T7-RNA polymerase mutant with high efficiency and low byproduct

Abstract

The invention provides a phage T7RNA (Ribonucleic Acid) polymerase (T7RNAP) mutant with high performance. The phage T7RNA polymerase (T7RNAP) mutant has mutation at a 744th amino acid residue corresponding to a wild type T7RNA polymerase; further, the T7RNAP mutant comprises an amino acid residue mutation occurring at at least one site selected from the following groups: a 62 site, a 68 site, a 72 site, a 152 site, a 173 site, a 535 site, a 538 site, a 539 site, a 633 site, a 723 site, a 767 site, a 881 site and a 883 site. When the T7RNAP mutant is applied to an in-vitro transcription (IVT) reaction, the yield of a target product in the IVT reaction can be remarkably increased, and / or the formation of a double-stranded RNA (dsRNA) byproduct in the IVT reaction can be reduced, so that the T7RNAP mutant can provide selection and adaptive industrial application for the IVT reaction of mRNA, CircRNA or saRNA and other drug molecules.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a T7-RNA polymerase mutant, which has high efficiency in an in vitro transcription reaction and effectively reduces the generation of by-products. Background Art

[0002] Bacteriophage T7 RNA polymerase (T7 RNAP) is a DNA-templated RNA polymerase. In in vitro transcription (IVT), T7 RNAP specifically recognizes the double-stranded T7 promoter sequence and, using one strand of linear double-stranded DNA as a template, catalyzes mRNA synthesis from the 5' to the 3' direction. Transcriptional byproducts are generated during the three main steps of transcription initiation, elongation, and termination. T7 RNAP is a single-subunit enzyme composed of 883 amino acids (uniprot sequence number: P00573), including an N-terminal domain (1-266AA) and a C-terminal domain / polymerase domain (267-883AA). The N-terminal domain binds to the T7 promoter sequence to form a transcription initiation complex (IC). During this stage, due to IC instability, abortive initiation products of 2-10 bases in length are formed [1,2]. When the transcribed bases exceed 10 bases, the N-terminal domain releases the promoter, and T7 RNAP forms a more stable transcription extension complex (EC) with the mRNA and DNA template. The C-terminal polymerase domain of T7 RNAP has a high structural homology with the DNA polymerase of the pol I family. The two aspartic acid residues (D537, D812) in the polymerase domain bind to the cofactor Mg2+, and the two Z, Y helix structures play an important role in substrate binding and transcription elongation [2]. Transcription termination occurs at a specific signal sequence or at the end of the linearized DNA template, generating a full-length RNA (run-off) product. However, there are two types of byproducts in the IVT reaction that cause mRNA heterogeneity. One type is the RNA 3' end extension byproduct. The extra sequence at the 3' end of the RNA does not come from the DNA template. It may be produced by the RNA itself after folding back and pairing or by the failure to terminate transcription in time [3,4,5]. Another type of byproduct is double-stranded RNA (dsRNA). T7 RNAP may be independent of the T7 promoter sequence and transcribe to form an RNA antisense strand, which pairs with the target mRNA product to form dsRNA. When the linear DNA template contains a 3' overhang sequence, promoter-independent transcription is likely to occur. However, there are also reports that antisense RNA and dsRNA were detected in IVT products of a 512-base fully complementary double-stranded DNA template sequence [6,7,8]. dsRNA has been shown to be the main factor triggering the body's immune response. As a therapeutic mRNA, it is necessary to avoid the production of dsRNA from the source, or to remove dsRNA by separating and purifying the product [9].

[0003] Circular RNA (circRNA) is a covalently closed RNA molecule that is widely present in eukaryotes. Artificially prepared circRNAs are typically obtained through an IVT reaction to obtain linear precursor RNA (pre-mRNA), which is then circularized by a ligase or ribozyme. The formation of circRNA by ribozyme circularization is an example of ribozyme circularization, where pre-mRNA self-cleaves through group I and group II introns

[10] . Byproducts of the IVT reaction can adversely affect the circularization efficiency of the pre-mRNA.

[0004] mRNA and circRNA have broad application prospects as vaccines and therapeutic drugs. The main issues of concern in industrial production include dsRNA byproducts in the IVT product mRNA that can trigger an immune response; the circularization efficiency of circRNA, etc. Patents and literature have reported some T7 RNAP mutants with improved performance. For example, Chinese patent CN 102177236 B introduces at least one amino acid mutation at Q786, K179, and V685 in the amino acid sequence of T7 RNAP, and the thermal stability and / or specific activity of the mutant are improved. The literature reports that the IVT reaction product mRNA of the (G47A+884G) T7 RNAP mutant does not contain byproducts that can trigger an immune response

[11] . At present, there are no reports in the literature on T7 RNAP mutants that are beneficial to the circularization efficiency of circRNA. In industrial applications, IVT reactions still require high-performance, low-byproduct T7-RNAP mutants.

[0005] In addition, for emerging self-amplifying RNA (saRNA), since linear DNA templates are usually 8-12k bases, high-performance, matching T7 RNAP mutants are also required for IVT transcription of long sequence templates such as saRNA. Summary of the Invention

[0006] The object of the present invention is to provide a bacteriophage T7 RNA polymerase (T7 RNAP) mutant with high efficiency and low by-products.

[0007] In a first aspect of the present invention, there is provided an isolated bacteriophage T7 RNA polymerase (T7 RNAP) mutant, wherein the T7 RNAP mutant has an amino acid residue mutation at position 744 corresponding to that of the wild-type T7 RNA polymerase and comprises an amino acid residue mutation at at least one position selected from the group consisting of:

[0008] amino acid residues corresponding to positions 62, 68, 72, 152, 173, 535, 538, 539, 633, 723, 767, 881, and 883 of wild-type T7 RNA polymerase;

[0009] Optionally, the T7 RNAP mutant further comprises an amino acid residue mutation at at least one position selected from the group consisting of:

[0010] corresponding to amino acid residues 43 and 880 of wild-type T7 RNA polymerase;

[0011] The amino acid numbering is based on the wild-type bacteriophage T7 RNA polymerase sequence shown in SEQ ID NO: 1.

