Method for detecting Poly A tail length of mRNA (messenger ribonucleic acid) based on programmable enzyme method

Abstract

The invention provides a method for detecting Poly A tail length of mRNA (messenger ribonucleic acid) based on a programmable enzyme method, which comprises the following steps: carrying out enzyme digestion on mRNA to be detected by adopting programmable ribonuclease MbpAgo to obtain an enzyme digestion product; carrying out affinity purification on the enzyme digestion product; and analyzing the purified product to detect the Poly A tail length. The method can be used for representing length distribution of mRNA PolyA tails, can effectively detect segmented PolyA with higher stability, and is wider in application range compared with an existing universal RNase T1 enzyme digestion method.

Description

Technical Field

[0001] The present invention relates to a method for detecting the length of the Poly A tail of mRNA, and specifically to a method for analyzing and detecting the length of the Poly A tail of mRNA after cutting and affinity purification using a programmable ribonuclease (MbpAgo), and belongs to the technical field of genetic engineering and enzyme engineering. Background Art

[0002] The mRNA synthesized by in vitro transcription (IVT) obtains a poly A tail (Poly A tail) by encoding a poly A sequence onto the template DNA. A longer Poly A tail can improve the translation efficiency of mRNA and enhance the stability of mRNA. However, during the in vitro transcription process, the mRNA will slip when synthesizing the Poly A tail, resulting in the extension or shortening of the Poly A tail. When the length of the Poly A tail is less than 60 nt, the expression ability of the mRNA will be severely weakened. Therefore, it is necessary to analyze the length of the Poly A tail of mRNA.

[0003] Beverly M et al. ("Poly A tail length analysis of in vitro transcribed mRNA by LC-MS", Anal Bioanal Chem, 2018) proposed a method for quantitatively analyzing the length of the poly A tail of mRNA. This method is based on the fact that the endonuclease RNase T1 can specifically cleave the phosphodiester bond between the guanine ribonucleotide (G) and the adjacent ribonucleotide at the 3' end of single-stranded RNA, and the poly A tail is purified by specific binding to oligo dT magnetic beads, and then a high-resolution liquid chromatography-mass spectrometer is used to quantitatively analyze the length distribution of the poly A tail of mRNA. This analysis method is currently a commonly used method in the current in vitro preparation process of mRNA.

[0004] However, the above analysis method is only applicable to the detection of the poly length distribution of pure ribonucleotide A. For segmented polyA tails, that is, two or more segments of pure ribonucleotide A poly A tails are connected by one or more linkers, where the linkers may carry guanine ribonucleotides. If RNase T1 is used for cutting, the poly A tail will undoubtedly be divided into multiple segments, making it difficult to determine the complete polyA tail length distribution. Summary of the invention

[0005] An object of the present invention is to provide a method for detecting the length of the Poly A tail of mRNA.

[0006] Another object of the present invention is to provide a kit for detecting the length of the Poly A tail of mRNA.

[0007] In one aspect, the present invention provides a method for detecting the length of the Poly A tail of mRNA, the method comprising:

[0008] The programmable ribonuclease MbpAgo is used to digest the mRNA to be detected to obtain a digestion product;

[0009] Affinity purification is performed on the enzyme cleavage product to obtain a purified product;

[0010] The purified product was analyzed to determine the length of the poly A tail.

[0011] The method for detecting the length of the Poly A tail of mRNA of the present invention is based on MbpAgo enzyme digestion and affinity purification, and quantitative detection of the purified product can be used to characterize the length distribution of the mRNA Poly A tail, and can also effectively detect the more stable segmented Poly A. Compared with the currently common RNase T1 enzyme digestion method, it has a wider range of applications.

[0012] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, when performing enzyme cleavage, the reaction system includes guide DNA. Preferably, in the reaction system, the molar ratio of MbpAgo:guide DNA:mRNA=(4-6):(1-2):1.

[0013] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the enzyme cleavage is reacted at 37° C.-55° C. for 0.5 h-2 h.

[0014] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, when performing enzyme cleavage, each 150 μL reaction system includes:

[0015] mRNA (100 pmol) 30 μL;

[0016] Guide DNA 40 μL;

[0017] 10× Buffer 15 μL;

[0018] MbpAgo 400-600 pmol;

[0019] RNase Free Water balance.

