5 '-terminal sequence rearrangement RNA molecule and application thereof in detection of capping rate of co-transcriptional capped target RNA
By designing RNA molecules with specific 5' end sequence rearrangements, the problem of preparing uncapped RNA standards in the co-transcriptional capping process has been solved, achieving efficient and accurate capping rate detection, which is suitable for the industrial production of mRNA and saRNA.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF HEALTH & MEDICINE HEFEI COMPREHENSIVE NAT SCI CENT
- Filing Date
- 2025-12-30
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies struggle to efficiently prepare uncapped RNA standards, resulting in poor accuracy and comparability in capping rate detection. This is especially true in co-transcriptional capping processes, where the yield of uncapped RNA is extremely low and there are significant batch-to-batch variations, making it impossible to obtain uncapped RNA standards that are consistent with the target mRNA or saRNA.
A specific 5' end sequence rearrangement RNA molecule was designed, with the nucleotide sequence of the 5' end 1-5 positions of its 5' UTR being GN2N3N4N5, where at least one base in N2-N5 is G. It was prepared by an in vitro transcription system without the addition of cap analogs, resulting in high yield and small batch-to-batch variability. It was used as an uncapped RNA standard to detect the capping rate.
This method enables the efficient preparation of uncapped RNA standards that are consistent with the target RNA under uncapped conditions, improving the accuracy and comparability of capping rate detection, reducing preparation costs, and making it suitable for industrial-scale production.
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Figure CN122012495A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, and in particular to a 5' end sequence rearranged RNA molecule and its application in detecting the capping rate of co-transcribed capped target RNA. Background Technology
[0002] The mRNA sequence from the 5' end to the 3' end includes: a 5' non-coding sequence (UTR), a target protein coding sequence, a 3' non-coding sequence (UTR), and a polyA tail.
[0003] Self-replicating RNA (saRNA) sequences, from the 5' to the 3' end, include: a 5' untranslated sequence (UTR), an open reading frame encoding RNA replicase, a subgenomic promoter (SGP), a target protein coding sequence, a 3' untranslated sequence (UTR), and a polyA tail. The target protein coding sequence is derived from the coding region of a viral structural protein, while the other sequences are derived from the viral genome; this is the current mainstream design approach for saRNA sequences. Due to its characteristics of low dosage, high expression, and self-amplification, saRNA has received widespread attention in recent years in the fields of tumor vaccines, infectious disease vaccines, and gene therapy.
[0004] Currently, industrialized mRNA and saRNA production commonly employs co-transcriptional capping. This process involves adding capping analogs (such as ARCA and CleanCap) to the in vitro transcription system, enabling the RNA to acquire a 5' cap structure simultaneously during transcription, thereby improving its stability and translation efficiency. This method is relatively simple and efficient.
[0005] In co-transcriptional capping systems, a high proportion of capped RNA products can usually be obtained due to the influence of factors such as the template promoter, 5' end sequence characteristics, and the participation of capping analogs in transcription initiation. However, without the addition of capping analogs, obtaining high-yield, reproducible uncapped RNA from the same template is challenging because some sequences (e.g., the 5' UTR nucleotide sequence of the target RNA sequence, as shown in SEQ ID NO: 5) are limited by factors such as transcription initiation efficiency, 5' end structure, and RNA stability, resulting in extremely low uncapped RNA yields and large batch-to-batch variability. Since the presence or absence of capping analogs has a significant impact on transcription efficiency, it is speculated that in co-transcriptional capping IVT reactions, capping analogs are added to the 5' end with an absolute majority.
[0006] In the preparation of mRNA or saRNA using co-transcriptional capping technology, it is necessary to monitor the capping rate of the final product for quality control. Commonly used methods for detecting the capping rate include HPLC, LC-MS, and capillary electrophoresis.
[0007] Currently, LC-MS is commonly used to qualitatively determine the capping rate using normalization. This typically involves determining the molecular weights of capped and uncapped RNA based on the 5' end splice sequence of the target RNA, then determining the peak positions of capped and uncapped RNA in the LC-MS spectrum based on their molecular weights, and finally calculating the relative capping rate using normalization. Theoretically, this method does not require the use of standards. However, in actual testing, some samples may show undetectable results for uncapped RNA, raising questions about the suitability of the chromatographic conditions and the ability to detect uncapped RNA. Therefore, it is necessary to further verify the accuracy of the results by testing "uncapped RNA standards."
