Method for measuring length of poly a tail of mRNA based on programmable enzyme method

Abstract

A method for measuring the length of the poly A tail of mRNA based on a programmable enzyme method. The method comprises: using programmable ribonuclease MbpAgo to perform enzyme digestion on mRNA under test to obtain an enzyme digestion product; performing affinity purification on the enzyme digestion product; and analyzing the purified product to measure the length of the poly A tail. The method can be used to represent a length distribution of the Poly A tail of mRNA, can also implement effective measurement for segmented Poly A having higher stability, and has a wider application range than existing universal RNase T1 enzyme digestion methods.

Description

A method for detecting the length of the Poly A tail of mRNA based on a programmable enzymatic method Technical Field

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

[0002] In vitro transcription (IVT) produces mRNA synthesized by encoding a polyadenylation sequence onto the template DNA, resulting in a poly A tail (Poly A tail). A longer Poly A tail can improve mRNA translation efficiency and enhance mRNA stability. However, during in vitro transcription, the Poly A tail can slip during mRNA synthesis, leading to lengthening or shortening of the Poly A tail. When the Poly A tail is less than 60 nt in length, mRNA expression is severely impaired. Therefore, it is necessary to analyze the length of the mRNA Poly A tail.

[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 polyA tail of mRNA. This method is based on the ability of the endonuclease RNase T1 to specifically cleave the phosphodiester bond between the guanine ribonucleotide (G) and the adjacent ribonucleotide at the 3' end of single-stranded RNA. The polyA tail is purified by specific binding to oligo dT magnetic beads, and the polyA tail length distribution of mRNA is then quantitatively analyzed using high-resolution liquid chromatography-mass spectrometry. This analysis method is currently commonly used in in vitro mRNA preparation processes.

[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, poly A tails with two or more segments of pure ribonucleotide A connected by one or more linkers, where the linkers may contain 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.

[0005] Summary of the Invention

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

[0007] Another object of the present invention is to provide a kit for detecting the length of the Poly A tail of mRNA.

[0008] In one aspect, the present invention provides a method for detecting the length of the Poly A tail of an mRNA, the method comprising:

[0009] The programmable ribonuclease MbpAgo is used to enzymatically digest the mRNA to be detected to obtain the digestion product;

[0010] Affinity purification is performed on the enzyme digestion product to obtain a purified product;

[0011] The purified product was analyzed to determine the length of the poly A tail.

[0012] The method of the present invention for detecting the length of the Poly A tail of mRNA is based on MbpAgo enzymatic digestion and affinity purification, and quantitative detection of the purified product. It 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 commonly used RNase T1 enzymatic digestion method, it has a wider range of applicability.

[0013] According to a specific embodiment of the present invention, in the method for detecting the length of the poly A tail of mRNA, the reaction system includes guide DNA during enzyme digestion. Preferably, the molar ratio of MbpAgo:guide DNA:mRNA in the reaction system is (4-6):(1-2):1.

[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, the enzyme digestion reaction is carried out at 37° C.-55° C. for 0.5 h-2 h.

[0015] 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 digestion, each 150 μL reaction system includes:

[0016] mRNA (100 pmol) 30 μL;

[0017] Guide DNA 40 μL;

[0018] 10× Buffer 15 μL;

[0019] MbpAgo 400-600 pmol;

[0020] RNase Free Water Remaining Volume

[0021] In the present invention, the preparation of the 10× Buffer is as follows: 100 mM Tris-HCl (pH 7.5), 1 M NaCl, 50% glycerol, and 50 mM MnCl 2 .

[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 guide DNA is a phosphorylated guide DNA.

[0023] 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 according to conventional procedures in the relevant field. Specifically, the guide DNA can be phosphorylated according to the following method:

[0024] Prepare the reaction system. Each 50 μL reaction system includes:

[0025] Guide DNA (100 μM) 5 μL;

[0026] ATP (10 mM) 5 μL;

[0027] T4 PNK phosphorylation kinase 1 μL;

[0028] T4 PNK buffer (10×) 5 μL;

[0029] RNase-free water 34 μL;

[0030] The reaction system was incubated at 37°C for 30 min and then immediately inactivated at 65°C for 20 min.

[0031] According to a specific embodiment of the present invention, in the method for detecting the length of the poly A tail of mRNA, the length of the guide DNA is 15-20 nt, preferably 16-17 nt. The specific sequence of the guide DNA can be determined based on the sequence of the RNA fragment to be cleaved.

