RNA polymerase variant and application thereof in circRNA synthesis

Abstract

The present disclosure provides RNA polymerase variants having thermal stability and methods of making the same. In addition, the invention also provides a method for synthesizing circRNA in in-vitro transcription, and by utilizing the method provided by the invention, the ring formation rate of a circRNA product can be obviously improved.

Description

Field of the Invention

[0001] The present disclosure relates to the field of biotechnology, and in particular to RNA polymerase variants and applications thereof in preparing circRNA. Background of the Invention

[0003] circRNA is a covalently closed single-stranded circular molecule formed by connecting the 3' and 5' ends of exons and introns during RNA molecule splicing. Its transcription length is hundreds to thousands of nucleotides. Due to its special covalently closed circular structure, it has stronger exonuclease tolerance and is more stable in the cytoplasm. Since the discovery of circRNA in 1976 by studying plant viruses, circRNA has been found in mice, rats, fungi and humans. CircRNA was originally considered to be an abnormal byproduct produced during RNA splicing, but with the rapid development of high-throughput sequencing technology and bioinformatics, the role of circRNA in gene transcription, post-transcriptional modification and translation regulation has gradually been confirmed. In recent years, people have become increasingly interested in developing circRNA synthesis technology and exploring the application of synthetic circRNAs in disease treatment.

[0004] With the popularity of circRNA research in recent years, the underlying preparation process has gradually attracted the attention of researchers. How to achieve efficient and accurate circularization of circRNA is a key issue that needs to be broken through. Unlike traditional linear mRNA, circRNA has a closed ring structure and does not have a 5' cap and a 3' polyA tail. In the process of IVT preparing circRNA, the biggest difference from mRNA is that it needs to be circularized after synthesizing linear RNA. Currently, there are three main methods for artificially preparing circRNA: chemical synthesis, T4 DNA ligase ligation, and intron self-cleavage. Recently, Suzhou Coremade published an article on the pre-public platform bioRxiv, disclosing a new Clean-PIE circRNA circularization strategy, which realizes RNA circularization without the introduction of exogenous sequences (such as Figure 1 shown).

[0005] Similar to synthesizing mRNA, circRNA can also be synthesized by in vitro transcription using T7 RNA polymerase. The current common circulogenesis process is to carry out RNA synthesis and circulogenesis simultaneously, but the circulogenesis efficiency of wild-type T7 RNA polymerase is poor. When reacting at the optimal temperature of wild-type T7 RNA polymerase at 37°C, the one-step circulogenesis efficiency of wild-type T7 RNA polymerase is less than 40%, resulting in a large amount of RNA raw material waste. Therefore, it is of great practical significance to develop T7 RNA polymerase variants that are suitable for RNA circulogenesis. Summary of the invention

[0006] Based on this, the present disclosure provides a T7 RNA polymerase variant and its use in synthesizing circRNA in in vitro transcription, wherein the variant can increase the circularization rate of circRNA in in vitro transcription; in addition, a method for synthesizing circRNA in one step in in vitro transcription is provided.

[0007] In the first aspect, the present disclosure provides RNA polymerase variants, which are obtained by mutation based on the RNA polymerase (M0) shown in SEQ ID NO: 2. In some embodiments, the variant comprises a substitution of at least one amino acid at a site selected from V236, K329, K389, L446, G788 or C530 relative to SEQ ID NO: 2, wherein the substituted amino acid can be selected from A, G, R, H, L, Y, S, E, D, W, F, I, M, V, P, K, T, C, N, Q. In some embodiments, the substituted amino acid at the V236 site is A, E, F, L or P. In some embodiments, the substituted amino acid at the K329 site is I. In some embodiments, the substituted amino acid at the G788 site is A. In some embodiments, the substituted amino acid at the C530 site is S. In some embodiments, the substituted amino acid at the K389 site is A. In some embodiments, the substituted amino acid at the L446 site is F.

