linear DNA of the previous stage as a template by an in vitro transcription technology, and the prepared mRNA structure comprises a 5 '-end capping structure and a 3' -end poly A tailing structure; 3) The liposome is encapsulated, mRNA and nano delivery materials are self-assembled to form nano ions by adopting different technologies at the stage, and the nano ions are filled after concentration, purification and sterile filtration. The 3 '-terminal poly A is a non-templated adenosine added at the 3' -end of the mRNA molecular structure, and plays a key role in translation and stability of mRNA. It is therefore necessary to detect the proportion of intact mRNA having a polyA tail to ensure the quality of the mRNA product.

There are two main methods for detecting nucleic acids in common use at present: the real-time quantitative PCR detection method and the digital PCR detection method have higher accuracy in quantitatively detecting the copy number, and are more convenient because no standard curve is required to be designed or prepared. The current digital PCR technology uses micro-chamber/microwell or microdroplet as PCR reactor to make the template randomly distributed in microwell or microdroplet, uses high-precision camera to analyze each microwell after PCR amplification of microwell by using thermal cycler, and obtains copy number of corresponding fragment in solution by calculating number of positive microdroplet. mRNA products require detection of the proportion of mRNA having a poly A tail to ensure their translation level and stability. However, no method for stably and accurately detecting the proportion of mRNA having a poly A tail in an mRNA product has been found in the prior art.

Disclosure of Invention

The present invention provides a method for detecting the proportion of mRNA having a polyA tail in an mRNA product, comprising the steps of:

1) Obtaining RNA with a connector at the 3' end;

2) Performing reverse transcription to obtain cDNA; reverse transcription primers are designed based on the sequence of the adaptor;

3) And respectively designing a synthetic primer and a probe aiming at the 5 'end of the cDNA, respectively designing the synthetic primer and the probe aiming at the 3' end of the cDNA, and preparing a digital PCR reaction system for determination. The 5' end of the cDNA is respectively designed with a synthetic primer and a probe which are used as internal references; the 3' end of the cDNA is respectively designed with a synthetic primer and a probe for detecting a poly A tail signal. The internal reference is that in actual detection, the internal references of different RNAs to be detected need not be identical, as specified by the need. In the present invention, fragments having both 5 'and 3' signals are defined as complete sequences in the assay. The invention also allows detection of the proportion of RNA containing incomplete poly A by targeted design of the primers. The length of poly A of the RNA to be detected is not limited in the present invention.

Preferably, the method further comprises calculating the proportion of mRNA having poly a tails based on the copy number of the digital PCR assay. Preferably, the proportion of mRNA with poly a tail is: at the same time, the copy number of the signals of the poly A and the reference gene/the copy number of the reference gene is multiplied by 100 percent.

According to a specific embodiment of the present invention, wherein the method further comprises a process of ligating mRNA with a linker to obtain the RNA having the linker at the 3' end;

preferably, the molar ratio of mRNA to linker is 1:5-20.

According to a specific embodiment of the present invention, wherein the method further comprises purifying, and the RNA having a linker at the 3' end is the purified RNA.

According to a specific embodiment of the present invention, wherein the step of reverse transcribing the RNA molecule further comprises reverse transcribing the purified mRNA with specific primers.

According to a specific embodiment of the present invention, the step of preparing a digital PCR reaction system further comprises subjecting the cDNA to purity and concentration analysis followed by appropriate dilution.

According to a specific embodiment of the present invention, the 5' end of the linker has one or more phosphorylation modifications;

preferably, the 3' end of the linker carries one or more ddC markers. The 5 'phosphorylation modification allows the linker to be attached to the 3' end of the mRNA, and the 3'ddC tag ensures that the mRNA will not be attached to the 3' end of the linker.

According to a specific embodiment of the present invention, wherein the probe is modified with a fluorophore, the modification comprising:

the 5 '-end base of the probe is modified by adopting a fluorescent reporter group, and the 3' -end base of the probe is modified by adopting a fluorescent quenching group.