[0032] 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.

[0033] Preferably, the affinity purification process of the present invention comprises:

[0034] Oligo dT beads are used to purify the cleavage product, and the purified product is obtained by complementary binding of the ribonucleotide A in the poly A tail with the nucleotide T on the oligo dT beads; the purified product includes the cleavage product containing the poly A tail and the uncleaved full-length mRNA.

[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, the analysis of the purified product adopts quantitative analysis. Preferably, the purified product is analyzed by LC-MS.

[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 components in the purified product are distinguished by differences in molecular mass and retention time.

[0037] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of mRNA, during LC-MS analysis, mobile phase A contains triethylamine and hexafluoroisopropanol; mobile phase B comprises methanol. Triethylamine in mobile phase A can improve peak shape and eliminate tailing; the triethylamine concentration is preferably 8-9 mM. Hexafluoroisopropanol can dissolve most polymers, and its addition to the mobile phase facilitates mRNA analysis; the hexafluoroisopropanol concentration is preferably 100-500 mM. Preferably, mobile phase B is 100% methanol.

[0038] According to a specific embodiment of the present invention, the method of detecting the length of the Poly A tail of mRNA of the present invention further comprises: searching the data results of the 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.

[0039] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of an mRNA, the mRNA to be detected may be modified or unmodified. The modification may include modified nucleosides, for example, including but not limited to one or more of 1-methylpseudouridine modification, 5' capping modification, and the like.

[0040] According to a specific embodiment of the present invention, in the method for detecting the length of the Poly A tail of an mRNA, the Poly A tail of the mRNA to be detected can be a single-stranded and / or multi-stranded Poly A tail. The single-stranded Poly A tail is a poly A sequence fragment composed entirely of A nucleotide residues. The multi-stranded Poly A tail includes two or more Poly A sequence fragments, with adjacent Poly A sequence fragments connected by linkers. The nucleotide sequence can be expressed as: A1-L1-A2-L2-A3-L3-...-Lm-An, wherein A1, A2, A3,...An represent poly A sequence fragments; L1, L2, L3,...Lm represent linker sequence fragments; n represents the number of poly A 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 1-24 nucleotides, not all of which are A nucleotide residues; and each Poly A sequence fragment independently consists of 2-100 nucleotides of consecutive A residues. Preferably, the multi-segmented Poly A tail includes 2-20 segments (e.g., 2, 3, 4, 5, 6, 7, 8, 9, etc.) of poly A sequence segments. Preferably, the total number of A in all Poly A 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.

[0041] 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. In particular, mRNA containing modified nucleosides can be normally cleaved and detected, and even multi-segmented A tail sequences can be accurately detected. Specifically, the length of the mRNA (excluding the length of the poly A tail) is not particularly limited, and can be, for example, 24-5000 nt, preferably 100-3000 nt.

[0042] 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:

[0043] a phosphorylated guide DNA or a reagent capable of phosphorylating a guide DNA; and

[0044] Programmable ribonuclease MbpAgo.

[0045] The present invention is based on MbpAgo enzymatic digestion, affinity purification, and quantitative detection, and can be used to characterize the length distribution of mRNA PolyA tails. It can also effectively detect more stable segmented PolyA. Compared with the currently common RNase T1 enzymatic digestion method, it has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1: Schematic diagram of electrophoretic detection of cleavage of RNA with and without nucleotide modifications.

[0047] Figure 2: Schematic diagram of electrophoresis detection of MbpAgo cleaving higher-order structure mRNA with different nucleotide modifications.

[0048] Figure 3: Schematic diagram of electrophoresis detection of mRNA containing PolyA tail in the purified enzyme digestion system.

[0049] Figure 4: Schematic diagram of electrophoresis detection of optimized enzyme digestion and purification of PolyA.

[0050] Figure 5: Schematic diagram of enzyme digestion purification electrophoresis results.

[0051] Figure 6: LC-MS analysis of the purified product. DETAILED DESCRIPTION

[0052] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solutions of the present invention are now described in detail in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of use of the present invention. In the embodiments, the raw materials, unless otherwise specified, are commercially available. The experimental methods for which specific conditions are not specified are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0053] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.

[0054] Expression and purification of programmable ribonuclease (MbpAgo)

[0055] Cell culture:

[0056] The nucleotide sequence encoding MbpAgo (for the specific sequence, see SEQ ID NO: 9) was ligated into the pET-28a vector to construct the pET-28a-MbpAgo plasmid. 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 culture medium reached 0.8. IPTG with a final concentration of 0.5 mM was then added to induce cell expression. The culture medium was then transferred to an 18°C ​​incubator and incubated for 16 h. After overnight culture, the cells were collected at 7000g for 10 min.