[0020] In the present invention, the 10×Buffer is prepared as follows: 100 mM Tris-Hcl (pH 7.5), 1 M NaCl, 50% glycerol, and 50 mM MnCl2.

[0021] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the guide DNA is a phosphorylated guide DNA.

[0022] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the phosphorylation of the guide DNA can be performed with reference to conventional operations in the relevant field. Specifically, the guide DNA can be phosphorylated according to the following method:

[0023] Prepare the reaction system. Each 50 μL reaction system includes:

[0024] Guide DNA (100 μM) 5 μL;

[0025] ATP (10 mM) 5 μL;

[0026] T4 PNK phosphorylation kinase 1 μL;

[0027] T4 PNK buffer (10×) 5 μL;

[0028] RNase Free Water 34 μL;

[0029] The reaction system was reacted at 37°C for 30 min and then immediately inactivated at 65°C for 20 min.

[0030] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the length of the guide DNA is 15-20nt, preferably 16-17nt. The design of the specific sequence of the guide DNA can be determined according to the sequence of the RNA fragment to be cut.

[0031] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, affinity purification can be performed with reference to conventional operations in the relevant field.

[0032] Preferably, the process of affinity purification in the present invention comprises:

[0033] Oligo dT beads are used to purify the enzyme cleavage products, and the purified products are purified by complementary binding of ribonucleotide A in the Poly A tail with nucleotide T on the Oligo dT beads; the purified products include the cleavage products containing the Poly A tail and the full-length mRNA that has not been cleaved.

[0034] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the analysis of the purified product adopts quantitative analysis. Preferably, the purified product is analyzed by LC-MS.

[0035] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, during the LC-MS analysis, the components in the purified product are distinguished by the differences in molecular mass and retention time.

[0036] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, during the LC-MS analysis, the mobile phase A contains triethylamine and hexafluoroisopropanol; the mobile phase B includes methanol. The triethylamine in the mobile phase A can improve the peak shape and eliminate tailing; the concentration of triethylamine is preferably 8-9mM. Hexafluoroisopropanol can dissolve most polymers, and adding the mobile phase facilitates the analysis of mRNA; the concentration of hexafluoroisopropanol is preferably 100-500mM. Preferably, the mobile phase B is 100% methanol.

[0037] According to a specific embodiment of the present invention, the method for detecting the length of the Poly A tail of mRNA of the present invention further comprises: searching in the data results of LC-MS detection based on the molecular weight of the purified product to analyze the distribution of the overall Poly A tail on the mRNA.

[0038] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the mRNA to be detected may be modified or unmodified. The modification may include modified nucleosides, for example, the modification includes but is not limited to one or more of 1-methyl pseudouridine modification, 5' capping modification, etc.

[0039] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA of the present invention, the Poly A tail of the mRNA to be detected can be a one-segment and / or multi-segment Poly A tail. The one-segment Poly A tail is a polyA sequence fragment composed entirely of A nucleotide residues. The multi-segment Poly A tail includes more than two Poly A sequence fragments, and adjacent Poly A sequence fragments are connected by linkers, and their nucleotide sequences can be expressed as: A1-L1-A2-L2-A3-L3-...-Lm-An, wherein A1, A2, A3,...An represent polyA sequence fragments; L1, L2, L3,...Lm represent linker sequence fragments; n represents the number of polyA sequence fragments, n≥2; m represents the number of linker sequence fragments, m≥1; and n=m or n=m+1. Preferably, each linker is composed of 1-24nt and not all A nucleotide residues; each Poly A sequence fragment is independently composed of 2-100nt continuous A. Preferably, the multi-segmented Poly A tail includes 2-20 segments (e.g., 2, 3, 4, 5, 6, 7, 8, 9, etc.) of polyA sequence segments. Preferably, the total number of A in all PolyA sequence segments in the multi-segmented Poly A tail is 30-800, preferably 60-240, and more preferably 100-200. Preferably, the total length of the multi-segmented Poly A tail is 42-1000nt, preferably 62-500nt, and more preferably 101-248nt. Preferably, the total number of bases in all Linker sequence segments is 1-200, preferably 1-100nt, further preferably 1-48nt, and more preferably 1-24nt or 2-24nt.