[0008] In some embodiments, the variant comprises at least one substitution selected from V236L, V236E, V236A, V236F, V236P, K329I, G788A, C530S, K389A / L446F, G788A / K389A / L446F, or G788A / K389A / C530S relative to SEQ ID NO:2.

[0009] In some embodiments, the amino acid sequence of the RNA polymerase variant is selected from SEQ ID NOs: 3-13.

[0010] In some embodiments, the variant has increased thermal stability and / or enzymatic activity at a temperature of at least 40°C, at least 41°C, at least 42°C, at least 43°C, at least 44°C, at least 45°C, at least 46°C, at least 47°C, at least 48°C, at least 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C or at least 55°C compared to the RNA polymerase whose amino acid sequence is set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In some embodiments, the variant still has at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or more of the initial enzyme activity after being stored at 45°C for a period of time (e.g., at least 20 min, at least 40 min, at least 60 min, at least 80 min, at least 100 min or at least 120 min).

[0012] In a second aspect, the present disclosure provides polynucleotides encoding RNA polymerase variants. Due to the degeneracy of codons or the preference of codons of host cells expressing polypeptides, the polynucleotide sequence is any polynucleotide sequence encoding a variant without changing the amino acid sequence. In some embodiments, the polynucleotide sequence encoding the RNA polymerase variant of the present disclosure can be selected from SEQ ID NO: 16-26.

[0013] In a third aspect, the present disclosure provides a method for preparing an RNA polymerase variant, comprising introducing a polynucleotide described in the present disclosure into a host cell to generate at least one RNA polymerase variant described in the present disclosure. In some embodiments, the host cell contains an expression vector carrying a polynucleotide encoding the RNA polymerase variant described in the present disclosure.

[0014] In a fourth aspect, the present disclosure provides a method for synthesizing circRNA, comprising contacting a DNA template with at least one RNA polymerase variant described in the present disclosure, and incubating them in an in vitro transcription reaction system to obtain circRNA.

[0015] In some embodiments, the in vitro transcription reaction system comprises one or more buffer components. In some embodiments, the buffer component can be selected from Tris-HCl, Hepes, citric acid or commercially available buffer components.

[0016] In some embodiments, the in vitro transcription reaction system contains nucleoside triphosphates. In some embodiments, the nucleoside triphosphates can be selected from modified or unmodified nucleoside triphosphates and their analogs. In some embodiments, the nucleoside triphosphates can be selected from unmodified ATP, GTP, CTP, UTP. In some embodiments, the nucleoside triphosphates can be selected from modified nucleoside triphosphates, including but not limited to m1A (N1-methyladenosine), m6A (N6-methyladenosine), m5C (5-methylcytidine), 5moU (5-methoxyuridine), ψ (pseudouridine), m1ψ (N1-methyl-pseudouridine), nucleoside triphosphates with markers (the markers can be biotin, fluorescent substances, digoxin, radioactive elements, etc.).

[0017] In some embodiments, the in vitro transcription reaction system further comprises RNase inhibitors, inorganic pyrophosphatase, and magnesium ions. In some embodiments, the in vitro transcription reaction system further comprises water (such as DEPC-water, RNase-free water, DNase-free water, sterile purified water, deionized water, distilled water, etc.).

[0018] In some embodiments, the incubation temperature of the in vitro transcription reaction can be selected from 30 to 60°C, preferably 40 to 55°C, and more preferably 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C.

[0019] In some embodiments, the circRNA product obtained by the method for synthesizing circRNA described in the present disclosure has a significantly increased circularization efficiency compared to using RNA polymerase (M0, SEQ ID NO: 2) or wild-type T7 RNA polymerase (SEQ ID NO: 1). In some embodiments, the residual rate of RNA precursor in the circRNA product does not exceed 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0020] In a fifth aspect, the present disclosure provides a composition comprising at least one RNA polymerase variant described in the present disclosure. In some embodiments, the composition further comprises at least one in vitro transcription system component, wherein the in vitro transcription component can be selected from one or more buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc. In some embodiments, the composition further comprises template DNA.

