A method for detecting mRNA sequence coverage using RNA endonuclease gradient dilution method

Abstract

The present invention relates to the field of biotechnology, specifically a method for detecting mRNA sequence coverage using an RNA endonuclease gradient dilution method, the detection method being as follows: S1, after buffer replacement, the sample is enzymatically hydrolyzed in a solution using a diluted RNase T1 enzyme to obtain a fragmented sample; S2, the fragmented sample is subjected to data acquisition by a liquid chromatography-mass spectrometer; S3, the molecular weight of the corresponding nucleic acid fragment is calculated according to the mass-to-charge ratio and the charge obtained by the primary mass spectrometer; S4, based on the fragment information provided by the secondary mass spectrometer of the peptide segment, whether the nucleic acid sequence of the fragment is consistent with the theoretical sequence; S5, all the enzyme-cut nucleic acid fragments after identification are combined to infer whether the entire nucleic acid sequence is correct, thereby confirming whether the nucleic acid sequence is correct. Compared with the prior art, the present invention has the advantages that the sequence coverage calculation of the primary sequence analysis of mRNA is more efficient, the cost is lower, and the result is more stable and reliable.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for detecting mRNA sequence coverage by using an RNA endonuclease gradient dilution method. Background Art

[0002] Currently, RNA sequence confirmation is based on molecular biology and biochemical methods. For example, RNA is reverse transcribed into cDNA and then PCR amplified for microarray studies or sequencing. It is well known that there is a risk of sequence mutations during mRNA reverse transcription and PCR amplification, which may hinder the accurate characterization of RNA sequences. In addition, the entire sequencing cycle takes a long time, up to about 3 weeks, and less than 20% of mutations in the sample cannot be detected by Sanger sequencing. In addition, molecular biology-based methods cannot characterize post-transcriptional modifications of nucleotides, which are essential for RNA biogenesis and function. In recent years, there has been an increasing number of studies on the confirmation of mRNA sequences based on mass spectrometry. Literature reports that the combined use of endoribonuclease to cut long mRNA sequences into oligonucleotide fragments can achieve full coverage of mRNA sequences (Anal. Chem. 2023, 95,1366−1375).

[0003] At present, the detection of mRNA sequence coverage based on mass spectrometry urgently needs to solve the repeatability problem. Since endoribonucleases recognize single bases, such as RNase A cytosine recognizes the 3' end of the pyrimidine residue on RNA, cutting the phosphodiester bond formed by cytosine C or uracil U with the adjacent nucleotide; RNase T1 specifically acts on the 3' end phosphate of guanine nucleotide G and cuts the phosphodiester bond connected to the adjacent nucleotide; since mRNA is composed of only four bases, after endoribonucleases act on mRNA, the product nucleic acid fragments are extremely short and are not conducive to coverage calculation. At present, researchers have reported the use of other ribonucleases for enzyme cleavage. These nucleases recognize combined bases, so that the fragments after enzyme cleavage are longer and more suitable for mass spectrometry analysis. For example, Human RNase 4 recognizes the UR combination and cleaves between UR, where R is adenine A and guanine G (Nucleic Acids Research, 2022, Vol. 50, No. 18); Colicin E5 recognizes the GU combination and cleaves in the middle of GU, and mazF recognizes the ACA combination and cleaves at the 3' end of ACA (Anal. Chem. 2019, 91, 8500−8506). However, these enzymes are only expressed and purified in local laboratories and are not commercially produced.

[0004] Therefore, the commercial enzyme that is currently used for mRNA sequence coverage detection based on mass spectrometry is RNaseT1 / A. However, RNase T1 / A has high enzyme activity and can fragment mRNA in just a few minutes, and the endoribonuclease is extremely stable and difficult to inactivate by heating or other means. Therefore, some researchers immobilized RNase T1 on beads, and during the sample digestion process, they quickly achieved physical separation of RNase T1 and the sample by controlling the digestion time, but the sequence coverage obtained by this method was only more than 70% (Anal. Chem. 2019, 91, 8500−8506). However, the cost of this reagent is too high, which is not conducive to high-throughput promotion and use. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for detecting mRNA sequence coverage using the RNA endonuclease gradient dilution method, which can be used to directly analyze the primary sequence of mRNA using a high-resolution mass spectrometer in the mRNA sequence coverage detection based on mass spectrometry to obtain better result repeatability.

[0006] In order to achieve the above purpose, a method for detecting mRNA sequence coverage using RNA endonuclease gradient dilution method is designed, and the detection method is as follows: S1. After the sample is replaced with buffer, it is enzymatically hydrolyzed in the solution using diluted RNase T1 enzyme to obtain a fragmented sample; S2. The fragmented sample is subjected to data acquisition by liquid chromatography-mass spectrometry; S3. The molecular weight of the corresponding nucleic acid fragment is calculated based on the mass-to-charge ratio and charge obtained by the primary mass spectrometry; S4. Whether the nucleic acid sequence of the fragment is consistent with the theoretical sequence is confirmed based on the fragment information provided by the secondary mass spectrometry of the peptide segment; S5. All the identified enzyme-cut nucleic acid fragments are combined to infer whether the entire nucleic acid sequence is correct, thereby confirming whether the nucleic acid sequence is correct through the primary and secondary mass spectrometry data.