[0008] As mentioned earlier, some sequences, when prepared into uncapped RNA using in vitro transcription without the addition of capping analogs, have extremely low yields, making it difficult to obtain uncapped RNA standards.
[0009] However, obtaining uncapped RNA standards through chemical synthesis is costly, and natural base RNA is easily degraded and contains many impurities during chemical synthesis and purification, which is not conducive to large-scale application, nor to obtaining standards that are highly consistent with the actual product process.
[0010] When it is not possible to obtain uncapped RNA standards that are highly consistent with the target mRNA or target saRNA in length and molecular weight but do not carry a cap structure, only indirect or relative quantitative methods can be used to detect the capping rate, which will significantly reduce the accuracy and comparability of the capping rate assessment.
[0011] Therefore, there is an urgent need for an uncapped RNA standard that can be efficiently prepared by in vitro transcription while maintaining a total length and theoretical molecular weight that is basically consistent with the target mRNA or target saRNA. Summary of the Invention
[0012] Based on the technical problems existing in the background technology, this invention proposes a 5' end sequence rearranged RNA molecule and its application in detecting the capping rate of co-transcribed capped target RNA. This invention designs the nucleotide sequence of the 5' UTR of RNA to obtain a specific 5' UTR sequence. 5' end sequence rearranged RNA molecules can be obtained by transcription in an in vitro transcription system without adding cap analogs. The yield is high, with minimal batch-to-batch variation, excellent process stability, and reproducible preparation of 5' end sequence rearranged RNA molecules. Furthermore, the 5' end sequence rearranged RNA molecules have the same total length and theoretical molecular weight as the target RNA, or are substantially the same, and can be used as a standard for uncapped RNA to detect the capping rate of co-transcribed capped target RNA.
[0013] This invention proposes a 5' end sequence rearranged RNA molecule, wherein the 5' end sequence rearranged RNA molecule has a 5' UTR, and the nucleotide sequence of the 5' end of the 5' end from position 1 to 5 is: GN2N3N4N5; wherein N2 is G or A, N3 is G, A or T, N4 is G, A or T, and N5 is G or T; and at least one base in N2-N5 is G, and the bases in N3-N5 are not all G at the same time.
[0014] Preferably, the nucleotide sequence at positions 1-5 of the 5' end of the 5' UTR of the 5' end rearranged RNA molecule is one of the following: GATGG, GGATG, GGGAT, or GAGTG.
[0015] Preferably, the nucleotide sequence of the 5'UTR of the 5' end sequence rearranged RNA molecule is shown in SEQ ID NO: 1-4.
[0016] Preferably, the nucleotide sequence of the 5'UTR of the 5' end sequence rearranged RNA molecule is shown in SEQ ID NO: 3.
[0017] The nucleotide sequence of SEQ ID NO: 1 is as follows: GATGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA.
[0018] The nucleotide sequence of SEQ ID NO: 2 is as follows: GGATGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA.
[0019] The nucleotide sequence of SEQ ID NO: 3 is as follows: GGGATCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA.
[0020] The nucleotide sequence of SEQ ID NO: 4 is as follows: GAGTGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA.
[0021] Preferably, the 5' end sequence rearranged RNA molecule is either an mRNA molecule or a saRNA molecule.
[0022] The aforementioned saRNA molecules may or may not have self-replication capabilities.
[0023] This invention also proposes a method for preparing the above-mentioned 5' end sequence rearranged RNA molecule. Without adding a capping analogue, the DNA sequence template of the above-mentioned 5' end sequence rearranged RNA molecule is transcribed using an in vitro transcription system to obtain the 5' end sequence rearranged RNA molecule.
[0024] Preferably, the method includes the following steps: constructing a DNA sequence template for the 5' end sequence rearranged RNA molecule; and, without adding a capping analogue, performing a transcription reaction on the DNA sequence template using an in vitro transcription system to obtain the 5' end sequence rearranged RNA molecule.
[0025] The above-mentioned in vitro transcription system is a conventional in vitro transcription system in this field.
[0026] After the transcription reaction was completed, the RNA molecules with rearranged 5' ends were obtained by purification; LiCl precipitation was preferred for purification.
[0027] This invention also proposes the application of the above-mentioned 5' end sequence rearranged RNA molecule and the 5' end sequence rearranged RNA molecule prepared according to the above method as an uncapped RNA standard.