[0040] The method of the present invention can be applied to detect the length of the Poly A tail of mRNA of any structure and / or length, especially mRNA containing modified nucleosides can be normally cut and detected, and multi-segmented A tail sequences can also be accurately detected. Specifically, the length of the mRNA (excluding the length of the Poly A tail) is not particularly limited, for example, it can be 24-5000nt, preferably 100-3000nt.

[0041] On the other hand, the present invention also provides a kit for detecting the length of the Poly A tail of mRNA, the kit comprising:

[0042] a phosphorylated guide DNA or a reagent capable of phosphorylating a guide DNA; and

[0043] Programmable ribonuclease MbpAgo.

[0044] The present invention is based on MbpAgo enzyme digestion, affinity purification and quantitative detection, and can be used to characterize the length distribution of mRNA PolyA tails. It can also effectively detect segmented PolyA with stronger stability. Compared with the currently common RNase T1 enzyme digestion method, it has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Schematic diagram of electrophoresis detection of cutting RNA containing nucleotide modifications and unmodified RNA.

[0046] Figure 2 :Schematic diagram of electrophoresis detection of MbpAgo cutting higher-structure mRNA with different nucleotide modifications.

[0047] Figure 3 : Schematic diagram of electrophoresis detection of mRNA containing PolyA tail in the purified enzyme digestion system.

[0048] Figure 4 :Schematic diagram of optimized enzyme digestion and purification of PolyA electrophoresis detection.

[0049] Figure 5 : Schematic diagram of enzyme digestion purification electrophoresis results.

[0050] Figure 6 : LC-MS detection bar graph of the enzyme-cleaved purified product. DETAILED DESCRIPTION

[0051] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail in conjunction with specific embodiments, and it should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of use of the present invention. In the embodiments, each raw reagent material, unless otherwise specified, can be commercially obtained, and the experimental method without specifying specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0052] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the relevant art.

[0053] Expression and purification of programmable ribonuclease (MbpAgo)

[0054] Cell culture:

[0055] The nucleotide sequence encoding MbpAgo (for the specific sequence, see SEQ ID NO: 9) was connected to the pET-28a vector to construct the pET-28a-MbpAgo plasmid, and the plasmid pET-28a-MbpAgo was transformed into Escherichia coli Rosetta (DE3), and a single colony was picked and cultured in LB medium containing 50 μg / ml kanamycin at 37°C until the OD600 of the medium reached 0.8, and then IPTG with a final concentration of 0.5 mM was added to induce cell expression, and then the culture medium was transferred to an 18°C ​​incubator for incubation for 16 h, and the bacteria were collected after overnight culture at 7000g for 10 min.

[0056] SEQ ID NO.9(5'-3'):

[0057]

[0058] Protein purification:

[0059] Buffer A: 20mM Tris-HCl (pH 7.4), 500mM NaCl, and 20mM imidazole;

[0060] Buffer B: 20mM Tris-HCl (pH 7.4), and 500mM NaCl;

[0061] Buffer C: 2M imidazole.

[0062] The purification method uses nickel column purification: resuspend the bacteria in Buffer A, then use an ultrasonic disruptor to break the cells, centrifuge at 10000g for 30 minutes, take the supernatant and incubate with the activated nickel column for 1 hour, then add 10mL Buffer B for elution twice. Buffer B and Buffer C are combined to contain 10mM, 20mM, 50mM, 100mM, 200mM, 300mM, and 400mM imidazole, and then use the mixed buffer configured above for gradient elution. Finally, 10% polyacrylamide gel electrophoresis is performed to detect the protein expression results, and the mixed buffer eluate with a concentration of 100mM and above is ultrafiltered and concentrated using a 30kd ultrafiltration tube, and the concentration of purified protein is determined using a BCA kit.

[0063] Poly A tail length detection pre-treatment

[0064] (1) Preparation before mRNA digestion:

[0065] Phosphorylation of guide DNA (gDNA): refer to the instructions of the phosphorylation kit. Specifically, taking a 50 μL reaction system as an example, the system is mixed according to Table 1, reacted at 37°C for 30 minutes, and then immediately inactivated at 65°C for 20 minutes.

