Method for detecting length of polya tail of mRNA

Abstract

Provided is a method for detecting the length of an mRNA polyA structure. The method comprises the following steps: 1) treating mRNA using a DNA ribozyme with an RNA shearing activity to generate an RNA fragment with a polyA structure and an RNA fragment without a polyA structure; 2) extracting the RNA fragment with the polyA structure; and 3) detecting and reading the length of the RNA fragment with polyA using capillary electrophoresis. Further provided are a deoxyribozyme DNAzyme(8-17) and a kit comprising the deoxyribozyme DNAzyme(8-17) for detecting the length of an mRNA polyA structure.

Description

A method for detecting the length of polyA tail of mRNA Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for detecting the length of the polyA tail of mRNA. Background Art

[0002] In eukaryotes, most mRNA molecules are polyadenylated at the 3' end. The resulting polyA tail and the proteins bound to it help protect the mRNA from degradation by exonucleases. 3' tailing is also important for transcription termination, mRNA export from the nucleus, and translation. PolyA length has been found to be closely related to mRNA translation and stability (Nicholson, Angela L.; Pasquinelli, Amy E. Trends in Cell Biology (2019), 29(3), 191-200). In the early days, people believed that the longer the polyA, the better it could protect mRNA from degradation. However, with the deepening of research, especially the development of sequencing technology for polyA, people found that the truth may not be as simple as imagined. Some studies have shown that relatively short polyA usually appears in highly expressed mRNAs with higher translation efficiency, while longer polyA appears more frequently in mRNAs with lower abundance and lower translation efficiency. At the same time, the length of polyA seems to be negatively correlated with its half-life in the cell.

[0003] When preparing mRNA in vitro, two methods are commonly used to add polyA to the 3' end of RNA: (1) designing a fixed-length polyA sequence on the in vitro transcription template and directly adding it through in vitro transcription; (2) adding it using polyA polymerase after transcription is complete. Due to the characteristics of the enzymes used in these two methods, the length of polyA in the RNA product will inevitably vary. Given the importance of polyA length for mRNA drugs, the mRNA drug guidelines issued by drug regulatory agencies in various countries stipulate that the polyA length of mRNA must be strictly tested.

[0004] Chinese patent CN105734053B discloses “a method for constructing a library for high-throughput sequencing analysis of polyA tail length”. The patent provides a library construction method for measuring the length of the polyA tail, that is, using a kit to add a 3' linker to the sample to be tested, and then transcribe it into cDNA, and then use the library construction kit to enrich the 3' end information of the sample sequence to be tested, and then use the high-throughput sequencer IlluminaNextSeq500SR300 to measure the polyA tail length of the mRNA. However, this method does not directly measure the polyA tail length of the mRNA, but rather it undergoes reverse transcription of the mRNA to generate complementary cDNA, and then DNA polymerase synthesizes the second chain to form a double-stranded DNA before library construction and sequencing. During the sequencing process, DNA polymerase needs to be used for sequencing while synthesizing. Due to the reverse transcription or PCR polymerase synthesis of the long poly A tail, the reverse transcriptase and DNA polymerase often misread, resulting in the generation of extra A or missing A, thereby introducing more systematic errors in sequencing. Moreover, the entire process involves numerous steps and is affected by factors such as the efficiency of multiple test kits, the proficiency of personnel, and the quality decline of mRNA samples after multi-step processing, which will affect the accuracy of the final test results.

[0005] Chinese patent CN110452951B discloses a "method and application for monitoring the length of mRNA polyA tail". The patent provides a method for monitoring the length of mRNA polyA tail. The method measures the ATP consumption and / or PPi generation at different reaction time points in the standard tailing reaction, draws a standard curve of standard mRNA tail length and reaction time, and then determines the length of mRNA polyA tail by the ATP consumption in the mRNA reaction solution to be tested. However, the principle of this method is to indirectly infer the number of polyA based on statistical calculations of biochemical reaction kinetics. The result is the average result of the chemical reaction, which makes it difficult to directly determine the length range of polyA in the sample. It is only applicable to mRNA synthesis or production processes that require a separate tailing reaction, and is not suitable for the generation strategy of directly placing polyA into the transcription template plasmid commonly used in current industrial production. Moreover, during the use of this method, once the various materials in the system change, the detection results will be affected.

[0006] Other current methods for measuring mRNA polyA tail length include Northern blotting and LC-MS, each of which has its own advantages and limitations. For example, Northern blotting is relatively simple, but it is a semi-quantitative method and lacks accuracy. LC-MS measures molecular weight with high accuracy, but is expensive, and its results are based on statistical calculations, making it difficult to directly determine the length range of polyA in a sample. Therefore, developing a simple, cost-effective experimental process that accurately and directly quantifies the length range of polyA tails in mRNA samples is one of the challenges facing mRNA drug research and drug production quality control methodology.