[0012] In another preferred embodiment, the T7 RNAP mutant has an amino acid residue mutation at position 744 corresponding to the wild-type T7 RNA polymerase, and comprises amino acid residue mutations at at least two positions selected from the group consisting of:

[0013] corresponding to amino acid residues 62, 68, 72, 152, 173, 535, 538, 539, 633, 723, 767, 881, and 883 of wild-type T7 RNA polymerase; and, wherein one of the positions is position 723;

[0014] Optionally, the T7 RNAP mutant further comprises an amino acid residue mutation at at least one position selected from the group consisting of:

[0015] Corresponding to amino acid residues 43 and 880 of wild-type T7 RNA polymerase.

[0016] In another preferred embodiment, the T7 RNAP mutant has a Q744R mutation corresponding to the wild-type T7 RNAP.

[0017] In another preferred embodiment, the T7 RNAP mutant corresponds to the wild-type T7 RNAP with a Q744R mutation and comprises at least one amino acid residue mutation selected from the group consisting of:

[0018] G62S, A68K or A68R, P72K, G152T, R173C, A535G, G538A, S539T, S633P, C723S, S767K, A881F, A883S, or A883T.

[0019] In another preferred embodiment, the T7 RNAP mutant corresponds to the wild-type T7 RNAP with a Q744R mutation and comprises at least two amino acid residue mutations selected from the group consisting of:

[0020] G62S, A68K or A68R, P72K, G152T, R173C, A535G, G538A, S539T, S633P, C723S, S767K, A881F, A883S or A883T; and, one of the amino acid residues is mutated to C723S.

[0021] In another preferred embodiment, the T7 RNAP mutant corresponds to the wild-type T7 RNAP with a Q744R mutation and comprises at least two amino acid residue mutations selected from the group consisting of:

[0022] G62, A68K or A68R, P72K, G152T, R173C, C723S, S767K; and, one of the amino acid residues is mutated to C723S.

[0023] In another preferred embodiment, the T7 RNAP mutant corresponds to the wild-type T7 RNAP with a Q744R mutation and comprises at least two amino acid residue mutations selected from the group consisting of:

[0024] G62S, P72K, G152T, C723S, A535G, G538A, S539T, S663P, A881F, A883S or A883T; and, one of the amino acid residues is mutated to C723S.

[0025] In another preferred embodiment, the T7 RNAP mutant further comprises at least one amino acid residue mutation selected from the group consisting of S43Y and F880Y.

[0026] In another preferred embodiment, the T7 RNAP mutant has G152T, C723S and Q744R mutations corresponding to the wild-type T7 RNAP (G152T / C723S / Q744R T7 RNAP mutant).

[0027] In another preferred embodiment, the amino acid sequence of the T7 RNAP mutant (G152T / C723S / Q744R T7 RNAP mutant) is shown in SEQ ID NO: 2.

[0028] In another preferred embodiment, the T7 RNAP mutant corresponds to the wild-type T7 RNAP and has P72K, C723S and Q744R mutations (P72K / C723S / Q744R T7 RNAP mutant).

[0029] In another preferred embodiment, the amino acid sequence of the T7 RNAP mutant (P72K / C723S / Q744R T7 RNAP mutant) is shown in SEQ ID NO:13.

[0030] In another preferred embodiment, the T7 RNAP mutant is used for in vitro transcription (IVT) reaction, and its performance in the IVT reaction is better than that of wild-type T7 RNAP, and the performance includes:

[0031] (a) increasing the yield of the target product (i.e., single-stranded RNA) in an IVT reaction; and / or

[0032] (b) Reduce the formation of by-products in IVT reactions, including double-stranded RNA (dsRNA).

[0033] In another preferred embodiment, when used in an IVT reaction to prepare circular RNA (CircRNA), the T7 RNAP mutant significantly improves the cyclization efficiency of the precursor mRNA (pre-mRNA) reaction product prepared by the IVT reaction in the subsequent cyclization reaction compared to the wild-type T7 RNAP.

[0034] In another preferred embodiment, when used for preparing an IVT reaction for self-amplifying RNA (saRNA), the T7 RNAP mutant increases the yield of the target product (ie, long-chain saRNA) in the IVT reaction relative to the wild-type T7 RNAP.

[0035] The second aspect of the present invention provides an isolated polynucleotide encoding the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to the first aspect of the present invention.

[0036] In another preferred embodiment, the polynucleotide encodes the G152T / C723S / Q744R T7 RNAP mutant with amino acids as shown in SEQ ID NO: 2.

[0037] In another preferred embodiment, the polynucleotide comprises the sequence shown in SEQ ID NO: 3.

[0038] In another preferred embodiment, the polynucleotide encodes a P72K / C723S / Q744R T7 RNAP mutant whose amino acids are as shown in SEQ ID NO:13.

[0039] In another preferred example, the polynucleotide comprises the sequence shown in SEQ ID NO: 14.

[0040] The third aspect of the present invention provides a vector comprising the polynucleotide according to the second aspect of the present invention.

[0041] In another preferred embodiment, the vector is an expression vector.

[0042] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0043] The fourth aspect of the present invention provides a genetically engineered host cell, which contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0044] In another preferred embodiment, the host cells include prokaryotic cells and eukaryotic cells.