According to a specific embodiment of the present invention, the sequence of the forward primer designed for the 5' end of the cDNA is: 5'-TGTTTGTGTTCCTGGTGCTG-3' (SEQ ID NO. 1);

preferably, the sequence of the reverse primer designed for the 5' end of the cDNA is: 5'-CGGAGCTCCTGAACACCTTA-3' (SEQ ID NO. 2);

preferably, the sequence of the probe designed for the 5' end of the cDNA is: 5 'ACTCCTCACCCCGGGCGTGT-3' (SEQ ID NO. 3);

preferably, the forward primer designed for the 3' end of the cDNA has the sequence: 5'-CTTGCCTTCTGGCCATGC-3' (SEQ ID NO. 4);

preferably, the sequence of the reverse primer designed for the 3' end of the cDNA is: 5'-AGTCATATGCTTTTTTTTTT-3' (SEQ ID NO. 5);

preferably, the sequence of the probe designed for the 3' end of the cDNA is: 5'-CCTTGCACCTGTACCTCTTGGTCTTT-3' (SEQ ID NO. 6).

According to a specific embodiment of the present invention, wherein the 5 'end of the probe designed for the 5' end of the cDNA is modified with VIC; the 3 'end of the probe designed for the 5' end of the cDNA is modified by BHQ-1;

preferably, the 5 'end of the probe designed for the 3' end of the cDNA is provided with FAM modification; the 3 'end of the probe designed for the 3' end of the cDNA is provided with BHQ-1 modification.

According to a specific embodiment of the present invention, wherein the sequence of the linker comprises the sequence shown in SEQ ID NO.7: SEQ ID NO.7:5'-TCGTATGCCGTCTTCTGCTTG-3'.

According to a specific embodiment of the present invention, wherein the sequence of the reverse transcribed primer comprises the sequence shown as SEQ ID NO.8, SEQ ID NO.8:5'-AGCAGAAGACGGCATACGA-3'.

According to a specific embodiment of the present invention, wherein the digital PCR is a titration type digital PCR;

preferably, the digital PCR is a dual probe digital PCR.

According to a specific embodiment of the present invention, the ligation reaction is performed as follows:

the mRNA product is denatured for 10 minutes at 60-80 ℃ and then rapidly placed on ice for cooling;

the prepared ligation system was incubated at 25℃for 2-6 hours.

According to a specific embodiment of the present invention, the digital PCR reaction step comprises:

1) Pre-denaturation at 95℃for 2-5 min, 1 cycle;

2) Denaturation at 95℃for 5-60 seconds, annealing at 50℃for 5-60 seconds, elongation at 72℃for 5-120 seconds, and a total of 30-40 cycles.

The invention also provides a kit for detecting the proportion of mRNA having a polyA tail in an mRNA product, wherein the kit comprises:

a linker comprising the sequence shown as SEQ ID NO.7 and/or a reverse transcription primer comprising the sequence shown as SEQ ID NO. 8.

The invention has the beneficial effects that:

the invention designs a corresponding primer and a probe according to the requirement of mRNA product quality control, and provides a method for effectively, stably and accurately detecting the proportion of mRNA with poly A tail in an mRNA product by matching with a digital PCR method.

The invention analyzes the sequence characteristics of the produced mRNA according to the requirement of mRNA product quality control, and designs a primer, a probe, a method and a PCR program which are applicable to detecting the proportion of complete mRNA containing poly A tail in the mRNA product with similar sequence. The digital PCR technology is matched with the primer and the probe to accurately quantify the proportion of the complete mRNA with the polyA tail in the mRNA product, thereby providing guarantee for the effectiveness and the safety of the mRNA product.

Drawings

FIG. 1 is a schematic diagram of the steps for detecting mRNA tailing rate by digital PCR.

FIG. 2 is data from the detection of mRNA tail-rate space-time white wells using digital PCR, where green represents the copy number of poly A and yellow represents the copy number of the reference fragment.

FIG. 3 shows the result of digital PCR of mRNA after ligation and reverse transcription after the addition of the tail rate of mRNA by digital PCR, wherein green represents the copy number of poly A and yellow represents the copy number of the reference fragment.

Detailed Description

The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.

Instrument and equipment

PCR instrument, desk centrifuge, digital PCR instrument, nanodrop ultra-micro spectrophotometer, vortex suspension, ultra-clean bench, etc.

Reagent(s)

Primers and probes (organisms) (SEQ ID NO. 1-SEQ ID NO. 6), adaptors (Suzhou Bei Xin) (SEQ ID NO. 7), T4 RNA library 1 (NEB), QIAcuity TM Probe PCR Kit Kit (Qiagen), MEGAclear Transcription Clean-Up Kit (Invitrogen).