[0021] In a sixth aspect, the present disclosure provides a kit comprising at least one RNA polymerase variant described in the present disclosure. In some embodiments, the kit further comprises one or more in vitro transcription system components, wherein the in vitro transcription components can be selected from one or more buffer components, nucleoside triphosphates, RNase inhibitors, inorganic pyrophosphatase, magnesium ions, etc. In one embodiment, the in vitro transcription system components can be selected from commercially available RNA in vitro transcription reagents.

[0022] A seventh aspect is the use of the RNA polymerase variant described in the present disclosure in RNA synthesis.

[0023] Other implementations:

[0024] 1. An RNA polymerase variant, wherein the amino acid sequence of the variant comprises a substitution of at least one amino acid selected from the group consisting of V236, K329, K389, L446, G788 and C530 relative to SEQ ID NO: 2.

[0025] 2. The variant as described in item 1, wherein the amino acid substitution is selected from V236L, V236E, V236A, V236F, V236P, K329I, G788A, C530S, K389A / L446F, G788A / K389A / L446F or G788A / K389A / C530S.

[0026] 3. An RNA polymerase variant, wherein the amino acid sequence of the variant is selected from SEQ ID NO: 3-13.

[0027] 4. A polynucleotide, characterized in that the nucleotide encodes an RNA polymerase variant as described in any one of items 1-3.

[0028] 5. The polynucleotide according to item 4, wherein the nucleotide sequence is selected from SEQ ID NO: 16-26.

[0029] 6. A method for preparing an RNA polymerase variant, characterized in that it comprises introducing the polynucleotide described in item 4 or 5 into a host cell.

[0030] 7. A method for synthesizing circRNA, comprising contacting a DNA template with any variant described in items 1-3 and incubating the mixture in an in vitro transcription system; wherein the in vitro transcription system comprises a buffer component and nucleoside triphosphates.

[0031] 8. A composition, characterized in that it comprises:

[0032] 1) An RNA polymerase variant as described in any one of items 1 to 3; and

[0033] 2) template DNA; and / or

[0034] 3) One or more buffer components.

[0035] 9. A kit, characterized in that it comprises the RNA polymerase variant described in any one of items 1 to 3.

[0036] 10. Use of any RNA polymerase variant as described in items 1 to 3 in RNA synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the Clean-PIE looping strategy;

[0039] Figure 2 Schematic diagram of the construction of recombinant plasmid;

[0040] Figure 3 shows the enzyme activity of RNA polymerase variants at different temperatures: Figure 3A is the enzyme activity of M0-M6 polymerase at 45℃ and 48℃, Figure 3Bis the activity of M7, M8, M9, M10 and M11 polymerases at 37-50°C, Figure 3C The enzyme activities of RNA polymerase and its variants at different incubation times at 45°C;

[0041] Figure 4 To test the stability of RNA polymerase and its variants in in vitro transcription system;

[0042] Figure 5 shows the circularization of RNA polymerase and its variants in in vitro circRNA synthesis: Figure 5A is the catalytic ring formation of wild-type (WT) RNA polymerase at 48°C, Figure 5B The catalytic ring formation of M1 variant polymerase at 48°C is shown in Figure 2. Figure 5C The catalytic circularization of M7 variant polymerase at different temperatures;

[0043] The correspondence between the polymerase and its variant names and amino acids in the figure is shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] Beneficial Effects

[0053] The RNA polymerase variant disclosed in the present invention has improved thermal stability relative to RNA polymerase M0 (SEQ ID NO: 2) or wild-type RNA polymerase (SEQ ID NO: 1). Using it to synthesize circRNA in in vitro transcription can significantly improve the circularization efficiency of RNA products, effectively avoid the waste of RNA raw materials, and is of great significance for the preparation of circRNA. DETAILED DESCRIPTION

[0054] The technical solution of the present disclosure is further described below in conjunction with specific embodiments. However, the following embodiments are merely examples of the present disclosure and do not represent or limit the scope of protection of the present disclosure. The scope of protection of the present disclosure shall be subject to the claims. In the following embodiments, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0055] In the disclosed embodiments, enzyme activity is defined as the amount of enzyme required to generate 1 μmol of product or convert 1 μmol of substrate within 1 minute under specific reaction conditions.