[0045] In another preferred embodiment, the host cell is a prokaryotic cell, preferably Escherichia coli.

[0046] A fifth aspect of the present invention provides a method for preparing a bacteriophage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention, comprising the following steps:

[0047] (1) introducing the polynucleotide according to the second aspect of the present invention into a host cell to obtain a genetically engineered host cell;

[0048] (2) culturing the genetically engineered host cell under conditions suitable for protein expression, thereby obtaining a culture containing the bacteriophage T7 RNA polymerase (T7 RNAP) mutant;

[0049] (3) isolating and / or purifying the bacteriophage T7 RNA polymerase (T7 RNAP) mutant from the culture species.

[0050] The sixth aspect of the present invention provides a use of the bacteriophage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention for in vitro transcription reaction (IVT), or for preparing a reagent or kit for in vitro transcription (IVT) reaction.

[0051] In a seventh aspect, the present invention provides a reagent for in vitro transcription (IVT) reaction, wherein the reagent comprises the bacteriophage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention.

[0052] The eighth aspect of the present invention provides a kit, which comprises the reagent as described in the seventh aspect of the present invention.

[0053] In another preferred embodiment, the kit is used for in vitro transcription (IVT) reaction, which further comprises other reagents required for in vitro transcription (IVT) reaction, including: DNA template, NTPs, in vitro transcription reaction buffer, and optionally RNase inhibitor.

[0054] In another preferred embodiment, the kit is used to prepare circular RNA (CircRNA), which further comprises reagents required for in vitro cyclization reaction.

[0055] In another preferred embodiment, the kit is used to prepare self-amplifying RNA (saRNA).

[0056] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to produce mRNA, circRNA or saRNA.

[0057] In a ninth aspect, the present invention provides a method for performing an in vitro transcription (IVT) reaction, comprising the step of performing a reaction using the bacteriophage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention.

[0058] In another preferred embodiment, the method has one or more advantages selected from the following group relative to the method using wild-type bacteriophage T7 RNA polymerase:

[0059] (a) Increase the yield of the target product (i.e., single-stranded RNA) in the IVT reaction;

[0060] (b) Reduce the formation of by-products in IVT reactions, including double-stranded RNA (dsRNA).

[0061] In another preferred embodiment, when the purpose of the method is to prepare circular RNA (CircRNA), the target product in the IVT reaction is the precursor mRNA (pre-mRNA) of CircRNA; and the method can significantly improve the cyclization efficiency of the reaction product containing pre-mRNA prepared by the IVT reaction in the subsequent cyclization reaction.

[0062] In another preferred embodiment, when the purpose of the method is to prepare self-amplifying RNA (saRNA), the target product in the IVT reaction is long-chain saRNA.

[0063] In a tenth aspect, the present invention provides a method for preparing circular RNA (circRNA), comprising the following steps:

[0064] (i) performing an in vitro transcription (IVT) reaction using the bacteriophage T7 RNA polymerase (T7 RNAP) mutant described in the first aspect of the present invention to prepare a reaction product comprising a precursor mRNA (pre-mRNA);

[0065] (ii) performing an in vitro cyclization reaction on the reaction product containing the pre-mRNA obtained in step (i), thereby obtaining CircRNA.

[0066] In another preferred embodiment, the method has the following advantages over the method using wild-type bacteriophage T7 RNA polymerase: the cyclization efficiency of the precursor mRNA (pre-mRNA) reaction product prepared by the IVT reaction in the cyclization reaction is significantly improved.

[0067] In an eleventh aspect of the present invention, a method for preparing self-amplifying RNA (saRNA) is provided, comprising the following steps:

[0068] The bacteriophage T7 RNA polymerase (T7 RNAP) mutant described in the first aspect of the present invention is used to perform an in vitro transcription (IVT) reaction to obtain the target product, a long-chain saRNA.

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shown, where A shows the polyacrylamide gel electrophoresis (SDS-page) and quantitative calibration coefficients of some T7 RNAP mutants, WT and competitors; B and C show the yield and dsRNA by-product content of some T7 RNAP mutants, WT and competitors used in IVT reactions, respectively.

[0071] Figure 2 The figure shows the IVT reaction with UTP, modified nucleotides N1-Me-pUTP, and Pseudo-UTP (Ψ). Equal volumes of purified IVT products were loaded onto the sample, and the 6% TBE electrophoresis gel was stained with acridine orange to qualitatively evaluate the amount of dsRNA and single-stranded mRNA in the IVT reaction products of different T7RNAP mutants.

[0072] Figure 3 The figures show: A shows the yields of two T7 RNAP mutants, KT-p3 and KT-p4x, at two NTP concentrations of 5 mM and 10 mM, two temperature conditions of 37°C and 39°C, and seven IVT reaction time conditions of 1, 1.5, 3, 5, 8, 24, and 26 hr; B shows the yields of two T7 RNAP mutants, KT-p3 and KT-p4x, at a 3-hour IVT reaction at a concentration range of 5 mM-12.5 mM NTP, and two temperature conditions of 37°C and 39°C; C shows the hydrolysis reaction using MDA5-ΔN ATPase, with equal masses of purified IVT products added to the reaction system, to compare the amount of dsRNA in the IVT products under different T7 RNAP enzyme mutants and reaction conditions.

[0073] Figure 4The results of testing T7 RNAP mutants suitable for CleanCap co-transcriptional capping are shown. A shows the mRNA yield in traditional IVT reactions using WT and T7 RNAP mutants with the DNA template set forth in SEQ ID NO:5; B shows the mRNA yield in IVT reactions using WT and T7 RNAP mutants with the co-transcriptional capped DNA template set forth in SEQ ID NO:9; and C shows the amount of dsRNA in the products of IVT reactions using WT and T7 RNAP mutants with the co-transcriptional capped DNA template set forth in SEQ ID NO:9.