[0007] Summary of the Invention

[0008] In one aspect, the present invention provides a method for detecting RNA polyA length, comprising the steps of:

[0009] 1) DNA ribozymes with RNA cleavage activity are used to specifically cleave mRNA to generate RNA fragments with polyA structures and RNA fragments without polyA structures;

[0010] mRNA includes natural mRNA with a polyA tail or artificially synthesized mRNA with a polyA tail. UTR (Untranslated Regions) are non-coding segments at both ends of the messenger RNA (mRNA) molecule. The 5'-UTR extends from the methylated guanine nucleotide cap at the start of the mRNA to the AUG start codon, and the 3'-UTR extends from the stop codon at the end of the coding region to before the poly A tail (poly-A). Non-coding regions (UTRs) exist in the mRNA of both prokaryotes and eukaryotes, but their length and composition vary. In prokaryotes, the 5' untranslated region is usually 3 to 10 nucleotide residues in length. However, in eukaryotes, the 5' untranslated region can grow to hundreds or even thousands of nucleotide residues in length. Compared to prokaryotes, the genomes of eukaryotes are more complex, and the length of the 3' untranslated region also varies.

[0011] Since the 3' UTR region of mRNA is not fixed, in order to make the detection results more accurate, for different 3' UTR regions, it is necessary to design a DNA ribozyme with RNA cleavage activity at a specific site and / or screen the cleavage site to obtain the optimal DNA ribozyme. The specific 3' UTR region and the corresponding designed DNA ribozyme provided by the present invention do not limit the scope of protection of the present invention. Any 3' UTR region and any DNA ribozyme that can specifically and effectively cleave the 3' UTR region can be used in the detection method provided by the present invention.

[0012] The detection method provided herein screens for DNA ribozymes capable of site-specific mRNA cleavage, with the cleavage site located within 200 bases of the 3'-UTR, including the polyA tail. When the number of bases in the polyA-containing RNA fragment after cleavage exceeds 200, the accuracy of the capillary electrophoresis detection results gradually decreases as the number of bases increases.

[0013] In some embodiments, the shearing enzyme is a deoxyribozyme capable of specifically cutting RNA, specifically, it can be a deoxyribozyme DNAzyme (10-23) or DNAzyme (8-17). In some embodiments, the shearing enzyme is DNAzyme (10-23), and in some embodiments, the shearing enzyme is DNAzyme (8-17). In some embodiments, the arm length of the two arms of DNAzyme (10-23) or DNAzyme (8-17) can be in the range of 7 to 15 nt, preferably 14 nt in this embodiment. The ribozyme provided in this application does not limit this application. Any sequence that is not identical or similar to the UTR sequence provided in this application can also be screened out as a suitable ribozyme by the method provided in this application and subjected to the next step of detection.

[0014] In some embodiments, the UTR region at the 3' end of the mRNA includes the sequence shown in SEQ ID NO: 1, and the cleavage enzyme is DNAzyme (8-17) or DNAzyme (10-23).

[0015] The sequence shown in SEQ ID NO: 1 is 295 nt long and is a relatively common UTR sequence. In actual applications, the 3' UTR of mRNA samples may also vary. A similar screening process can be performed to obtain suitable DNAzymes. In some embodiments, a poly A can be attached to the end of the 3' UTR region.

[0016] In some embodiments, the polyA linked to the end of the 3' UTR region is 110 nt in length, as shown in SEQ ID NO: 2:

[0017] In some embodiments, the polyA linked to the end of the 3' UTR region is 92 nt in length, as shown in SEQ ID NO: 3:

[0018] In some embodiments, the polyA linked to the end of the 3' UTR region is 73 nt in length, as shown in SEQ ID NO: 4:

[0019] In some embodiments, the polyA linked to the end of the 3' UTR region is 32 nt in length, as shown in SEQ ID NO: 5:

[0020] In some embodiments, the UTR region connected to the polyA is cleaved, and preferably, the RNA fragment with the polyA fragment after cleavage is no longer than 200 nt.

[0021] The DNAzyme employed in the present invention can specifically cleave a fixed site in mRNA. Therefore, the UTR sequence portion of the RNA fragments containing the polyA structure obtained after cleavage is fixed and known. After accurately measuring the total length range of the fragments by capillary electrophoresis, the length of the fixed UTR sequence in the fragments can be subtracted to obtain the length of the polyA in the mRNA. Furthermore, because the enzyme employed in the present invention to cleave RNA samples is a DNAzyme, it can be easily removed by digestion with DNase I after the cleavage reaction. This minimizes interference with subsequent analysis of the cleaved short RNA fragments by proteases (which often contain RNase residues that degrade RNA) or RNAzymes (which also produce short RNA fragments), thereby ensuring the high-precision requirements for fragment length analysis.