[0056] Example 1 Preparation of RNA polymerase variants and assay of their enzyme activity

[0057] (1) A His tag was added to the front end of the amino acid sequence of RNA polymerase and its variants (SEQ ID NOs: 1 to 13), and after DNA sequence synthesis, PCR amplification was performed (DNA sequence SEQ ID NOs: 14 to 26), and then the BseRI and HindIII restriction sites of the expression vector pQE-80L were introduced to obtain a recombinant expression vector, which was transformed into E. coli BL21 (Novagen Inc.), and cloned after antibiotic resistance plate coating screening; after successful recombination was verified, the obtained strain was activated overnight in LB medium at 37°C and added to fermentation liquid (LB medium), cultured to an OD600 value of 0.6 to 0.8, IPTG with a final concentration of 0.5 mol / L was added and continued to be cultured for 4 to 6 hours, and then the strain was collected by centrifugation at 12000 rpm and 5°C, and the collected strain was washed with a 0.2 M PBS buffer with a pH value of 7.0 to obtain the bacterial cells; after ultrasonic disruption, affinity chromatography was performed to purify the RNA polymerase and its variants.

[0058] (2) The enzyme activity of the purified enzyme is detected by the following method:

[0059] Take two rows of eight strips and place them in an ice box. Assume that the activity of the purified enzyme is 500U / μL, and perform gradient dilution to an enzyme activity of 0.1U / μL, 0.2U / μL, 0.3U / μL, 0.4U / μL, 0.5U / μL, 0.6U / μL, and add a blank control of 0U / μL; dilute the T7 RNA polymerase standard (Vazyme, catalog number DD4101) to 0.1U / μL, 0.2U / μL, 0.3U / μL, 0.4U / μL, 0.5U / μL, 0.6U / μL according to its accurate enzyme activity, and add a blank control of 0U / μL. The enzyme activity determination system is shown in Table 2.

[0060] Table 2: Enzyme activity detection system

[0061]

[0062]

[0063] The above solution was mixed and centrifuged for 15 seconds, and reacted at 37℃-50℃ for 30 minutes (ABIPCR instrument); a fluorescent dye mixture (Vazyme, fluorescent dye component in product number EQ212) was prepared, and 180μL of the fluorescent dye mixture was added to the eight strips after the reaction and mixed, and 180μL was added to the black ELISA plate, and the ELISA instrument was used for detection, and the parameters were set to an excitation wavelength of 630nm and an emission wavelength of 680nm. The regression equation (slope is k) of the enzyme activity of the T7 RNA polymerase standard and enzyme variant samples was calculated using a graphing software.

[0064] Y(standard product)=k2X+b

[0065] Y(sample)=k1X+b

[0066] Actual enzyme activity: Actual enzyme activity of sample = k1 / k2×500U

[0067] Test results see Figure 3A , Figure 3B , among which at a reaction temperature of 45°C, the unit activity (enzyme specific activity) of the M1, M3, M4, M6, M7, M8, M9, M10 and M11 variants was higher than that of M0; at a reaction temperature of 48°C, the enzyme activity (enzyme specific activity) of the M1-M11 variants was higher than that of M0. At a reaction temperature above 45°C, the wild-type T7 RNA polymerase of the WT group was almost inactive.

[0068] (3) Dilute RNA polymerase and its variants to 100 U / μL using 10× RNA reaction buffer, incubate at 45°C for 20 min, 40 min, 60 min, 80 min, and 100 min, and perform enzyme activity detection according to step (2). The detection results are shown in Figure 3C , after incubation at 45°C for 80 min, the M7 polymerase variant still had at least 60% of the initial enzyme activity, while M0 had almost no residual enzyme activity.