[0074] Figure 5 It shows that when T7 promoters are present at both ends of the DNA template, pre-mRNA forms complementary double-stranded dsRNA, and the ribozyme cannot circularize to form circRNA

[0075] Figure 6 This image shows the use of a 4% polyacrylamide-urea gel to characterize in vitro synthesized circRNA products. A two-step IVT group I intron cyclization reaction was performed in the presence and absence of GTP to produce varying amounts of cirRNA and linear RNA, allowing for the identification of the apparent positions of the various RNA components on the gel.

[0076] Figure 7 The results show that the production of circRNA and the content of linear RNA were qualitatively observed on urea gel after the IVT two-step circularization reaction of WT and different T7 RNAP enzyme mutants.

[0077] Figure 8 Figure 2 shows Northern blot analysis of circRNA synthesis products in vitro. Figure 2 shows the IVT two-step group I intron cyclization reaction. (-) indicates completion of only step one, and (+) indicates completion of both steps. A and B show probe primers designed at the 3'-intron and 5'-intron, respectively, resulting in Northern blot detection of only linear RNA. C shows probe primers designed at the EGFP sequence, resulting in Northern blot detection of both linear RNA and circRNA. D shows probe primers designed at the circularization adapter sequence, resulting in Northern blot detection of only circRNA. Figure 2 shows a 4% polyacrylamide urea gel. Based on the Northern blot results in Figures A, B, C, and D, the components of the circRNA synthesis products in vitro can be annotated on Figure 2.

[0078] Figure 9The results of the IVT two-step cyclization reaction of WT, preferred T7 RNAP mutants, and three competitors are shown. A shows a urea gel image of the IVT two-step cyclization reaction products of WT, preferred T7 RNAP mutants, and three competitors; B shows the quantitative comparison of dsRNA by MDA5 ATPase assay using equal masses of cyclized products; C shows the comparison of cell viability after transient transfection of equal masses of cyclized products into HEK293 mammalian cells; D shows the FACS fluorescence signal generated by the expression of EGFP protein encoded by the circRNA in HEK293 cells transfected with equal masses of cyclized products.

[0079] Figure 10 The distribution of product molecules of different lengths in the IVT reaction products obtained by using WT, a preferred T7 RNAP enzyme mutant, and three competitors using 8.5k bp long-chain DNA templates for IVT preparation of saRNA on the capillary electrophoresis gel is shown. DETAILED DESCRIPTION

[0080] The present invention introduces amino acid mutation points through modeling and rational design of the T7 RNAP protein structure, including 6 amino acid mutation sites in the N-terminal domain, including S43, G62, A68, P72, G152, and R173; there are 10 mutation sites in the polymerase domain, including A535, G538, S539, S633, C723, Q744, S767, F880, A881, and A883. The present invention adds one, two or more mutant amino acid combinations of S43Y, G62S, A68K or A68R, P72K, G152T, R173C, A535G, G538A, S539T, S633P, C723S, L752R, G753K, S767K, F880Y, A881F, A883S or A883T on the basis of the Q744R mutation point to obtain 28 T7 RNAP mutants (KT-2q, KT-2r, KT-2t, KT-h2, KT-J, KT-K, KT-L, KT-p3, KT-u0, KT-m0, KT-pr, KT-qt, KT-rt, KT-pt, KT-prt, KT-t2, KT-t3, KT-u2, KT-u4, KT-uc, KT-vt1, KT-pt1, KT-qf, KT-qu, KT-qu7, KT-ua, KT-p4x, KT-p4y) were experimentally verified to be different from wild-type T7 RNAP (WT T7). Compared with T7 RNAP, T7 RNAP has one or more advantages selected from the following group: (1) significantly reducing the production of dsRNA byproducts in in vitro transcription reactions, (2) increasing the mRNA yield produced by in vitro transcription reactions, (3) improving the circularization efficiency of in vitro prepared circRNAs, (4) improving the level of target protein translation and expression of in vitro prepared circRNAs, and (5) increasing the yield of in vitro prepared saRNAs. Among them, the specific T7 RNAP mutant is particularly suitable for in vitro co-transcription and capping with CleanCap AG. On this basis, the present invention was completed.

[0081] the term

[0082] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0083] Unless defined 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 this invention belongs.

[0084] As used herein, the terms "bacteriophage T7 RNA polymerase mutant", "bacteriophage T7 RNAP mutant", "T7 RNAP mutant", "mutant T7 RNAP", and "T7 RNAP mutant protein" are used interchangeably and refer to mutant proteins having at least two (one of which is Q744) or more amino acid substitutions, deletions, or insertions compared to the amino acid sequence of wild-type bacteriophage T7 RNA polymerase.

[0085] As used herein, the terms "wild-type bacteriophage T7 RNA polymerase" or "wild-type T7 RNAP" or "WT T7 RNAP" all include a protein having an amino acid sequence as shown in SEQ ID NO: 1, and its uniprot sequence number is P00573.

[0086] As used herein, the term "corresponding to" has the meaning commonly understood by persons of ordinary skill in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in another sequence after alignment for homology or sequence identity. Thus, for example, "corresponding to wild-type T7 RNAP" means that an amino acid sequence is aligned with the amino acid sequence of wild-type T7 RNAP to identify a site in the amino acid sequence that corresponds to wild-type T7 RNAP.

[0087] Mutants of the present invention

[0088] As used herein, the term "mutant of the present invention" refers to a bacteriophage T7 RNA polymerase mutant.