[0022] In some embodiments, the sequence of the UTR region at the 3' end of the mRNA is as shown in SEQ ID NO: 1; further, the cutting enzyme is DNAzyme (8-17); in some embodiments, the sequence of DNAzyme (8-17) is as shown in SEQ ID NO: 11. The UTR region shown in SEQ ID NO: 1 used in this application is a relatively common UTR sequence. Any UTR sequence shown in SEQ ID NO: 1 or a similar UTR sequence can be cut using the ribozyme shown in SEQ ID NO: 11 provided in this application. The UTR sequence and corresponding ribozyme provided in this application do not limit this application. Any sequence that is not identical or similar to the UTR sequence provided in this application can also be screened for ribozymes by the method provided in this application and subjected to the next step of detection.

[0023] In some embodiments, the sequence of DNAzyme (8-17) is as shown in SEQ ID NO: 11, and any tailed mRMA whose UTR region contains the DNAzyme (8-17) ribozyme targeting sequence as shown in SEQ ID NO: 11 can be cleaved using the ribozyme shown in DNAzyme (8-17), and the targeting sequence is shown in SEQ ID NO: 13.

[0024] In some embodiments, a cleavage enzyme is designed and screened based on the 3' UTR region of the mRNA, and the cleavage enzyme is an enzyme that can specifically cleave the mRNA;

[0025] 2) Extract RNA fragments with polyA structures;

[0026] The method for detecting the length of mRNA polyA provided by the present invention can be applied to mRNA of various lengths, especially mRNA of longer length, ranging from several hundred to several thousand. The RNA fragments with polyA structure after shearing generally have significant length differences from RNA fragments without polyA structure, and the RNA fragments with polyA structure can be purified by various common methods (such as magnetic bead method, column chromatography, electrophoresis, etc.). In some embodiments, the magnetic bead method is used to extract RNA fragments with polyA structure. In some embodiments, 1 μm paramagnetic microspheres coupled with Oligo (dT) are used to separate polyA+RNA fragments from the purified total RNA mixture. Magnetic separation technology can separate complete mRNA from small volume samples, avoiding the step of mRNA precipitation. The entire operation process can be completed within 1 hour.

[0027] 3) Using capillary electrophoresis to detect and read the length of the RNA fragment with polyA.

[0028] Capillary electrophoresis (CE) technology is a highly sensitive and precise method for separating and analyzing biomolecules (nucleic acids, proteins, peptides, sugars, etc.) based on differences in charge and molecular weight. Because CE can achieve high precision (up to 1 nt for short nucleic acid fragments) and sensitivity, it is often used to accurately analyze relatively short DNA or RNA fragments, such as primer amplification products or small RNA molecules. Using an electric field to move charged biomolecules within a charged capillary, molecules of different sizes, charges, and shapes can be separated. mRNA is a single-stranded biomacromolecule composed of linearly linked nucleotides and has negative charges. Therefore, under the action of an electric field, mRNA can move within the capillary and be differentially separated based on differences in size, morphology, and charge density. The present invention uses capillary electrophoresis to directly read the sheared short fragments containing the polyA tail, and then a simple calculation can be performed to obtain the length of the polyA tail.

[0029] In another aspect, the present invention further provides an mRNA polyA length detection kit, which comprises the above-mentioned deoxyribozyme DNAzyme (8-17), and the sequence of DNAzyme (8-17) is shown in SEQ ID NO:11.

[0030] The method for detecting RNA polyA length provided by the present invention first designs and screens a cleavage enzyme that can specifically cleave RNA based on the 3' UTR region of mRNA. The mRNA is then treated with the cleavage enzyme to generate RNA fragments with and without polyA structures. The RNA fragments with polyA structures are then extracted. Finally, capillary electrophoresis is used to determine the length of the RNA fragments with polyA structures. This method is not only simple, economical, and efficient, but also allows for accurate readings, directly determining the range of polyA lengths in mRNA samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to better understand the present invention and more clearly show how to implement the present invention, the features of the embodiments according to the present invention are now described by way of example with reference to the accompanying drawings, in which:

[0032] Figure 1A: Schematic diagram of the principle of 10-23 DNAzyme cleavage of mRNA; Figure 1B: Schematic diagram of the principle of 8-17 DNAzyme cleavage of mRNA;

[0033] Figure 2: Capillary electrophoresis patterns of mRNA 3'-UTR cleavage by DNAzyme (10-23) x-43 and DNAzyme (8-17) y-42;

[0034] Figure 3: Capillary electrophoresis patterns of DNAzyme (8-17) 144-42 after cleavage of the 3'-UTR regions of mRNAs with different poly A lengths. DETAILED DESCRIPTION

[0035] definition

[0036] In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0037] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Similarly, the word "another" can mean at least a second or many.