[0069] Example 2 Stability of RNA polymerase and its variants in in vitro transcription system

[0070] (1) Prepare in vitro transcription reaction solution according to the reaction system in Table 3, react at 48°C for 1-7h, dilute the reaction solution 200 times and detect the fluorescence value; the specific method is as follows: prepare a fluorescent dye (Vazyme, fluorescent dye component in product number EQ212) mixed solution, add 180 μL of the fluorescent dye mixed solution to the eight strips after the reaction and mix well, draw 180 μL and add it to the black ELISA plate, and use an ELISA reader for detection.

[0071] Table 3: Reaction system ratio

[0072]

[0073] Test results see Figure 4 , T7 RNA polymerase variants M1 and M7 can stably exist in the in vitro transcription reaction system at 48°C.

[0074] Example 3 One-step in vitro transcription into circular

[0075] (1) Dilute the enzyme stock solution of the M1 variant with storage buffer to an enzyme activity of 300U / μL. Prepare the MIX solution (except T7 RNA polymerase) according to the reaction system (20μL) in Table 4 and put it into an EP tube; dispense the MIX solution into eight strips, and then take 1μL of the diluted T7 RNA polymerase into each strip containing the MIX solution, mix well, and centrifuge; place the eight strips on a PCR instrument, react at 48℃ for 1.5h, add 36μL of magnetic beads, mix well, and incubate at room temperature for 2-5min; place the mixed solution on a magnetic rack to purify circRNA (Vazyme, catalog number: N412), and transfer it to an RNase-free centrifuge tube after purification to obtain purified circRNA.

[0076] Table 4: Reaction system ratio

[0077]

[0078] The purified circRNA was tested for circularization rate using the Qsep400 ultra-micro nucleic acid protein analyzer R1 card (injection and separation 4KV, 20ntMarker). Figure 5A and Figure 5B , RNA in the WT group was almost not circularized, while the residual rate of RNA precursor in the M1 group was only 5%.

[0079] (2) The ability of the M7 variant to catalyze the synthesis of circRNA in vitro at reaction temperatures of 37°C, 40°C, 45°C, 48°C, and 50°C was tested according to the steps in (1). The test results are shown in Figure 5C , As the temperature increased, the circulization rate of circRNA in the M7 group gradually increased. When the reaction temperature was raised to 45°C, the residual precursor rate (the ratio of the residual precursor to the total RNA product) was only 7.5%. When the temperature was further raised to 48°C, the residual precursor rate dropped to 5.2%.

[0080] The above description is only a preferred embodiment of the present invention and does not limit the technical solution of the present invention in any form. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An RNA polymerase variant, characterized in that The amino acid sequence of the variant comprises, relative to SEQ ID NO: 2, substitution of at least one amino acid at a position selected from V236, K329, K389, L446, C530 or G788.

2. The variant of claim 1, wherein the amino acid substitution is selected from V236L, V236E, V236A, V236F, V236P, K329I, C530S, G788A, K389A / L446F, G788A / K389A / L446F or G788A / K389A / C530S.

3. An RNA polymerase variant, characterized in that The amino acid sequence of the variant is selected from SEQ ID NOs: 3-13.

4. A polynucleotide, characterized in that The polynucleotide encodes the RNA polymerase variant according to any one of claims 1-3. The polynucleotide according to claim 4 , wherein the sequence of the nucleotide is selected from SEQ ID NO: 16-26.

6. A method for preparing an RNA polymerase variant, characterized in that: The method comprises introducing the polynucleotide according to claim 4 or 5 into a host cell.

7. A method for synthesizing circRNA, characterized in that: The method comprises contacting a DNA template with the variant according to any one of claims 1 to 3, and incubating the mixture in an in vitro transcription system; wherein the in vitro transcription system comprises a buffer component and nucleoside triphosphates.

8. A composition, characterized in that Include: 1) The RNA polymerase variant according to any one of claims 1 to 3; and 2) template DNA; and / or 3) One or more buffer components.

9. A kit, characterized in that Comprising the RNA polymerase variant according to any one of claims 1 to 3.

10. Use of any RNA polymerase variant according to claim 1 in RNA synthesis.

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