[0028] Preferably, it is used as a standard for detecting the capping rate of target RNA.
[0029] Preferably, it is used as a standard for detecting the capping rate of target RNA produced by co-transcriptional capping.
[0030] The above-mentioned in vitro transcription (IVT) and co-transcription capping are commonly used methods in this field for producing mRNA and saRNA.
[0031] In vitro transcription refers to a technique that simulates the in vivo RNA synthesis process in a cell-free system using DNA as a template by adding components such as RNA polymerase and nucleoside triphosphates. Co-capping transcription refers to the process of directly synthesizing capped RNA by adding a cap analogue during the transcription reaction in an in vitro transcription system, using DNA as a template. This simplifies subsequent purification steps and improves product homogeneity. Apart from the cap analogue, the in vitro transcription system for co-transcriptional capping to produce target RNA is the same as the in vitro transcription system for preparing 5' end sequence rearranged RNA molecules.
[0032] Preferably, the target RNA has a 5' cap structure.
[0033] Preferably, both the target RNA and the 5' end sequence rearranged RNA molecule have a 5' UTR, and the lengths of their 5' UTR sequences are the same; the nucleotide sequences starting from the 6th position at the 5' end of their 5' UTR sequences are the same.
[0034] Preferably, except for the 5' cap structure and the 5' UTR region, the target RNA and the other regions of the 5' end sequence rearranged RNA molecule have the same sequence.
[0035] Preferably, the nucleotide sequence of the 5'UTR of the target RNA is as shown in SEQ ID NO: 5.
[0036] The nucleotide sequence of SEQ ID NO: 5 is as follows: ATGGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA.
[0037] When the above RNA is mRNA, the target mRNA and the uncapped mRNA molecule have the same target protein coding sequence, 3'UTR, and polyA tail sequence.
[0038] When the above RNA is saRNA, the open reading frame, subgenomic promoter, target protein coding sequence, 3'UTR, and polyA tail sequence of the target saRNA and the uncapped saRNA molecule are the same.
[0039] The aforementioned promoter can be a T7 promoter, etc., and the 5' cap structure can be a GAU cap analogue, etc.
[0040] This invention also proposes a method for detecting the capping rate of target RNA, comprising the following steps: detecting the target RNA sample produced by co-transcriptional capping using high performance liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry or capillary electrophoresis, and qualitatively detecting the capping rate using uncapped RNA standards; Among them, the uncapped RNA standard is the 5' end sequence rearranged RNA molecule mentioned above or the 5' end sequence rearranged RNA molecule prepared according to the above method.
[0041] Preferably, the target RNA has a 5' cap structure.
[0042] Preferably, both the target RNA and the 5' end sequence rearranged RNA molecule have a 5' UTR, and the lengths of their 5' UTR sequences are the same; the nucleotide sequences starting from the 6th position at the 5' end of their 5' UTR sequences are the same.
[0043] Preferably, except for the 5' cap structure and the 5' UTR region, the target RNA and the other regions of the 5' end sequence rearranged RNA molecule have the same sequence.
[0044] Preferably, the nucleotide sequence of the 5'UTR of the target RNA is as shown in SEQ ID NO: 5.
[0045] The above-mentioned qualitative detection of the capping rate can be performed using the normalization method.
[0046] The formula for calculating the capping rate using the normalization method is as follows: Hat-on rate = A 加帽RNA / (A 加帽RNA + A 不加帽RNA )*100%, A 加帽RNA A represents the response value of capped RNA in the target RNA sample. 不加帽RNA The response value is the uncapped RNA in the target RNA sample.
[0047] In the above detection method, the preparation method of the test solution can be as follows: the biotinylated nucleic acid probe is bound to the test sample sequence by PCR annealing, then enzyme digestion is performed to specifically cut and obtain the 5' end fragment, the 5' end fragment is bound to streptavidin magnetic beads, then elution buffer is added, the probe is heated to denature, purified, and the test solution is obtained.
[0048] The present invention also proposes a kit for detecting the capping rate of target RNA, comprising: uncapped RNA standard, wherein the uncapped RNA standard is the above-mentioned 5' end sequence rearranged RNA molecule or the 5' end sequence rearranged RNA molecule prepared according to the above method.
[0049] Preferably, the target RNA is the target RNA produced by co-transcriptional capping.
[0050] Preferably, the target RNA has a 5' cap structure.