[0038] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "including" and "comprising"), "having" (and any form of having, "having", "including" and "containing") are inclusive and open-ended and do not exclude additional, unrecited elements or processing steps.

[0039] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably to refer to a single-stranded or double-stranded RNA or DNA polymer, optionally containing synthetic, non-natural, or altered nucleotide residues. In a sequence of a single-stranded nucleic acid (or one of the strands of a double-stranded nucleic acid) (usually starting with a 5'-terminal nucleotide residue in the form of a 5'-monophosphate and ending with a 3'-hydroxy nucleotide residue), each nucleotide residue is designated by its base single letter as follows: "A" or "a" represents an adenylate residue or a deoxyadenylate residue (representing A in RNA or DNA, respectively), "C" or "c" represents a cytidylate residue or a deoxycytidylate residue, "G" or "g" represents a guanosine residue or a deoxyguanosine residue, and "U" or "h" represents a guanosine residue or a deoxyguanosine residue. " or "u" represents a uridine residue, "T" or "t" represents a deoxythymidylate residue, "R" or "r" represents a purine (A or G) nucleotide residue (deoxy or not deoxy), "Y" or "y" represents a pyrimidine (C or T) nucleotide residue (deoxy or not deoxy), "K" or "k" represents G or T, "H" or "h" represents A or C or T, "I" or "i" represents an inosinic acid residue (deoxy or not deoxy), and "N" or "n" represents any nucleotide residue (deoxy or not deoxy). Although the nucleotide sequences herein may be expressed as DNA sequences (including T(s)), when referring to RNA, those skilled in the art can readily determine the corresponding RNA sequence (i.e., replacing T with U).

[0040] As used herein, the terms "polyA" or "poly-A" or similar expressions are used interchangeably and all refer to polyadenosine. Polyadenosine is a long chain molecule containing dozens to hundreds of adenosine residues, formed by multiple adenosine molecules linked together by phosphodiester bonds. This polymer is characterized by being composed of adenosine residues, wherein the adenosine residues contain an adenine base and a ribose sugar, and a portion of the phosphate group after forming the phosphodiester bond. One of the most common applications of "poly A" refers to the polyadenylic acid tail on ribonucleic acid (RNA), commonly referred to as the "poly-A tail", "polyA structure" or "polyadenylation tail". In eukaryotic RNA, the 3' end of the mRNA (messenger RNA) molecule usually has a string of polyadenylic acids, which is called the "poly-A tail". This poly-A tail is added to the 3' end of the mRNA molecule by specific enzymes during the post-transcription process of RNA. The poly-A tail plays an important role in many biological processes. In in vitro mRNA synthesis technology, a polyA tail consisting of a small number of (generally 5 to 8) non-A bases is also widely used. All "tails" or "tails" in this article refer to the above-mentioned "poly-A tail".

[0041] As used herein, the term "DNA ribozyme with RNA cleavage activity" refers to a class of DNA ribozymes (ribozymes) that have RNA cleavage activity, capable of recognizing and cleaving phosphodiester bonds at specific locations within RNA molecules. These enzymes play important roles in biology, including RNA splicing, gene regulation, and genome editing. The following are some common DNA ribozymes with RNA cleavage activity, including 10-23, 8-17, Dz13, hgd70, GR-5, 17E, E5, E6, G4, 10–12opt, and Ag10c.

[0042] As used herein, the term "DNAzyme (10-23)" is a DNA ribozyme with RNA cleavage activity, which was first 14 A type of DNA ribozyme was screened from the 23rd clone in the 10th cycle of in vitro screening of random sequences, so it was named DNAzyme (10-23). ​​The active center of the enzyme consists of a sequence of 15 deoxyribonucleotide residues, which is the "10-23 motif" - 5'-GGCTAGCTACAACGA-3'. The two ends of the active center are substrate binding regions (binding arms), which are generally 7 or more nucleotide residues complementary to the target RNA (such as N in Figure 1A). Their function is to bind to the target RNA through Watson-Cr The cleavage site is located at the phosphodiester bond between an unpaired purine nucleotide and a paired pyrimidine nucleotide on the mRNA molecule. Due to the varying tail lengths of different mRNAs, the number of nucleotide residues in the polyA-containing RNA fragments in the mRNA cleavage products varies, resulting in different peak times on capillary electrophoresis. The capillary electrophoresis instrument used in this study is the Qsep1 Plus portable capillary electrophoresis instrument, and the detection software is Q-Analyzer for Qsep1 (software version: 3.4.3). The instrument's built-in software can automatically calculate the length range of the RNA fragments being tested based on the peak time of the test sample and by comparing it with the peak time of a molecular weight standard. The ratio of different lengths can also be calculated based on the peak area. The cleavage principle is shown in Figure 1A.