[0089] In the present invention, a preferred class of mutants is a T7 RNAP mutant having an amino acid residue mutation corresponding to the Q744 position of the wild-type T7 RNAP (amino acid sequence as shown in SEQ ID NO: 1), and comprising at least one of the G62, A68, P72, G152, R173, A535, G538, S539, C723, S767, A881 and A883 positions, and a combination of two or more (e.g., three, four, five, six, etc.) amino acid residue mutations; optionally, further comprising at least one amino acid residue mutation selected from positions S43 and F880. Such mutants have optimized performance relative to wild-type T7 RNAP, specifically, when used in an in vitro transcription (IVT) reaction, the mutants increase the yield of the target product (i.e., single-stranded RNA) in the IVT reaction and / or reduce the formation of byproducts in the IVT reaction, including double-stranded RNA (dsRNA); further, specific T7 RNAP mutants are suitable for IVT reactions containing T7 promoter-AG linear DNA templates, thereby producing higher yields of the target product and are suitable for cleancap AG co-transcriptional capping; further, in some application scenarios, in the circularization reaction for preparing circRNA after the IVT reaction, the circularization efficiency of the circularization reaction is improved, thereby increasing the protein translation expression of the circRNA; further, specific T7 RNAP mutants are adapted to IVT reactions with long linear DNA templates, thereby obtaining higher yields of saRNA.

[0090] More preferably, the mutant of the present invention is based on the Q744R mutation, and further introduces at least one amino acid residue mutation selected from the following group: G62S, A68K or A68R, P72K, G152T, R173C, A535G, G538A, S539T, C723S, S767K, A881F, A883S or A883T; optionally, it also contains at least one amino acid residue mutation selected from the following group: S43Y, F880Y; wherein the site is based on the wild-type phage T7 RNA polymerase sequence shown in SEQ ID NO: 1. In some specific embodiments, the mutant of the present invention, based on the Q744R mutation point, further comprises at least two or more (for example, three, four, five, six, etc.) amino acid residue mutations selected from the following group: G62S, A68K or A68R, P72K, G152T, R173C, A535G, G538A, S539T, C723S, S767K, A881F, A883S or A883T; optionally, further comprises at least one amino acid residue mutation selected from the following group: S43Y, F880Y.

[0091] In one specific embodiment, the mutant is a G152T / C723S / Q744R T7 RNAP mutant (referred to herein as the KT-2r mutant), comprising the amino acid sequence set forth in SEQ ID NO: 2. In another specific embodiment, the mutant is a P72K / C723S / Q744R T7 RNAP mutant (referred to herein as the KT-2q mutant), comprising the amino acid sequence set forth in SEQ ID NO: 13. The term also encompasses variant forms and derivative polypeptides of the amino acid sequence of the polypeptide (e.g., SEQ ID NO: 2 or 13) that have the same function as the polypeptide. These variations include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus.

[0092] As used herein, "isolated protein (or polypeptide)" means that the protein is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the protein using standard protein purification techniques. A substantially pure protein will produce a single major band on a non-reducing polyacrylamide gel. The purity of the protein can also be further analyzed using techniques such as amino acid sequencing.

[0093] The present invention also includes fragments, derivatives and analogs of the protein (or polypeptide). As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity of the polypeptide.

[0094] The polypeptide fragments, derivatives, or analogs of the present invention can be (i) polypeptides having a substitution group in one or more amino acid residues, or (ii) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a fusion protein / tag sequence used to purify the polypeptide). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0095] The amino or carboxyl termini of the proteins of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag may be used in the present invention. For example, the tag may be FLAG, HA, c-Myc, Poly-His, Poly-Arg, StrepII, etc. These tags can be used to purify the protein.

[0096] Polynucleotides, vectors and host cells of the present invention

[0097] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0098] Taking the G152T / C723S / Q744R T7 RNAP mutant as an example, the coding region sequence encoding the mature polypeptide can be identical to the coding region sequence shown in SEQ ID NO:3 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a protein having SEQ ID NO:2 but differing from the coding region sequence shown in SEQ ID NO:3. Polynucleotides encoding the mature polypeptide of SEQ ID NO:2 include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0099] The present invention also relates to variants of the aforementioned polynucleotides, which encode polypeptides having the same amino acid sequence as the present invention, or fragments, analogs, and derivatives of the polypeptides. Variants of these polynucleotides may be naturally occurring variants or non-naturally occurring variants, but do not substantially alter the function of the encoded polypeptide.

[0100] The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably purified to homogeneity. The full-length nucleotide sequence of the T7 RNAP mutant of the present invention, or fragments thereof, can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed herein, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences.

[0101] Once the relevant sequence is obtained, bioengineering methods can be used to produce recombinant proteins. This usually involves cloning the polynucleotide sequence encoding the recombinant protein into an expression vector, then transferring the expression vector into cells for expression, and then harvesting and purifying the recombinant protein.

[0102] Currently, DNA sequences encoding the proteins of the present invention (or their fragments or derivatives) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or vectors) known in the art, transferred into cells, and purified to obtain the protein.

[0103] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0104] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0105] Transforming host cells with recombinant DNA and recombinantly expressing proteins are conventional techniques well known to those skilled in the art. Recombinantly expressed proteins can be isolated and purified by conventional techniques well known to those skilled in the art, which will not be described in detail here.

[0106] Applications and Methods

[0107] The present invention also provides applications of the bacteriophage T7 RNA polymerase mutant of the present invention, especially applications in in vitro transcription (IVT) reactions.