[0066] Table 1

[0067] Element Volume (50 μL) Guide DNA (100 μM) 5μL ATP(10mM) 5μL T4 PNK phosphorylation kinase 1μL T4 PNK buffer (10×) 5μL RNase Free Water 34μL

[0068] The 10× buffer in the above and various examples is prepared by: 100 mM Tris-Hcl (pH 7.5), 1 M NaCl, 50% glycerol, and 50 mM MnCl2.

[0069] (2) Enzyme digestion and purification of polyA tail:

[0070] Enzyme digestion of mRNA: In a 150 μL system, enzymatic digestion and purification were performed at 37-55°C according to the molar ratio of MbpAgo:guide DNA:mRNA=(4-6):2:1 in Table 2.

[0071] Table 2

[0072] Element Volume or amount of substance mRNA 0~200pmol guide DNA 200 pmol 10×Buffer 15μL MbpAgo 400~600pmol RNase Free Water Make up to 150 μL

[0073] The enzyme digestion reaction was carried out for 1 h, followed by purification of the Poly A tail.

[0074] Purification of Poly A tail: Using Dynabeads TM The mRNA Purification Kit was used according to the instructions of the kit to purify the Poly A tail fragment by complementary binding of the ribonucleotide A in the Poly A tail with the nucleotide T on the Oligo dT beads in the purification kit. This method removes the mixed protein components in the cleavage product and the cleavage product without the Poly A tail, and finally purifies the short cleavage product containing the Poly A tail and the full-length mRNA without cleavage, which is the injection solution for LC-MS.

[0075] Poly A tail length detection mass spectrometry analysis

[0076] Using LC-MS, we can distinguish Dynabeads by differences in molecular mass and retention time without the need for radiolabeling. TM The purified product separated and purified by the mRNA Purification Kit. The mobile phase A used in the LC-MS analysis process is composed of 200mM hexafluoroisopropanol and 8.15mM triethylamine (pH 7.9), and the mobile phase B is 100% methanol. The main function of triethylamine is to improve the peak shape and eliminate tailing; hexafluoroisopropanol is a highly polar solvent that can be effectively mixed with water and methanol, has good thermal stability, can dissolve most polymers, and is added to the mobile phase for easy analysis of mRNA. Based on the different molecular weights of purified products of different lengths, the molecular weights of purified products of different lengths can be searched in the data results of LC-MS detection. At the same time, the size of the search range is set. Taking the characterization of the PolyA tail with a length of 120nt as an example, the size requirement of the search target can be set to be less than 50,000 Dalton, that is, the molecular weight of the cleavage products of different lengths from 2 to 149nt, so as to accurately represent the distribution of the overall Poly A tail on the mRNA.

[0077] In the present invention, mRNA has a negative charge due to its phosphate group, and there is a sodium ion buffer system in the MbpAgo cleavage system. At this time, the mRNA molecule can form an ionic bond with the sodium ion through electrostatic interaction. Therefore, during the enzyme cleavage process of the present invention, one sodium ion modification is added to all possible cleavage products. At the same time, since the mobile phase contains triethylamine during the LC-MS detection process, there may be (triethylamine + H) + Therefore, the present invention adds 1 (triethylamine+H) to all possible cleavage products. + Thus, all retrieval of cleavage products of different lengths from 2 to 149 nt can be completed.

[0078] Example 1

[0079] This example verifies that the purified MbpAgo can effectively and specifically cleave nucleotide-modified RNA, and the following mRNA sequences and DNA sequences (as shown in Table 3) are synthesized for enzyme cleavage verification. The preparation before enzyme cleavage is carried out according to the aforementioned "phosphorylation guide DNA".

[0080] Table 3: DNA and RNA sequences for verification of MbpAgo cleavage activity

[0081]

[0082] The enzyme activity was verified according to the enzyme system shown in Table 4. The following mixed system was placed in a PCR instrument and reacted at 37°C for 1 hour. The detection product was detected by 10% TBE-PAGE electrophoresis. After 1 hour of electrophoresis, the detection result was as follows: Figure 1 shown.