[0043] As used herein, the term "DNAzyme (8-17)" is a DNA ribozyme with RNA cleavage activity, which was first 14The 17th clone obtained in the 8th cycle of in vitro screening of random sequences was named DNAzyme (8-17). The enzyme active center sequence is a specific DNA sequence contained in the entire DNAzyme molecule, which is used to recognize and catalyze specific RNA or DNA reactions. It consists of a short stem-loop structure and a downstream unpaired 4-5nt region. The stem is generally composed of 3 base pairs, at least 2 of which are GC paired. The loop sequence is mostly 5'-AGC-3', and in this patent it is 5'-GCG-3'. If the length of the stem is increased or the sequence of the loop is changed, the 8-17 type DNAzyme will lose its activity. The unpaired region is connected to the 3' end of the stem-loop structure, and the sequence is 5'-WCGAA-3' or 5'-WCGR-3' (W=A or T, R=A or G). In addition to the core sequence, the ribozyme also contains seven or more nucleotide residues on either side that are complementary to the target RNA (N and GT in Figure 1B), allowing the ribozyme to specifically recognize and cleave the complementary site on the RNA. The cleavage mechanism is shown in Figure 1B.

[0044] "Binding arm," "substrate binding arm," "substrate binding domain," "substrate binding region," "homologous arm," or similar descriptions refer to a portion of a DNAzyme that is capable of binding to a portion of its substrate or reporter gene through complementarity. Preferably, this complementarity is 100%, but it can be lower if desired. For example, as few as 10 of the 14 bases (7 on the left and 7 on the right) at either end of the cleavage site can base pair (see, for example, Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31). In other words, the sequences of these arms contained within the DNAzyme are intended to specifically bind the DNAzyme to the substrate, such as RNA, through complementary base pairing interactions. The DNAzymes of the present invention may have binding arms that are continuous or discontinuous and may have varying lengths. The length of the binding arm of the DNA ribozyme on one side of the cleavage site is generally greater than or equal to 7 nucleotide residues and has sufficient length to stably interact with the target substrate, and 14 nt is preferred in the present invention. The lengths of the left and right binding arms of the DNAzyme can be symmetrical (i.e., each binding arm has the same length; for example, 7 and 7 nucleotide residues, or 8 and 8 nucleotide residues, or 9 and 9 nucleotide residues long), etc., or asymmetrical (i.e., the binding arms have different lengths; for example, 7 and 8 nucleotide residues; 8 and 7 nucleotide residues long; 8 and 9 nucleotide residues long; 9 and 8 nucleotide residues long; 7 and 9 nucleotide residues long, etc.).

[0045] Example

[0046] The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and are not to be construed as limiting the scope of the invention in any way.

[0047] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0048] Unless otherwise defined or the context clearly dictates otherwise, all materials and instruments used herein were purchased from commercial sources.

[0049] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all fall within the scope defined by the claims of the present invention.

[0050] The experimental materials and instruments in the embodiment of the present invention include:

[0051] 1. T7 HighYield RNA Transcription Kit, Vazyme (Nanjing Novozyme Biotechnology Co., Ltd.);

[0052] 2.GTP, Vazyme;

[0053] 3. DNase I (deoxyribonuclease 1), Vazyme;

[0054] 4. Co-transcription capping T7 in vitro transcription reagent (Cap GAG) Wuhan Hanhai New Enzyme Biotechnology Co., Ltd.;

[0055] 5. UltraPure TM DNase / RNase-free distilled water, Invitrogen TM ;

[0056] 6. Tris-HCl (Tris(hydroxymethyl)aminomethane), Invitrogen TM ;

[0057] 7.LiCl precipitation solution (7.5 M), Invitrogen TM ;

[0058] 8.MgCl2, Ambion TM ;

[0059] 9.VAHTS mRNA Capture Beads, Vazyme TM ,

[0060] 10. High-resolution card holder (S1), product number C105202, Guangding Biotechnology (Jiangsu) Co., Ltd.

[0061] 11.Qsep1-Plus portable biological fragment analyzer, Guangding Biotechnology (Jiangsu) Co., Ltd.

[0062] Materials and instruments involved in the present invention, unless otherwise specified, were purchased commercially. All DNAzymes and oligonucleotides were synthesized by General Biotechnology (Anhui) Co., Ltd.

[0063] Example 1: Synthesis of mRNA containing a polyA tail

[0064] 1. Synthesis of mRNA with polyA tail

[0065] Four groups of mRNAs, A, B, C, and D, were set up. The sequences of the mRNAs in each group were consistent except for the polyA tail. The sequence of the mRNA without the tail was shown in SEQ ID NO: 6:

[0066] The above mRNA has a total of 1074 nucleotide residues, of which the 3' untranslated region sequence and the 5' untranslated region sequence are both represented by bold horizontal lines.