The sequences of the mRNAs involved in the examples are as follows:

the cap structure is as follows: in the present invention, the cap structures used are m7G (5 ') ppp (5') (2 'OMeA) pG, m7G (5') ppp (5 ') (2' OMeA), G (5 ') ppp (5') G, m G (5 ') ppp (5') G, 3'-O-Me-m7G (5') ppp (5 ') G or m7 (3' OMeG) (5 ') ppp (5') (2 'OMeA) pG, preferably m7G (5') ppp (5 ') (2' OMeA) pG.

"T" is used in the present invention to represent uracil (U) in RNA, specifically, mRNA sequence:

examples

This example provides a method for detecting the proportion of mRNA having a polyA tail in an mRNA product. The schematic of the steps for detecting mRNA tailing rate by digital PCR is shown in FIG. 1.

The method comprises the following specific steps:

1. obtaining RNA with a linker at the 3' end:

(1) Ligation reaction

1) The mRNA was heat denatured at 65℃for 10 minutes and then placed on ice for rapid cooling;

2) A20. Mu.l ligation reaction was established:

1×T4 RNA Ligase Reaction Buffer(50mM Tris-HCl,pH 7.5,10mM MgCl ₂ ,1mM DTT)

15～25％(wt/vol)PEG 8000

10％(v/v)DMSO

0.5μl RNase inhibitor(40U/μl)

1mM ATP

20pmol RNA product

40～200pmol RNA adaptor

1μl(10units)T4 RNA ligase；

3) After incubation at 25℃for 4 hours, thermal denaturation at 65℃for 10 minutes.

(2) And (3) RNA separation and purification:

1) The samples subjected to the ligation reaction were filled up to 100. Mu.l with an Elutation Buffer, and gently mixed with a pipette tip;

2) Adding 350 μl Binding Buffer, and gently mixing with a suction head;

3) 250 μl of 100% ethanol was added and gently mixed with a suction head;

4) Inserting an RNA micro adsorption column into a collecting pipe, lightly mixing the sample obtained in the step 3) by using a suction head, transferring the mixture onto the RNA micro adsorption column, centrifuging at room temperature of 14000g for 1 minute, and discarding the filtrate, wherein the total volume of the mixture is not more than 700 mu l at a time (each adsorption column can bear 500 mu g mRNA at most);

5) Repeating the steps 3) -4) until all the samples are transferred to a micro adsorption column;

6) 550 μl of Washing Buffer was added;

7) Centrifuging 14000g for 1 min at room temperature, and discarding the filtrate;

8) Repeating steps 6) -7);

9) Centrifuging 14000g of the empty RNA micro adsorption column for 2 minutes at room temperature to dry column matrix;

10 Transferring the RNA micro adsorption column into a new collecting pipe, uncovering and standing for 2-3 minutes;

11 100 mu l RNase Free Water, and standing for 10 minutes;

12 14000g for 2 minutes at room temperature;

13 The micro adsorption column is discarded, the dissolved mRNA is transferred to a new 1.5ml centrifuge tube, fully mixed and marked, and a proper amount of mRNA is taken out for cDNA preparation.

2. Obtaining cDNA:

first, a reverse transcription reaction system was prepared as shown in Table 1.

TABLE 1

Buffer	Volume (mul)
		5×Prime Script Buffer	1
Prime Script RT Enzyme MixⅠ	0.25
		Primer (SEQ ID NO. 8)	0.25
RNase Free dH 2 O	2.5
		cDNA template	1

The prepared reaction system was subjected to reverse transcription according to the procedure shown in Table 2.

TABLE 2

And after the reverse transcription is finished, the PCR tube is taken out from the PCR instrument, and a proper amount of sterile deionized water is added for purity and concentration analysis and then used for subsequent digital PCR detection.

3. Digital PCR reaction:

1) DPCR reaction mixtures were prepared according to table 3.

TABLE 3 Table 3

2) Vortex mixing for 30 seconds;

3) Transferring the content of each well from a standard PCR tube into a nanopore well;

4) The nano-plate sealing film is used for correctly sealing the nano-plate;

5) The digital PCR reactions were performed as follows:

step 1:95 ℃ for 2 minutes

Step 2:95 ℃ for 15 seconds

Step 3:50 ℃ for 10 seconds

Step 4:72 ℃ for 15 seconds

Step 2 through Step 4 were performed for 39 cycles.