[0083] Table 4: Enzyme digestion reaction system of T124-1 and T124-2

[0084]

[0085] The electrophoresis results of cutting RNA containing nucleotide modifications and unmodified RNA are as follows Figure 1As shown, lanes 1-6 represent the unmodified RNA digestion verification results of MbpAgo, where lane 1 represents RNA target T124-1, lane 2 is the control without adding MbpAgo, lane 3 is the digestion control without adding guide DNA, and lanes 4-6 are digestion reactions using phosphorylated guide DNA (G1, G2, G3); lanes 7-12 represent the nucleotide-modified RNA digestion verification results of MbpAgo, where lane 7 represents RNA target T124-2, lane 8 is the control without adding MbpAgo, lane 9 is the digestion control without adding guide DNA, and lanes 10-11 are digestion reactions using phosphorylated guide DNA (G1, G2, G3). The results show that MbpAgo protein can effectively cut nucleotide-modified and unmodified RNA under the guidance of phosphorylated G1, G2, and G3 and produce RNA products with consistent trends.

[0086] Example 2

[0087] This example verifies that the purified MbpAgo can effectively and specifically cut nucleotide-modified higher-structure mRNA. Two mRNAs with different poly A tails and the same guide DNA (sequences are shown in Table 5) were selected. In a 150 μL system, 100 pmol of different mRNA targets were cut at 37°C according to the molar ratio of MbpAgo: guide DNA: mRNA = 4:2:1, and then 10% TBE-PAGE was performed. The test results are shown in Figure 5. Figure 2 shown.

[0088] Table 5: DNA and RNA sequences for verification of MbpAgo cleavage activity

[0089]

[0090]

[0091]

[0092]

[0093] like Figure 2 As shown, Luc-110 and Luc-120 mRNAs themselves did not produce obvious specific bands, but under the guidance of guide-R, MbpAgo could effectively cut mRNAs containing different Poly A tails and may produce bands of specific sizes.

[0094] Example 3

[0095] This example verifies that the band produced by cleavage is a tail containing Poly A. The cleavage product was combined with Oligo dT magnetic beads to perform the following experiment: In a 150 μL system, 100 pmol of Luc-110 target was cleaved at 37°C according to the molar ratio of MbpAgo: guide DNA: mRNA = 4:2:1, and Dynabead TM The mRNA was purified by using the mRNA Purification Kit (Oligo dT magnetic beads) and then detected by 10% TBE-PAGE. Figure 3 shown.

[0096] like Figure 3 As shown in the figure, there is a band of uniform size in both the flow-through and the eluate, which can be enriched and purified by OligodT magnetic beads, so this band can be considered to be the poly A tail produced after enzyme cleavage. There are also possible unbound poly A tails in the flow-through, proving that the magnetic beads are saturated; at the same time, in addition to the poly A tail, there is also residual mRNA in the eluate, and the proportion is relatively large, proving that the mRNA has not been completely cleaved.

[0097] Example 4

[0098] In order to effectively perform accurate analysis through LC-MS, it is necessary to improve the efficiency of enzyme cutting as much as possible to ensure that the enriched Poly A tail has a high purity. This example conducted an enzyme cutting system optimization experiment. In a 150μL system, the amount of enzyme used was gradually increased, and enzyme cutting and purification were performed at 37°C according to the following molar ratio of MbpAgo: guide DNA: mRNA = (4-6): 2: 1; at the same time, the temperature was only increased to 50°C according to the enzyme cutting ratio of MbpAgo: guide DNA: mRNA = 4: 2: 1 for enzyme cutting and purification detection. The test results are as follows Figure 4 shown.

[0099] like Figure 4 As shown, there are still possible unbound poly A tails in the flowthrough under the four enzyme digestion conditions, proving that the magnetic beads are saturated; after adjusting the enzyme digestion molar ratio (increasing the amount of enzyme used) and increasing the enzyme digestion temperature, poly A tail products with specific bands can be obtained.

[0100] Example 5

[0101] In order to verify the effectiveness of the method, the products enriched by enzyme digestion were quantitatively analyzed by LC-MS. In a 150 μL system, enzyme digestion and purification were performed at 37°C according to the following molar ratio of MbpAgo: guide DNA: mRNA = 5:2:1. The specific reaction system is shown in Table 6, and the enriched enzyme digestion products were then analyzed by LC-MS.