[0067] The sequence of the polyA tail at the 3' end of Group A is shown in SEQ ID NO: 2; the sequence of the polyA tail at the 3' end of Group B is shown in SEQ ID NO: 3; the sequence of the polyA tail at the 3' end of Group C is shown in SEQ ID NO: 4; and the sequence of the polyA tail at the 3' end of Group D is shown in SEQ ID NO: 5. mRNA containing a polyA tail was synthesized by in vitro transcription. After linearizing the transcription template plasmid with a restriction endonuclease, in vitro transcription of RNA was initiated by T7 RNA polymerase to obtain mRNA containing a polyA tail. The reaction system is shown in Table 1. Transcription was performed using the T7 High Yield RNA Transcription Kit, Vazyme (Nanjing Novozyme Biotechnology Co., Ltd.), catalog number: DD4201. For specific operations, refer to the instructions.

[0068] Table 1 mRNA in vitro transcription system

[0069] After the reagents in Table 1 are mixed evenly, incubate at 37°C for 2 hours. After the reaction is completed, add 1 μL of DNase I provided by the kit and digest at 37°C for 30 minutes to remove the template.

[0070] Post-transcription purification was performed as follows;

[0071] (1) Add 7.5 M LiCl at a volume ratio of 1:3, mix well, let stand at -20°C for 30 min, centrifuge at 12,000 rpm for 10 min, and remove the supernatant after observing the precipitate at the bottom;

[0072] (2) Add 1 ml of pre-chilled 70% ethanol to resuspend the precipitate. Centrifuge at 12,000 rpm for 5 min and discard the supernatant.

[0073] (3) Repeat the previous step;

[0074] (4) Remove the supernatant completely, open the lid and dry at 37°C for 5 min. After the precipitate is dry, add RNase-free ddH2O to fully dissolve the precipitate to obtain uncapped mRNA.

[0075] 2. Enzymatic two-step capping method to cap mRNA containing polyA tail

[0076] In this example, a two-step enzymatic capping method was used to cap mRNA containing a poly A tail. Vaccinia Capping Enzyme (Vazyme, Catalog No.: 10615) and mRNA Cap 2'-O-Methyltransferase (Vazyme, Catalog No.: DD4110-PC-01) were used to co-cap the mRNA to obtain capped mRNA.

[0077] 10 μg of the purified mRNA from Example 1 was denatured at 65° C. for 5 min, and then immediately placed on ice for 5 min. The capping reaction system was then prepared according to Table 2.

[0078] Table 2 Enzymatic two-step capping reaction system

[0079] After mixing the reagents in Table 2 above, incubate at 37°C for 120 minutes, and then add 1 μL of DNase I (Vazyme, Catalog No.: DD4104-PC-03)

[0080] Digest at 37°C for 30 minutes to remove the plasmid template (after digesting the plasmid template, ensure that DNase I is completely inactivated, such as by heating or inactivating it overnight with 70% ethanol as shown below. Otherwise, residual activity will cause degradation of the DNAzyme in the subsequent DNAzyme reaction).

[0081] Then perform post-transcription purification as follows:

[0082] (1) Add 7.5 M LiCl at a volume ratio of 1:3, mix well, let stand at -20°C for 30 min, centrifuge at 12,000 rpm for 10 min, and remove the supernatant after observing the precipitate at the bottom;

[0083] (2) Add 1 ml of pre-chilled 70% ethanol to resuspend the precipitate (it is recommended to incubate at -20 degrees Celsius overnight before subsequent centrifugation. 70% ethanol can effectively inactivate DNase I in the previous step). Centrifuge at 12,000 rpm for 5 minutes and discard the supernatant.

[0084] (3) Repeat the previous step;

[0085] (4) Remove the supernatant completely, open the lid and dry at 37°C for 5 min. After the precipitate is dry, add RNase-free ddH2O to fully dissolve the precipitate to obtain capped mRNA.

[0086] Example 2: Design and screening of deoxyribozymes targeting the UTR region

[0087] The binding arms at both ends of the DNAzyme were designed according to different cleavage sites. In this example, the mRNA of group A shown in Example 1 was used, the polyA tail sequence was shown in SEQ ID NO: 2, and the cleavage site was set within 200 bases of the 3' end.

[0088] The binding arms at both ends of the DNAzyme (10-23)xy were designed based on the cleavage site, where x represents the number of nucleotide residues in the mRNA short fragment produced after cleavage, and y represents the number of nucleotide residues in the deoxyribozyme itself, and y is an integer greater than or equal to 37. To enable qualitative comparison of the differences between different cleavage sites, y was fixed to 43 (y=43), i.e., the number of nucleotide residues in the deoxyribozyme itself was fixed to 43. Since the catalytic core of the DNAzyme (10-23) is fixed at 15 nt and the catalytic core of the DNAzyme (8-17) is fixed at 14 nt, the total length of the arms of the DNAzyme (10-23)x-43 and DNAzyme (8-17) designed in this example is 28 nt. The specifically designed sequences of the DNAzyme (10-23)x-43 and DNAzyme (8-17) are shown in Table 3.