[0057] SEQ ID NO.9(5'-3'):

[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] Purification was performed using a nickel column: cells were resuspended in Buffer A, then disrupted using an ultrasonic disruptor. After centrifugation at 10,000 g for 30 minutes, the supernatant was incubated with an activated nickel column for 1 hour, followed by elution twice with 10 mL of Buffer B. Buffer B and Buffer C were combined to contain 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM, and 400 mM imidazole, respectively, and gradient elution was performed using this mixed buffer. Protein expression was detected by 10% polyacrylamide gel electrophoresis. The eluate containing the mixed buffer at a concentration of 100 mM or higher was concentrated by ultrafiltration using a 30 kD ultrafiltration tube, and the purified protein concentration was determined using a BCA kit.

[0063] Poly A tail length detection pre-processing

[0064] (1) Preparation before mRNA digestion:

[0065] Phosphorylate guide DNA (gDNA): Follow the instructions of the phosphorylation kit. Specifically, using a 50 μL reaction system as an example, mix the system according to Table 1, react at 37°C for 30 minutes, and then immediately inactivate at 65°C for 20 minutes.

[0066] Table 1

[0067] The 10× buffer solution in the above and various examples is prepared as follows: 100 mM Tris-HCl (pH 7.5), 1 M NaCl, 50% glycerol, and 50 mM MnCl 2 .

[0068] (2) Enzymatic digestion and purification of polyA tail:

[0069] mRNA digestion: In a 150 μL system, 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.

[0070] Table 2

[0071] The enzyme digestion reaction was carried out for 1 h, followed by purification of the Poly A tail.

[0072] Purification of Poly A tail: Using Dynabeads TM The mRNA Purification Kit is used according to the kit instructions to purify poly A tail fragments through 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 mixed protein components and cleavage products without the poly A tail from the cleavage products. Ultimately, the purified product contains a short cleavage product containing the poly A tail and the intact full-length mRNA, which serves as the injection solution for LC-MS.

[0073] Poly A tail length detection mass spectrometry analysis

[0074] Using LC-MS, we can distinguish Dynabead-derived proteins by molecular mass and retention time without the need for radiolabeling. TMThe purified product was isolated and purified using the mRNA Purification Kit. The mobile phase A used in the LC-MS analysis consisted of 200mM hexafluoroisopropanol and 8.15mM triethylamine (pH 7.9), and the mobile phase B was 100% methanol. The primary function of triethylamine is to improve peak shape and eliminate tailing. Hexafluoroisopropanol is a highly polar solvent that mixes effectively with water and methanol, exhibits excellent thermal stability, and can dissolve most polymers. Adding it to the mobile phase facilitates mRNA analysis. Based on the varying molecular weights of purified products of varying lengths, molecular weights of purified products of varying lengths can be searched within the LC-MS data. The search range can also be set. For example, for characterizing a 120nt Poly A tail, the search target size requirement can be set to less than 50,000 Dalton, meaning the molecular weight of cleavage products of varying lengths from 2 to 149nt, thereby accurately representing the overall distribution of Poly A tails on the mRNA.

[0075] 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, the present invention adds a sodium ion modification to all possible cleavage products during the enzyme cleavage process. 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 cleavage products with different lengths of 2 to 149 nt can be searched.

[0076] Example 1

[0077] This example verifies that the purified MbpAgo can effectively and specifically cleave nucleotide-modified RNA. The following mRNA sequences and DNA sequences (as shown in Table 3) were synthesized for enzyme digestion verification. Preparation before enzyme digestion was carried out according to the aforementioned "phosphorylation guide DNA".

[0078] Table 3: DNA and RNA sequences used to verify MbpAgo cleavage activity

[0079] The enzymatic activity was verified according to the enzyme digestion 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. The detection result was shown in Figure 1 after 1 hour of electrophoresis.