[0108] In one aspect, the bacteriophage T7 RNA polymerase mutant of the present invention can be used as an RNA polymerase reagent in an in vitro transcription reaction, specifically recognizing a double-stranded T7 promoter sequence and catalyzing mRNA synthesis from the 5' to 3' direction using one strand of linear double-stranded DNA as a template. Therefore, the bacteriophage T7 RNA polymerase mutant of the present invention can be used as an RNA polymerase reagent and can be configured in a container together with other reagents required for the in vitro transcription reaction to form a commercial in vitro transcription reaction kit. Each reaction reagent in the kit can be provided in liquid form or in lyophilized form; each reaction reagent can be configured in several separate containers or configured in the same container as a reagent mixture; preferably, each reaction reagent is configured in several separate containers and provided separately.

[0109] Based on this, the present invention also provides an in vitro transcription method, which includes the step of using the bacteriophage T7 RNA polymerase mutant of the present invention to perform a reaction. Specifically, the method includes: mixing the reaction reagents, including a DNA template (its amount / concentration is sufficient to provide the required amount of RNA), the bacteriophage T7 RNA polymerase mutant of the present invention, NTPs, an in vitro transcription reaction buffer, and an RNase inhibitor to form a reaction mixture, and incubating the reaction mixture at about 37°C for 1 to 16 hours, for example, at 37°C for 3 hours. In some embodiments, the reaction mixture is incubated at about 37°C to about 40°C for about 1 to 16 hours.

[0110] In another aspect, the bacteriophage T7 RNA polymerase mutants of the present invention are highly suitable for in vitro transcription of circular RNA (circRNA). The precursor mRNA (pre-mRNA) obtained by the IVT reaction has a low content of dsRNA byproducts, which can be further used in cyclization reactions to improve cyclization efficiency. Therefore, the present invention provides a method for preparing circRNA, comprising the step of using the bacteriophage T7 RNA polymerase mutants of the present invention to perform an in vitro transcription reaction to produce pre-mRNA.

[0111] On the other hand, the bacteriophage T7 RNA polymerase mutant of the present invention is very suitable for IVT reaction of long linear DNA templates, and can obtain high yield of saRNA. Based on this, the present invention also provides a method for preparing saRNA, comprising the step of using the bacteriophage T7 RNA polymerase (T7 RNAP) mutant as described in the first aspect of the present invention to perform an in vitro transcription (IVT) reaction to obtain the target product, a long-chain saRNA.

[0112] Advantages of the present invention include:

[0113] 1. T7 RNAP mutants (Table 3) are 28 mutants with additional mutation points introduced based on Q744R (KT-2q, KT-2r, KT-2t, KT-h2, KT-J, KT-K, KT-L, KT-p3, KT-u0, KT-m0, KT-pr, KT-qt, KT-rt, KT-pt, KT-prt, KT-t2, KT-t3, KT-u2, KT-u4, Lower dsRNA levels were observed in most IVT reaction products compared to WT. KT-p4y, KT-vt1, KT-pt1, KT-qf, KT-qu, KT-qu7, KT-ua, KT-p4x, and KT-p4y were observed in most of the mutants (KT-uc, KT-vt1, KT-pt1, KT-qf, KT-qu, KT-qu7, KT-ua, KT-p4x, and KT-p4y). Among them, KT-p4y, KT-vt1, and KT-h2 had higher mRNA yields and lower dsRNA levels. KT-p4y also had higher mRNA product amounts and lower dsRNA byproducts than the WT T7 RNAP group. The IVT products of mutants such as KT-pt1, KT-qt, KT-p3, and KT-2r also had lower dsRNA byproducts. Different T7 RNAP mutants may exhibit better adaptability to specific DNA template sequences (not limited to the exemplary template sequences used in the examples of the present invention), template lengths, and IVT reaction systems.

[0114] 2. The T7 RNAP mutant of the present invention was compared with a T7 RNAP polymerase (C1) in the literature that can significantly reduce dsRNA byproducts. The mutant of the present invention showed advantages over C1 in the production of mRNA and circRNA.

[0115] 3. Compared with two competitive products (C2 and C3) with superior performance on the market, the T7 RNAP mutant of the present invention produced obvious dsRNA byproducts in the IVT reaction. Although no obvious advantage was observed in mRNA yield, the mutant of the present invention was able to significantly reduce the production of dsRNA byproducts in the IVT reaction compared with C2 and C3.

[0116] 4. When using the cleancap AG reagent for co-transcriptional mRNA capping, the T7 promoter-GG sequence on the DNA template needs to be changed to T7 promoter-AG. The mRNA yield in the IVT reaction is generally reduced. The KT-2q and KT-qf mutants have higher mRNA yields and lower dsRNA byproducts than the WT.

[0117] 5. Since double-stranded complementary dsRNA cannot undergo circularization reaction, T7 RNAP mutants with low dsRNA byproducts and high circularization efficiency, such as KT-uc, KT-pt, and KT-u0, are more suitable for the preparation of group I intron preRNA and circularization to obtain circRNA.

[0118] 6. The T7 RNAP mutant KT-uc has higher circRNA production and circularization efficiency, with a larger proportion of precursor RNA converted into circRNA, and the target protein translation efficiency is the highest after circRNA transfection into cells.

[0119] 7. The T7 RNAP mutants KT-uc and KT-u0 performed superiorly in IVT reactions with long-chain DNA templates. The Q-sepCE map revealed the distribution of RNAs of different molecular weights in the IVT product. The yield of the 8.5k-base-long target saRNA was significantly higher than that of WT, competitors, or other mutants.

[0120] 8. Matching a specific IVT DNA template sequence with a specific T7 RNAP mutant may yield optimal RNA transcription and protein expression. The 28 mutants listed herein, or further combinations of T7 RNAP mutants based on these, can provide industrially applicable T7 RNAP mutants that are selected and adapted for IVT reactions of specific mRNA, circRNA, or saRNA drug molecules.