[0089] Table 3 Sequences of DNAzyme (10-23) x-43 and DNAzyme (8-17) x-42

[0090] mRNA was cleaved using DNAzyme (10-23)x-43 and DNAzyme (8-17)x-42, respectively, as shown in Table 1. The specific annealing reaction system is shown in Table 4. The annealing procedure was 85°C pre-denaturation for 1 minute and 37°C reaction for 3 hours. After the reaction, the instrument was maintained at 4°C.

[0091] Table 4 Annealing reaction system

[0092] Add 1 μL of DNase I to the reaction system in Table 4 and react at 37°C for 1 hour to digest DNAzyme (10-23) or DNAzyme (8-17).

[0093] Magnetic bead purification steps for DNAzyme (10-23) x-43 and DNAzyme (8-17) x-42 cleavage products:

[0094] (1) Remove the mRNA Capture Beads from 2-8°C and allow them to equilibrate to room temperature.

[0095] (2) Mix the mRNA Capture Beads thoroughly by turning them upside down, and then pipette 25 μL into the sample in Table 5.

[0096] 25 μL Binging buffer and 30 μL Nuclease-free water. Total volume: 100 μL. Pipette up and down 6 times to mix thoroughly.

[0097] (3) Place the sample in a PCR instrument, incubate at 65°C for 5 min, 25°C for 5 min, and hold at 4°C to allow the mRNA to bind to the magnetic beads.

[0098] (4) Place the sample on a magnetic rack for 5 minutes to separate mRNA from total RNA; carefully remove the supernatant.

[0099] (5) Remove the sample from the magnetic rack, add 200 μL Beads Wash Buffer, pipette up and down 6 times to mix thoroughly, let it stand on the magnetic rack for 5 minutes, and carefully remove the supernatant.

[0100] (6) Remove the sample from the magnetic rack and add 50 μL Tris Buffer to resuspend the magnetic beads; pipette up and down 6 times to mix thoroughly.

[0101] (7) Place the sample in a PCR instrument, incubate at 80°C for 2 minutes, hold at 25°C, and elute the mRNA.

[0102] (8) Add 50 μL Beads Binding Buffer and pipette up and down 6 times to mix thoroughly.

[0103] (9) Incubate at room temperature for 5 minutes to allow the mRNA to bind to the magnetic beads.

[0104] (10) Place the sample on a magnetic rack for 5 minutes to separate mRNA from total RNA; carefully remove the supernatant.

[0105] (11) Remove the sample from the magnetic rack, add 200 μL Beads Wash Buffer, pipette up and down 6 times to mix thoroughly, let it stand on the magnetic rack for 5 min, and carefully remove all the supernatant.

[0106] (12) Remove the sample from the magnetic rack, add 20 μL RNase-free ddH2O, mix thoroughly by pipetting 6 times, incubate at 80°C for 2 min, and immediately place on the magnetic rack for 5 min. After the solution is clarified, carefully pipette 18 μL of supernatant into a new Nuclease-free PCR tube.

[0107] (13) Add 2 μL of 1*dillution buffer provided in the S1 cartridge to maintain a final concentration of 0.1*dillution buffer in the buffer system before sample injection.

[0108] The results are shown in Figure 2 (capillary electrophoresis). When the mRNA substrate mass and the dosages of DNAzyme (10-23) x-43 and DNAzyme (8-17) x-42 are the same, DNAzyme (8-17) 144-42 can significantly detect the 3'-end cleavage product peak. The recognition sequence corresponding to DNAzyme (8-17) 144-42 is the sequence shown in SEQ ID NO: 13:

[0109] Example 3: Cleavage of RNAs with Different PloyA Tail Lengths by DNAzyme (8-17) 144-42

[0110] Four experiments, a, b, c, and d, were set up: a: DNAzyme (8-17) 144-42 was used to cleave the 3' untranslated region of group A mRNA carrying the ployA tail shown in SEQ ID NO: 2; b: DNAzyme (8-17) 144-42 was used to cleave the 3' untranslated region of group B mRNA carrying the ployA tail shown in SEQ ID NO: 3; c: DNAzyme (8-17) 144-42 was used to cleave the 3' untranslated region of group C mRNA carrying the ployA tail shown in SEQ ID NO: 4; d: DNAzyme (8-17) 144-42 was used to cleave the 3' untranslated region of group D mRNA carrying the ployA tail shown in SEQ ID NO: 5. The expected products after cleavage are shown below:

[0111] Group a experimental cleavage products:

[0112] Group B experimental cleavage products:

[0113] C group experimental cleavage products:

[0114] Group d experimental cleavage products:

[0115] Example 4: Detection of RNA fragments with polyA structures

[0116] The cleavage products containing the ployA tail obtained from the four groups of experiments a, b, c, and d in Example 3 were further tested.