[0080] Table 4: Enzyme digestion reaction system of T124-1 and T124-2

[0081] The electrophoresis results of cleavage of modified and unmodified RNAs are shown in Figure 1. Lanes 1-6 demonstrate the MbpAgo enzyme digestion validation of unmodified RNAs, with lane 1 representing the RNA target T124-1, lane 2 a control without MbpAgo, lane 3 a control without guide DNA, and lanes 4-6, respectively, demonstrating digestion reactions with phosphorylated guide DNAs (G1, G2, and G3). Lanes 7-12 demonstrate the MbpAgo enzyme digestion validation of modified RNAs, with lane 7 representing the RNA target T124-2, lane 8 a control without MbpAgo, lane 9 a control without guide DNA, and lanes 10-11, respectively, demonstrating digestion reactions with phosphorylated guide DNAs (G1, G2, and G3). These results demonstrate that MbpAgo protein, guided by phosphorylated G1, G2, and G3, effectively cleaves both modified and unmodified RNAs, generating consistent RNA products.

[0082] Example 2

[0083] This example demonstrates that purified MbpAgo can effectively and specifically cleave nucleotide-modified higher-order mRNA structures. Two mRNAs with different poly A tails and the same guide DNA (sequence shown in Table 5) were selected. In a 150 μL system, 100 pmol of each mRNA target was cleaved at a molar ratio of MbpAgo:guide DNA:mRNA = 4:2:1 at 37°C. The cleavage was then performed on 10% TBE-PAGE. The results are shown in Figure 2.

[0084] Table 5: DNA and RNA sequences used to verify MbpAgo cleavage activity

[0085] As shown in Figure 2, Luc-110 and Luc-120 mRNAs themselves did not produce obvious specific bands. However, under the guidance of guide-R, MbpAgo could effectively cut mRNAs containing different Poly A tails and may produce bands of specific sizes.

[0086] Example 3

[0087] 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 conduct 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. TM The mRNA was purified using an mRNA Purification Kit (Oligo dT magnetic beads) and then detected by 10% TBE-PAGE. The detection results are shown in FIG3 .

[0088] As shown in Figure 3, a uniformly sized band is present in both the flow-through and the eluate, which is enriched and purified by the Oligo dT magnetic beads. Therefore, this band is considered to be the poly A tail produced after enzyme cleavage. The presence of possibly unbound poly A tails in the flow-through indicates saturation of the magnetic beads. Furthermore, the presence of a significant fraction of residual mRNA in the eluate, in addition to the poly A tail, indicates that the mRNA was not completely cleaved.

[0089] Example 4

[0090] To effectively perform accurate analysis via LC-MS, it was necessary to maximize enzyme digestion efficiency and ensure high purity of the enriched Poly A tail. This example conducted an enzyme digestion system optimization experiment. In a 150 μL system, the enzyme dosage was gradually increased, and digestion and purification were performed at 37°C using the following molar ratio of MbpAgo:guide DNA:mRNA = (4-6):2:1. Furthermore, enzyme digestion and purification were performed at an elevated temperature of only 50°C, using a digestion ratio of MbpAgo:guide DNA:mRNA = 4:2:1. The results are shown in Figure 4.

[0091] As shown in Figure 4, under the four enzyme digestion conditions, there were still possible unbound poly A tails in the flowthrough, indicating that the magnetic beads were saturated. After adjusting the enzyme digestion molar ratio (increasing the amount of enzyme used) and increasing the enzyme digestion temperature, a specific poly A tail product was obtained.

[0092] Example 5

[0093] To validate the method, the enriched products were quantitatively analyzed by LC-MS. Enzymatic digestion and purification were performed in a 150 μL system at 37°C using the following molar ratio of MbpAgo: guide DNA: mRNA = 5:2:1. The specific reaction system is shown in Table 6. The enriched digestion products were then analyzed by LC-MS.

[0094] Table 6: Enzyme Digestion Reaction Volume

[0095] The results of enzyme digestion and purification detected by 10% TBE-PAGE are shown in FIG5 , and the results of LC-MS detection are shown in FIG6 .

[0096] As shown in FIG5 , the results were consistent with those in Example 4, and both were able to effectively enrich and purify to obtain specific bands with high purity, demonstrating the effectiveness of the segmented Poly A tail enrichment method.

[0097] As shown in Figure 6, the LC-MS bar graph shows that the test results exhibit a normal distribution consistent with literature reports. The Ago cleavage method LC-MS test results show that 98.65% of tail lengths meet the standard. The Ago enzyme digestion method also produces a higher signal response value, reducing background noise. This shows that the Ago enzyme digestion method is consistent with the RNase T1 enzyme digestion method and can both be used to characterize the length distribution of mRNA PolyA tails. It can also effectively detect the more stable segmented PolyA, thus having a wider range of applications.

[0098] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection 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.

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