The digital PCR fluorescent signal acquisition results are shown in FIG. 2 and FIG. 3.

As can be seen from FIG. 2, when the blank wells were detected by digital PCR, only (-) positive droplets (aerosol contamination) were observed in the fluorescent droplets, i.e., only the reference fragment sequence and (-) positive droplets (aerosol contamination) were detected, i.e., only the Poly-A tail sequence, whereas (++) double positive droplets were almost zero, i.e., both the reference fragment sequence and the Poly-A tail sequence, and the primers and probes for detecting the reference fragment sequence and the Poly-A tail sequence were not interfered with. The detection accuracy is good.

As can be seen from FIG. 3, standard plasmids with complete reference sequence and Poly-A tail sequence were tested by digital PCR and were seen in fluorescent droplets: the proportion of positive droplets (- +) that is, only the reference fragment sequence and (-) that is, only the Poly-A tail sequence are detected is very small and the proportion of double positive droplets (++) that are the existing reference fragment sequence and Poly-A tail sequence to positive droplets is 94.6%, indicating that digital PCR is effective in detecting RNA having both the reference sequence and the Poly-A sequence.

4. Calculating the proportion of mRNA having a poly A tail

Copy number calculation from digital PCR assay: proportion of mRNA with polya tail = copy number of signal with both polya and reference gene/reference gene copy number. As can be seen from FIG. 3, the proportion of mRNA having a poly A tail in this example was 97.3% (97.3% = 13519/(369+13519). Times.100%).

The embodiments described above may be modified in various ways by those skilled in the art without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and not by the foregoing detailed description, and each implementation within the scope of which is defined by the present invention.

Claims (11)

1. A method of detecting the proportion of mRNA having a poly a tail in an mRNA product, the method comprising:

1) Obtaining RNA with a connector at the 3' end;

2) Performing reverse transcription to obtain cDNA;

3) Designing synthetic primers and probes for the 5 'end of the cDNA, designing synthetic primers and probes for the 3' end of the cDNA, and preparing a digital PCR reaction system for determination.

2. The method of claim 1, wherein the method further comprises calculating the proportion of mRNA having poly a tails based on the copy number of the digital PCR assay.

3. The method of claim 1 or 2, wherein the method further comprises the step of ligating mRNA with a linker to obtain the 3' -end-ligated RNA.

4. A method according to any one of claims 1 to 3 wherein the 5' end of the linker carries one or more phosphorylation modifications; preferably, the 3' end of the linker carries one or more ddC markers.

5. The method of any one of claims 1-4, wherein the probe is modified with a fluorophore, the modification comprising:

the base at the 5 '-end of the probe sequence is modified by adopting a fluorescent reporter group, and the base at the 3' -end of the probe sequence is modified by adopting a fluorescent quenching group.

6. The method of any one of claims 1-5, wherein the 5 'end of the probe designed for the 5' end of the cDNA has a VIC modification; the 3 'end of the probe designed for the 5' end of the cDNA is modified by BHQ-1;

the 5 'end of the probe designed for the 3' end of the cDNA is provided with FAM modification; the 3 'end of the probe designed for the 3' end of the cDNA is provided with BHQ-1 modification.

7. The method of any one of claims 1-6, wherein the sequence of the linker comprises the sequence set forth in SEQ ID No.7, SEQ ID No.7:5'-TCGTATGCCGTCTTCTGCTTG-3'.

8. The method of any one of claims 1-7, wherein the sequence of the reverse transcribed primer comprises the sequence set forth in SEQ ID No.8, SEQ ID No.8:5'-AGCAGAAGACGGCATACGA-3'.

9. The method of any one of claims 1-8, wherein the digital PCR is a titrated digital PCR.

10. The method of any one of claims 1-9, wherein the digital PCR reaction step comprises:

1) Pre-denaturation at 95℃for 2-5 min;

2) Denaturation at 95℃for 5-60 seconds, annealing at 50℃for 5-60 seconds and elongation at 72℃for 5-120 seconds.

11. A kit for detecting the proportion of mRNA having a poly a tail in an mRNA product, the kit comprising:

a linker comprising the sequence shown as SEQ ID NO.7 and/or a reverse transcription primer comprising the sequence shown as SEQ ID NO. 8.