[0121] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which no specific conditions are specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0122] Example 1: Preparation, quantitative calibration, and performance testing of T7 RNAP and its mutants in IVT reactions

[0123] 1.1 Preparation of T7 RNAP and its mutants

[0124] Taking the KT-2r mutant (SEQ ID NO: 2) as an example, the G152T / C723S / Q744R mutations were introduced into the amino acid sequence of the wild-type bacteriophage T7 RNA polymerase shown in SEQ ID NO: 1. The codon-optimized E. coli gene sequence (SEQ ID NO: 3) with an N-terminal his-tag was constructed downstream of the T5 promoter in the pQE80L (Kana+) vector. The correctly sequenced plasmid was transformed into the BL21 (DE3) expression strain, and the recombinant protein was expressed and purified according to the reference method

[12] .

[0125] Other mutants (KT-2q, KT-2t, KT-h2, KT-h3, KT-h4, KT-h5) with additional mutations (S43Y, G62S, A68K or A68R, P72K, G152T, R173C, A535G, G538A, S539T, S633P, C723S, L752R, G753K, S767K, F880Y, A881F, 1, 2, 3, 4, 5 or 6 of A883S or A883T) were introduced based on Q744R. KT-J, KT-K, KT-L, KT-p3, KT-u0, KT-m0, KT-pr, KT-qt, KT-rt, KT-pt, KT-prt, KT-t2, KT-t3, KT-u2, KT-u4, KT-uc, KT-vt1, KT-pt1, KT-qf, KT-qu, KT-qu7, KT-ua, KT-p4x, and KT-p4y were also obtained using the same expression and purification methods as described above. A total of 28 mutants, including KT-2r and KT-2q mutants, were generated.

[0126] Amino acid sequence of wild-type bacteriophage T7 RNA polymerase (uniprot sequence number: P00573) (SEQ ID NO: 1)

[0127] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0128] Amino acid sequence of the KT-2r mutant (SEQ ID NO:2):

[0129] MRGSHHHHHHGSNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFTRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGV QHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRSAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0130] Codon-optimized KT-2r mutant gene sequence (SEQ ID NO:3)

[0131]

[0132] Amino acid sequence of the KT-2q mutant (SEQ ID NO:13):

[0133] MRGSHHHHHHGSNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAKKLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKS PLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRSAVHWVTPDGFPVWQEYKKPIRTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA

[0134] KT-2q mutant gene sequence (SEQ ID NO: 14):

[0135]

[0136] 1.2 Quantitative calibration of mutant proteins

[0137] Before conducting the IVT experiment, 100 ng of all mutant proteins and competitor controls were subjected to SDS-page electrophoresis to calibrate the amount of protease. Figure 1 As shown in Figure A, some mutants prepared in Example 1 are shown. Wild-type phage T7 RNA polymerase (WT) and a competitor (C1) were quantitatively calibrated using the KT-m0 mutant as a benchmark and grayscale scanning was performed on a Biorad gel imaging analyzer to obtain the quantitative calibration coefficients shown in Table 1. This method was used to calibrate the enzyme amount in subsequent IVT experiments.

[0138] Table 1

[0139]

[0140] 1.3 IVT reaction performance test of mutants

[0141] The IVT reaction system is shown in Table 2. The quantitative calibration coefficients in Table 1 were used to calibrate the enzyme amount in each reaction system to be the same. 28 mutants, WT, and competitors were subjected to IVT reactions using the DNA template (SEQ ID NO: 5). After reacting at 37°C for 3 hr, 1 μL of DNAse I (40 U / μL) was added to digest the template at 37°C for 30 min. The product was purified by LiCl precipitation, and the mRNA product was resolubilized in 50 μL of RNase-free ddH2O. The RNA product concentration was determined using NanoDrop One (Thermo Fisher Scientific). Figure 1 Center B shows the RNA yields of some mutants, WT, and competitors.

[0142] Table 2 IVT transcription reaction system (20 μl)

[0143]

[0144] The MDA5-ΔN ATPase hydrolysis reaction was further used to assess the dsRNA byproduct content in the IVT product. MDA5 is a dsRNA receptor protein. The MDA5-ΔN protein (N-terminally truncated protein) can bind to dsRNA in vitro, stimulating the receptor ATPase activity, hydrolyzing ATP, and releasing phosphate groups. The released phosphate groups are detected using the Malachite Green (MG) Phosphate Assay. This assay uses a phosphate group to form a phosphomolybdic heteropolyacid association complex with ammonium molybdate under acidic conditions. This complex then reacts with malachite green to form a colorimetric complex. The absorbance intensity at a wavelength of 620 nm is measured, and the intensity is proportional to the amount of phosphate groups. This assay is used to assess the content of dsRNA byproducts.

[0145] The MDA5-ΔN protein preparation and ATPase assay were performed using the literature [8]. The Malachite Green (MG) Phosphate Assay working solution was prepared by mixing MG stock solution (13 mM malachite green oxalate dissolved in 3.6 M sulfuric acid), 7.5% ammonium molybdate aqueous solution, and 11% Tween 20 at a ratio of 1:0.25:0.02 (v:v:v), and then adding 4 times the volume of deionized water. The MG stock solution was stored in the dark, and the MG working solution was freshly prepared before use

[13] . The MDA5-ΔN ATPase hydrolysis reaction was carried out according to the following steps:

[0146] MDA5-ΔN protein (final concentration 0.1 mg / ml) and RNA from each IVT transcript (300 ng) were premixed in reaction buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, and 2 mM DTT). Equal amounts of RNA were used in each hydrolysis reaction (20 μl). The reaction was activated by the addition of 2.5 mM ATP and incubated at 37°C for 15 min. The reaction was terminated by the addition of 50 mM EDTA and placed on ice. Five μl of the reaction solution was added to 95 μl of MG working solution and color was developed at room temperature for 30 min. Absorbance was measured at 620 nm using a Multiskan microplate reader (Thermo Fisher). Figure 1 Center C shows the dsRNA byproduct content of some mutants, WT and competitors.