[0051] Preferably, both the target RNA and the 5' end sequence rearranged RNA molecule have a 5' UTR, and the lengths of their 5' UTR sequences are the same; the nucleotide sequences starting from the 6th position at the 5' end of their 5' UTR sequences are the same.
[0052] Preferably, except for the 5' cap structure and the 5' UTR region, the target RNA and the other regions of the 5' end sequence rearranged RNA molecule have the same sequence.
[0053] Preferably, the nucleotide sequence of the 5'UTR of the target RNA is as shown in SEQ ID NO: 5.
[0054] The above-mentioned uncapped RNA standards can be selected and designed according to different target RNA sequences.
[0055] The above-mentioned kits may also contain detection reagents such as buffer solutions; they may also contain instructions for use.
[0056] Through multiple experiments, this invention has discovered that by designing the nucleotide sequence of the 5'UTR of RNA and adjusting only the bases at positions 1-5 of the 5' end of the 5' UTR sequence, a 5' end sequence rearranged RNA molecule with the same molecular weight as the target can be obtained by transcription reaction using an in vitro transcription system without the addition of cap analogs. The yield is high, and the yield difference between batches is very small, with excellent process stability.
[0057] The resulting 5' end sequence rearranged RNA molecule has the same total length and theoretical molecular weight as the target RNA produced by co-transcription and capping, and the two can maintain structural comparability and molecular weight consistency.
[0058] The 5' end sequence rearranged RNA molecule described in this invention can be used as an uncapped RNA standard for qualitative detection of the capping rate of co-transcriptional capped RNA production, thereby improving the accuracy of LC-MS detection. It overcomes the technical bottleneck of "low yield or difficulty in obtaining uncapped control standards" in traditional co-transcriptional capping systems, and has good versatility and application prospects.
[0059] Furthermore, this invention uses conventional in vitro transcription to prepare 5' end sequence rearranged RNA molecules, which does not rely on expensive and difficult-to-scale chemical synthesis, greatly reducing the preparation cost. Moreover, the process is easy to integrate with actual production processes, which is conducive to industrial scale-up and batch preparation. It is highly versatile and easy to promote.
[0060] The method of preparing 5' end sequence rearranged RNA molecules by adjusting the bases at positions 1-5 of the 5' end of the 5' UTR sequence is applicable to RNA products with extremely low in vitro transcription yields under uncapped conditions, such as saRNA, and provides a new technical path for establishing a universal capping rate detection and release standard system for related products. Attached Figure Description
[0061] Figure 1 The graph shows the yield of saRNA prepared by in vitro transcription without the addition of capping analogs.
[0062] Figure 2 The image shows the LC-MS chromatogram of sample 2 in Example 4, where Base indicates uncapped and Base Cap1 indicates capped. Detailed Implementation
[0063] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.
[0064] Example 1
[0065] A method for preparing a 5' end sequence rearranged saRNA molecule includes the following steps: (1) Constructing a DNA sequence template: Based on the target saRNA template plasmid (the nucleotide sequence of the 5'UTR of the target saRNA is shown in SEQ ID NO: 5), the bases at positions 1-5 of the 5' end of the 5'UTR sequence were adjusted by point mutation to obtain the full-length saRNA template. The full-length saRNA template was linearized with MluI. The reaction system was as follows: 10 μg saRNA vector, 5 μL 10×cutsmart buffer, 1 μL NotI-HF enzyme, and ddH2O to make up to 50 μL. The mixture was thoroughly mixed and reacted at 37℃ for more than 12 h. 1 μL of the reaction product was taken for agarose gel electrophoresis to confirm that the plasmid was cut. The enzyme digestion product was purified with DNA purification magnetic beads to obtain the DNA sequence template. (2) Preparation of 5' end rearranged saRNA molecules by in vitro transcription without the addition of capping analogs: The DNA sequence template was used in the in vitro transcription system shown in Table 1. Transcription was performed at 37°C for 3 h. Then, 2 L of DNase I was added to the reaction system, and the reaction was continued at 37°C for 20 min. LiCl solution was then added until the final concentration of LiCl was 2.5 M. The mixture was placed on ice for 30 min or at -20°C for 30 min to precipitate. The mixture was then centrifuged at 4°C and ≥12000 g for 15 min. The supernatant was discarded, and the precipitate was washed with pre-cooled 75% ethanol aqueous solution. The supernatant was discarded after centrifugation. The tube cap was opened, and the precipitate was dried at room temperature for several minutes to obtain the 5' end sequence rearranged saRNA molecule. The precipitate was dissolved in DEPC-treated water or RNase-free water, aliquoted, and stored at -70°C.