[0117] 1. Instrument

[0118] Detection software: Q-Analyzer for Qsep1 software version: 3.4.3, fluorescence detector.

[0119] 2. Reagents and consumables

[0120] High-resolution cartridge (S1) cartridge, containing reagents: ① separation buffer ② 1X dilution buffer.

[0121] 3. Calibration conditions

[0122] Using current CE conditions, analysis was performed using five single-stranded oligonucleotides (synthesized with universal bioprimers) with sequence sizes of 20 nt, 66 nt, 107 nt, 126 nt, and 144 nt as standard size markers. The capillary electrophoresis instrument's built-in software was used to generate a standard sequence length calculation file. After obtaining capillary electrophoretic migration data for the test sample, this calculation file was applied to calculate the sequence length range corresponding to the fragment peak in the unknown sample based on its migration time.

[0123] 4. Sample preparation before loading

[0124] Sample processing: Denaturation procedure: Initially at 70°C for 5 minutes, for one cycle. Incubation: 4°C. Capillary electrophoresis calibration conditions: Alignment marker: 20 nt oligonucleotide standard, calibration voltage: 6 kV.

[0125] 5. Injection conditions

[0126] Time: 30 s, sample separation time 700 s, separation voltage 2 kV. Alignment Marker: 20 nt oligonucleotide standard.

[0127] The test results are shown in Table 5.

[0128] Table 5 Test results *Theoretical cleavage product length refers to the predicted length of the mRNA after cleavage, calculated based on the length of the polyA in the in vitro transcription template plasmid. In actual in vitro transcribed samples, the polyA length of the resulting mRNA sample may vary, due to the potential for transcriptase misreading and skipping at long polyA regions, resulting in a certain degree of heterogeneity in polyA length within the sample. **The measured length range is given by the signal peak start and end points in the spreadsheet generated by the capillary electrophoresis analysis software.

[0129] As shown in Figure 3, with the same mRNA substrate mass and DNAzyme (8-17) dosage, the 3'-end cleavage product peak increases with polyA tail length. The 3'-end cleavage product peak can not only be clearly separated on a capillary electrophoresis instrument, but the length of the cleaved fragment can also be directly read, thereby calculating the polyA length range of the sample being tested. As shown in Table 5, the polyA tail length obtained by the method provided by the present invention has a peak length within 5 nt of the theoretical length, with an accuracy exceeding 90%.

[0130] In summary, the present invention designs and screens the optimal DNAzyme (8-17) according to the mRNA sequence to be detected, uses DNAzyme (8-17) to efficiently and specifically cleave mRNA molecules in the 3'-UTR region, and uses capillary electrophoresis to detect the relative peak time of the RNA fragment with polyA at the 3' end after cleavage to determine the polyA length of the RNA, thereby achieving quantitative detection of the polyA tail length of the mRNA. This technology does not require the use of radioactive labels, is free of radioactive contamination, and does not require the use of expensive mass spectrometry equipment. It is simple to operate, low in cost, and easy to implement.

[0131] Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all fall within the scope defined by the claims of the present invention.

Claims

1. A method for detecting the length of the 3'-terminal polyA structure of mRNA, comprising the following steps: 1) Treat the mRNA with a DNA ribozyme having RNA cleavage activity to generate an RNA fragment with the polyA structure and an RNA fragment without the polyA structure; 2) Extract the RNA fragment with the polyA structure; 3) Detect the length of the RNA fragment with the polyA structure by capillary electrophoresis.

2. The method according to claim 1, wherein, The mRNA is selected from natural mRNA containing the polyA structure and synthetic mRNA containing the polyA structure.

3. The method according to claim 1, wherein The RNA fragment with the polyA structure is extracted by the magnetic bead method.

4. The method according to claim 1, wherein The DNA ribozyme having RNA cleavage activity is a deoxyribozyme capable of specifically cleaving mRNA.

5. The method according to claim 4, wherein, The deoxyribozyme is DNAzyme(10-23).

6. The method according to claim 4, wherein The deoxyribozyme is DNAzyme(8-17).

7. The method according to claim 1, wherein, When the UTR region at the 3'-end of the mRNA comprises the sequence shown in SEQ ID NO:1, the DNA ribozyme having RNA cleavage activity is the deoxyribozyme DNAzyme(8-17).

8. The method according to claim 7, wherein, The sequence of the deoxyribozyme DNAzyme(8-17) is as shown in SEQ ID NO:

11.

9. An mRNA polyA structure length detection kit, which comprises the deoxyribozyme DNAzyme(8-17) with the sequence shown in SEQ ID NO:

11.

10. The mRNA polyA structure length detection kit according to claim 9, wherein, The mRNA comprises the UTR region of the sequence shown in SEQ ID NO:1.

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