[0102] Table 6: Enzyme digestion reaction volumes

[0103] Element volume mRNA 100 pmol guide DNA 200 pmol 10×Buffer 15μL MbpAgo 500pmol RNase Free Water Refill to 150 μL

[0104] The results of enzyme cleavage and purification were detected by 10% TBE-PAGE. Figure 5 As shown, the LC-MS detection results are as follows Figure 6 shown.

[0105] like Figure 5 As shown, it is consistent with the purification results in Example 4, and both can effectively enrich and purify to obtain specific bands with higher purity, proving the effectiveness of the segmented Poly A tail enrichment method.

[0106] like Figure 6 As shown in the bar graph of LC-MS detection, the detection results show a normal distribution consistent with the literature reports, among which the LC-MS detection results of the Ago cleavage method: 98.65% of the tail lengths meet the standards. At the same time, the signal response value generated by the Ago enzyme cleavage method is higher, which reduces the background noise. It can be seen that the Ago enzyme cleavage method is consistent with the enzyme cleavage method using RNase T1, and can be used to characterize the length distribution of the mRNA PolyA tail, and can also effectively detect the more stable segmented PolyA, and has a wider range of applications.

[0107] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for detecting the length of the Poly A tail of mRNA, the method comprising: The programmable ribonuclease MbpAgo is used to digest the mRNA to be detected to obtain a digestion product; Affinity purification is performed on the enzyme cleavage product to obtain a purified product; The purified product was analyzed to determine the length of the poly A tail.

2. The method according to claim 1, wherein: When performing enzyme digestion, the reaction system includes guide DNA, and the molar ratio of MbpAgo:guide DNA:mRNA=(4-6):(1-2):1; Preferably, the enzyme digestion is carried out at 37°C-55°C for 0.5h-2h; Preferably, each 150 μL reaction system includes: mRNA (100 pmol) 30 μL; Guide DNA 40 μL; 10× Buffer 15 μL; MbpAgo 400-600 pmol; RNase Free Water balance.

3. The method according to claim 2, wherein: The guide DNA is a phosphorylated guide DNA; Preferably, the guide DNA is phosphorylated according to the following method: Prepare the reaction system. Each 50 μL reaction system includes: Guide DNA (100 μM) 5 μL; ATP (10 mM) 5 μL; T4 PNK phosphorylation kinase 1 μL; T4 PNK buffer (10×) 5 μL; RNase Free Water 34 μL; The reaction system was reacted at 37°C for 30 min and then immediately inactivated at 65°C for 20 min.

4. The method according to any one of claims 2 to 3, wherein: The length of the guide DNA is 15-20 nt, preferably 16-17 nt.

5. The method according to claim 1, wherein: The process of affinity purification involves: Oligo dT beads are used to purify the enzyme cleavage products, and the purified products are purified by complementary binding of ribonucleotide A in the Poly A tail with nucleotide T on the Oligo dT beads; the purified products include the cleavage products containing the Poly A tail and the full-length mRNA that has not been cleaved.

6. The method according to claim 1 or 5, wherein: The purified product was analyzed by LC-MS; Preferably, during LC-MS analysis, components in the purified product are distinguished by differences in molecular mass and retention time.

7. The method according to claim 6, wherein: During LC-MS analysis, mobile phase A contained triethylamine and hexafluoroisopropanol; mobile phase B included methanol; Preferably, the method further comprises: searching the data results of LC-MS detection based on the molecular weight of the purified product to analyze the distribution of the overall Poly A tail on the mRNA.

8. The method according to any one of claims 1 to 7, wherein: The mRNA to be detected is modified or unmodified; Preferably, the modification includes one or more of 1-methylpseudouridine modification and 5' capping modification.

9. The method according to any one of claims 1 to 8, wherein: The Poly A tail of the mRNA to be detected is a one-segment and / or multi-segment Poly A tail.

10. A kit for detecting the length of the Poly A tail of mRNA, the kit comprising: Phosphorylating guide DNA or a reagent capable of phosphorylating guide DNA; as well as Programmable ribonuclease MbpAgo.

Images