[0066] Table 1 In vitro transcription system
[0067] Four 5' end sequence rearranged RNA molecules were prepared according to the method in Example 1. The nucleotide sequences of their 5' UTRs are shown in SEQ ID NO: 1-4, and are respectively designated as optimization groups 1-4.
[0068] Furthermore, the target saRNA was prepared using the template plasmid of the target saRNA (the nucleotide sequence of the 5'UTR of the target saRNA is shown in SEQ ID NO: 5) without point mutation treatment, and was denoted as the original sequence group.
[0069] The production results for each group were statistically analyzed separately as follows: Figure 1 As shown. Figure 1The graph shows the yield of saRNA prepared by in vitro transcription without the addition of capping analogs.
[0070] Depend on Figure 1 It can be seen that: in the original sequence group, the target saRNA sequence was not mutated and had the lowest in vitro transcription yield without the addition of a capping analog; while in optimized groups 1-4, the bases at positions 1-5 of the 5' end of the 5'UTR sequence were adjusted by point mutation, and the in vitro transcription yield was much higher than that of the original sequence without the addition of a capping analog, with optimized group 3 having the highest yield.
[0071] Example 2
[0072] The 5' end sequence rearranged saRNA molecules were prepared five times according to the method in Example 1, and the nucleotide sequence of their 5' UTR is shown in SEQ ID NO: 3. The yield of the obtained 5' end sequence rearranged saRNA molecules was detected, and it was found that the yields of the 5' end sequence rearranged saRNA molecules obtained in the five times were 146, 148, 151, 147 and 155 mg respectively. The yields of the five times were similar, indicating that the present invention adjusts the bases at positions 1-5 of the 5' end of the 5' UTR sequence. Under the condition of not adding capping analogues, a stable product with good batch consistency can be obtained by in vitro transcription.
[0073] Example 3
[0074] A method for co-transcriptional capping to produce target saRNA includes the following steps: (1) Constructing a DNA sequence template: Take the same target saRNA template plasmid as in Example 1 (the nucleotide sequence of the 5'UTR of the target saRNA is shown in SEQ ID NO: 5), linearize it with MluI, and the reaction system is: 10 μg saRNA vector, 5 μL 10×cutsmartbuffer, 1 μL NotI-HF enzyme, and ddH2O to make up to 50 μL. Mix thoroughly and react at 37℃ for more than 12 h. Take 1 μL of the reaction product for agarose gel electrophoresis to confirm that the plasmid is cut. Purify the enzyme digestion product with DNA purification magnetic beads to obtain the DNA sequence template. (2) Add a capping analogue and co-transcribe and cap the target saRNA to prepare it: The DNA sequence template was used in the in vitro transcription system shown in Table 2. Transcription was performed at 37°C for 3 hours. Then, 2 L of DNase I was added to the reaction system, and the reaction was continued at 37°C for 20 minutes. LiCl solution was then added until the final concentration of LiCl was 2.5 M. The mixture was placed on ice for 30 minutes or at -20°C for 30 minutes to precipitate. The mixture was then centrifuged at 4°C and ≥12000 g for 15 minutes. The supernatant was discarded, and the precipitate was washed with pre-cooled 75% ethanol aqueous solution. The supernatant was discarded after centrifugation. The tube cap was opened, and the precipitate was dried at room temperature for several minutes to obtain the target saRNA. The precipitate was dissolved in DEPC-treated water or RNase-free water, aliquoted, and stored at -70°C.
[0075] Table 2 In vitro transcription system
[0076] Nucleotide sequencing was performed on the 5' end sequence rearranged saRNA molecules of optimized groups 1-4 obtained in Example 1 and the target saRNA obtained in Example 3. The nucleotide sequences of the 5' end sequence rearranged saRNA molecules of optimized groups 1-4 are shown in SEQ ID NO: 6-9, and the nucleotide sequence of the target saRNA is shown in SEQ ID NO: 10.