[0007] Preferably, the sample of the present invention is incubated with the enzyme at 37° C. for 30 minutes, and then incubated at 95° C. for 10 minutes to inactivate the RNase T1 enzyme.

[0008] Preferably, the liquid chromatography-mass spectrometry instrument of the present invention confirms the correctness of the nucleic acid sequence based on the combination of the primary mass spectrometry and the secondary mass spectrometry data during the mass spectrometry analysis process.

[0009] Compared with the prior art, the present invention has the following advantages: Through the technical solution of the present invention, the analysis of the primary sequence of mRNA can be carried out more simply and efficiently, because its pre-treatment process is simple and low-cost. Direct detection by a high-resolution mass spectrometer is not only easy to operate, but also can ensure the consistency and reliability of the results. The length of the nucleic acid fragment of the mRNA product after enzyme cleavage is improved and optimized, the length of the maintenance time of the mRNA product after enzyme cleavage is lengthened, the premature fragmentation of mRNA is avoided, and it is easier to inactivate the endonuclease by heating or other means, so that the sequence coverage calculation is more efficient, the cost is lower, and the results are more stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 , is a table of correspondence between LC-MS detection conditions; Figure 2 , is the BPC diagram (triplicate) of mRNA after RNaseT1 enzymatic digestion; Figure 3 , is the UV image (three-needle repetition) of mRNA after RNaseT1 enzymatic digestion; Figure 4 , is a chart of sequence coverage (three-needle repeat). DETAILED DESCRIPTION

[0011] In order to make the purpose, principle and structure of the present invention clearer, the following is further elaborated in conjunction with the accompanying drawings and specific embodiments. In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The reagents used in the following embodiments are purchased from ordinary commercial channels. The experimental operations and experimental conditions not specified refer to the conventional operations and conventional conditions in the art.

[0012] Example 1: Using the liquid chromatography-mass spectrometer of Thermo Company to detect the coverage of mRNA sequences. This example provides a method for detecting the coverage of mRNA sequences using the RNA endonuclease gradient dilution method, which is as follows: 1) Sample buffer replacement: Take 100ug sample, add anhydrous ethanol, 5 M ammonium acetate solution, vortex to mix, incubate on ice for 30 min, place the sample in a pre-cooled centrifuge, centrifuge at 15000 rpm for 15 min at 4°C, and discard the supernatant; add 1 ml of 70% ethanol solution pre-cooled at -20°C, turn upside down 10 times, centrifuge at 15000 rpm for 15 min at 4°C, and discard the supernatant; then repeat this step once; after drying at room temperature for 10 min, add 80 μl of 50mM Tris-HCl pH 7.6, let stand at room temperature for 10 min, and then use Nanodrop to measure the mRNA concentration after replacement (the sample concentration is ≥1mg / ml at this time).

[0013] 2) Enzyme digestion: Dilute RNase T1 10 times with 50 mM Tris (pH 7.6) 8 Take 20 μg of sample, add EDTA (final concentration 3.3 mM), make up the volume to 30 μL with sterile enzyme-free water, incubate at 90℃ for 10 min, cool on ice after incubation, then add 1 μL of diluted RNase T1 enzyme and incubate at 37℃ for 30 min, then incubate at 95℃ for 10 min to inactivate, and centrifuge at 15000 rpm for 5 min to take the supernatant.

[0014] 3) Sampling analysis: 20 μL of sample was injected for analysis. Data were collected using a Thermo Vanquish UPLC-Thermo Q Exactive Plus LC-MS / MS instrument (UPLC-UV-MS / MS).

[0015] LC-MS detection conditions, see Figure 1 The results obtained can be found in Figure 2-4 , the base peak chromatogram (BPC), ultraviolet chromatogram (UV) and sequence coverage table of the sample enzymatic hydrolysis product were detected.

[0016] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent substitutions or changes according to the technical solutions and novel concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting mRNA sequence coverage using RNA endonuclease gradient dilution method, characterized in that The detection method is as follows: S1. After the sample is replaced with buffer, it is enzymatically digested in the solution using diluted RNase T1 enzyme to obtain a fragmented sample; S2. The fragmented samples are collected by liquid chromatography-mass spectrometry; S3. Calculate the molecular weight of the corresponding nucleic acid fragment according to the mass-to-charge ratio and charge obtained by the primary mass spectrometry; S4. confirming whether the nucleic acid sequence of the fragment is consistent with the theoretical sequence based on the fragment information provided by the secondary mass spectrometry of the peptide segment; S5. All the identified enzyme-cut nucleic acid fragments are then combined to infer whether the entire nucleic acid sequence is correct, thereby confirming whether the nucleic acid sequence is correct through primary and secondary mass spectrometry data.

2. A method for detecting mRNA sequence coverage using RNA endonuclease gradient dilution method as claimed in claim 1, characterized in that After the samples were incubated with the enzyme at 37°C for 30 minutes, the RNase T1 enzyme was inactivated by incubation at 95°C for 10 minutes.

3. A method for detecting mRNA sequence coverage using RNA endonuclease gradient dilution method as claimed in claim 1, characterized in that During the mass spectrometry analysis, the LC-MS instrument confirms the correctness of the nucleic acid sequence based on the combination of the primary mass spectrometry and the secondary mass spectrometry data.

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