[0077] Comparing SEQ ID NO: 6-10, it can be seen that: except for the nucleotide sequence of positions 1-5 at the 5' end of the 5' UTR, the sequences of other regions of the target saRNA and the 5' end sequence rearranged saRNA molecule are the same; and the 5' end sequence rearranged saRNA molecules of optimized groups 1-4 have the same molecular weight as the target saRNA; the 5' end sequence rearranged saRNA molecules of optimized groups 1-4 can be used as uncapped saRNA standards to detect the capping rate of co-transcriptional capping production of target saRNA.
[0078] Example 4
[0079] A method for detecting the capping rate of target RNA, comprising the following steps: LC-MS analysis was performed using a Waters UNIFI instrument. The chromatographic conditions were as follows: a mixed aqueous solution of 8.15 mM triethylamine and 200 mM hexafluoroisopropanol was used as mobile phase A; methanol was used as mobile phase B, and gradient elution was performed. The gradient elution program was as follows: Within 0.0-2.0 min, the volume fraction of mobile phase B is 5.0%; within 2.0-10.0 min, the volume fraction of mobile phase B gradually changes to 50.0%; within 10.0-10.5 min, the volume fraction of mobile phase B gradually changes to 90.0%; within 10.5-12.5 min, the volume fraction of mobile phase B is 90.0%; within 12.5-13.0 min, the volume fraction of mobile phase B gradually changes to 5.0%; within 13.0-15.0 min, the volume fraction of mobile phase B is 5.0%. The mass spectrometry conditions were: negative ion mode; scan range: 400-5000 m / z; capillary: 0.8 kV; sampling cone: 40 V; acquisition rate: 2 Hz.
[0080] Test sample 1 solution: The target saRNA prepared in Example 3 was used as test sample 1; Then, 80 μg of test sample 1 was mixed with 2 μL of biotinylated nucleic acid probe (0.1 mmol / L), and the mixture was placed in a PCR instrument and the annealing program was run: 95℃ for 2 min, and then the temperature was decreased from 70℃ to 16℃ at 0.1℃ per second to allow the probe to bind to the test sample; after annealing, RNase H, 10×RNase H Reaction Buffer and enzyme-free water were added, mixed well, and incubated at room temperature for 2 h to excise the 5'UTR fragment and obtain the enzyme digest product; Take 20 µL of Streptavidin MagPoly Beads, wash once with enzyme-free water, then mix the enzyme digestion product with the Streptavidin MagPoly Beads, incubate at room temperature for 1 h, place on a magnetic rack for 30 sec to allow the magnetic beads to aggregate, and remove the supernatant; wash three times each with 200 µL of capping rate test rinsing buffer and enzyme-free water; then add 50 µL of elution buffer to the magnetic beads, denature at 85 °C in a metal bath for 5 min; then place the centrifuge tube on a magnetic rack for adsorption, allow the magnetic beads to aggregate, collect the elution buffer, and obtain test solution 1.
[0081] When the solution of test sample 1 was injected for testing, no uncapped saRNA was detected in test sample 1.
[0082] To verify the accuracy of the above detection results, uncapped RNA standards (i.e., the optimized 3 groups of 5' end sequence rearranged saRNA molecules prepared in Example 1) and target saRNA prepared in Example 3 were mixed at a mass ratio of 20:80 to form test sample 2; test sample 2 solution was prepared according to the method for preparing test sample 1 solution.
[0083] The sample solution 2 was injected for testing, and the results are as follows: Figure 2 As shown. Figure 2 The image shows the LC-MS chromatogram of sample 2 in Example 4, where Base indicates uncapped and Base Cap1 indicates capped.
[0084] The above Figure 2 The middle base has 4 peaks, and the base cap1 also has 4 peaks, all of which are green.
[0085] Depend on Figure 2 It can be seen that the above chromatographic conditions can simultaneously detect 5' end sequence rearranged saRNA molecules and target saRNA, and the above detection results are accurate. The capping rate of test sample 2 calculated by the normalization method is 82.57%, which is close to the content of target saRNA in test sample 2 of 80%, indicating that the chromatographic conditions have high detection accuracy.
[0086] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A 5' end sequence rearranged RNA molecule, characterized in that, The 5' end sequence rearranged RNA molecule has a 5' UTR, and the nucleotide sequence of the 5' end of the 5' end from position 1 to 5 is: GN2N3N4N5; where N2 is G or A, N3 is G, A or T, N4 is G, A or T, and N5 is G or T; and at least one base in N2-N5 is G, and the bases in N3-N5 are not all G at the same time.
2. The 5' end sequence rearranged RNA molecule according to claim 1, characterized in that, The nucleotide sequence of the 5' end of the 5'UTR of the 5' end rearranged RNA molecule is one of the following: GATGG, GGATG, GGGAT, and GAGTG; preferably, the nucleotide sequence of the 5'UTR of the 5' end rearranged RNA molecule is as shown in SEQ ID NO: 1-4; preferably, the nucleotide sequence of the 5'UTR of the 5' end rearranged RNA molecule is as shown in SEQ ID NO: 3; preferably, the 5' end rearranged RNA molecule is one of the following: mRNA molecule and saRNA molecule.
3. A method for preparing a 5' end sequence rearranged RNA molecule as described in claim 1 or 2, characterized in that, Without the addition of capping analogues, the DNA sequence template of the 5' end sequence rearranged RNA molecule as described in claim 1 or 2 is transcribed using an in vitro transcription system to obtain the 5' end sequence rearranged RNA molecule.
4. The method for preparing the 5' end rearranged RNA molecule according to claim 3, characterized in that, The method includes the following steps: constructing a DNA sequence template for a 5' end sequence rearranged RNA molecule as described in claim 1 or 2; and performing a transcription reaction on the DNA sequence template using an in vitro transcription system without adding a capping analogue to obtain a 5' end sequence rearranged RNA molecule.
5. The use of a 5' end sequence rearranged RNA molecule as described in claim 1 or 2, or a 5' end sequence rearranged RNA molecule prepared according to the method described in claim 3 or 4, as a standard for uncapped RNA; preferably, its use as a standard for detecting the capping rate of target RNA; preferably, its use as a standard for detecting the capping rate of target RNA produced by co-transcriptional capping.
6. The application according to claim 5, characterized in that, The target RNA has a 5' cap structure; preferably, both the target RNA and the 5' end sequence rearranged RNA molecule have a 5' UTR, and the lengths of their 5' UTR sequences are the same; the nucleotide sequences starting from the 6th position at the 5' end of their 5' UTR sequences are the same; preferably, except for the 5' cap structure and the 5' UTR region, the sequences of other regions of the target RNA and the 5' end sequence rearranged RNA molecule are the same.
7. The application according to claim 5 or 6, characterized in that, The nucleotide sequence of the 5'UTR of the target RNA is shown in SEQ ID NO:
5.
8. A method for detecting the capping rate of target RNA, characterized in that, The steps include: using high performance liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry or capillary electrophoresis to detect the target RNA samples produced by co-transcriptional capping, and using uncapped RNA standards to qualitatively detect the capping rate; The uncapped RNA standard is the 5' end sequence rearranged RNA molecule as described in claim 1 or 2, or the 5' end sequence rearranged RNA molecule prepared according to the method described in claim 3 or 4.
9. The method for detecting the capping rate of target RNA according to claim 8, characterized in that, The target RNA has a 5' cap structure; preferably, both the target RNA and the 5' end sequence rearranged RNA molecule have a 5' UTR, and the lengths of their 5' UTR sequences are the same; the nucleotide sequences starting from the 6th position at the 5' end of their 5' UTR sequences are the same; preferably, except for the 5' cap structure and the 5' UTR region, the sequences of other regions of the target RNA and the 5' end sequence rearranged RNA molecule are the same; preferably, the nucleotide sequence of the 5' UTR of the target RNA is as shown in SEQ ID NO:
5.
10. A kit for detecting the capping rate of target RNA, characterized in that, include: Uncapped RNA standards, wherein the uncapped RNA standards are 5' end sequence rearranged RNA molecules as described in claim 1 or 2 or 5' end sequence rearranged RNA molecules prepared according to the method described in claim 3 or 4; preferably, the target RNA is target RNA produced by co-transcriptional capping; preferably, the target RNA has a 5' cap structure; preferably, both the target RNA and the 5' end sequence rearranged RNA molecules have a 5' UTR, and the length of their 5' UTR sequences is the same; the nucleotide sequences starting from the 6th position at the 5' end of their 5' UTR sequences are the same; preferably, except for the 5' cap structure and the 5' UTR region, the sequences of other regions of the target RNA and the 5' end sequence rearranged RNA molecules are the same; preferably, the nucleotide sequence of the 5' UTR of the target RNA is as shown in SEQ ID NO: 5.