[0147] The RNA yields and dsRNA contents of T7 RNAP WT, 28 mutants, and C1, C2, and C3 competitor enzymes using the DNA template shown in SEQ ID NO: 5

[14] are summarized in Table 3. The mRNA yield of the C1 competitor enzyme is not high, and the dsRNA content is low. The mRNA yields of C2 and C3 are high, but the dsRNA content is relatively high. The T7 RNAP mutants KT-2r, KT-p3, and KT-t2 of this patent have low dsRNA content, which is higher than C1 but lower than WT, C2, and C3. The mutants KT-p4y, KT-vt 1, and KT-h2 have high yields, which are higher or equivalent to WT, C2, and C3, and the dsRNA content is significantly lower than WT, C2, and C3.

[0148] Table 3

[0149]

[0150]

[0151] Example 2: IVT reaction using UTP and modified nucleotides N1-Me-pUTP and Pseudo-UTP (Ψ) to test the content of dsRNA byproducts

[0152] In this example, T7p-512B (SEQ ID NO: 7) was used as a template, i.e., the 512B sequence containing the T7 promoter. The 512B DNA sequence was obtained from the literature [8]. UTP, modified nucleotides N1-Me-pUTP, and Pseudo-UTP (Ψ) were used for IVT reactions to test the dsRNA content in the IVT products of T7-RNAP WT, five mutant enzymes (KT-m0, KT-p3, KT-2q, KT-pt, KT-uc), and competitors C2 and C3. The reaction system experimental procedures were the same as in Example 1. The LiCl-precipitated IVT product was dissolved in 50 μl of RNase-free ddH₂O. 10 μl of the product was diluted 50-fold. 20 μl of the same diluted RNA was loaded onto a 6% polyacrylamide TBE gel. After electrophoresis, the gel was stained with 2.5 ng / ml acridine orange (Sigma) for 5 minutes, destained with deionized water for 10 minutes, and photographed using an iBright 1500 (Thermo Fisher) instrument. Acridine orange intercalates into dsRNA, resulting in green fluorescence detected at 530 nm. Acridine orange electrostatically binds to the phosphate groups of single-stranded mRNA, resulting in red fluorescence detected at 640 nm.

[0153] Figure 2 The TBE gel image in Figure A shows distinct dsRNA bands in the IVT reaction products of WT, C2, and C3. The highest amount of dsRNA byproduct was observed in the IVT reaction products using N1-Me-pUTP. The four mutant enzymes, KT-p3, KT-2q, KT-pt, and KT-uc, all showed low dsRNA levels in the UTP, modified nucleotide N1-Me-pUTP, and pseudo-UTP (Ψ) reaction systems. Figure 2 B is the quantitative detection of dsRNA by MDA5 ATPase assay. The performance of the five mutants in this example is consistent with that in Example 1.

[0154] Example 3: Effects of NTP dosage, reaction temperature and reaction time on yield in IVT reaction

[0155] In this example, two T7 RNAP mutants, KT-p3 and KT-p4x, were used, and the DNA template was SEQ ID NO: 5. The effects of different NTP amounts, IVT reaction temperatures (37° C., 39° C.), and reaction times on RNA yield were tested. Figure 3Center A shows that at 5mM and 10mM NTP dosages, at both 37°C and 39°C, a 3-hour reaction time achieves high yields. Extending the reaction time results in little or no increase in yield. With the same 3-hour reaction time and enzyme dosage, 10mM NTP yields higher yields than 5mM, and mutant KT-p4x exhibits higher yields than KT-p3.

[0156] T7 RNAP WT and mutant KT-p3 were further tested in the range of 5mM-12.5mM NTP dosage, reaction temperature (37℃, 39℃), and reaction time for 3 hours. The results showed that the mRNA yield of KT-p3 mutant enzyme was comparable to that of WT T7 RNAP at NTP dosages of 10mM and above ( Figure 3 The dsRNA content in the reaction products was also quantitatively determined using the MDA5 ATPase assay. The results showed that the amount of dsRNA byproducts in the IVT products of the KT-p3 mutant enzyme was significantly lower than that of the WT enzyme, and that the dsRNA content in the products produced at a reaction temperature of 39°C was lower than that at 37°C. Figure 3 Middle C).

[0157] Example 4: T7 RNAP mutants suitable for CleanCap co-transcriptional capping

[0158] The mRNA produced by the IVT reaction requires 5' capping to ensure the stability of the mRNA and its translation expression. The traditional T7 RNA polymerase reaction will initiate transcription with guanosine triphosphate to form 5'-triphosphate RNA. For example, the T7 promoter sequence (TAATACGACTCACTATA) in the DNA template shown in SEQ ID NO:5 is connected to GGG to initiate mRNA transcription. After the mRNA is obtained by the IVT reaction, it is capped in vitro with vaccinia capping enzyme. The new generation of CleanCap capping technology can use cap analogs in the IVT reaction to produce capped transcripts, which is called co-transcriptional capping. That is, the CleanCap trimer binds to the +1 and +2 nucleotides of the template through complementary base pairing and then incorporates the complementary NTP at the +3 position to initiate transcription downstream of the T7 promoter sequence. As shown in SEQ ID NO:8, the T7 promoter sequence is connected to AGG to initiate co-transcriptional capping

[15] .

[0159] In this example, a traditional IVT reaction was performed using the DNA template shown in SEQ ID NO: 5. Figure 4Center A shows the mRNA yields of T7RNAP WT and 11 mutants KT-m0, KT-p3, KT-2q, KT-u0, KT-uc, KT-pt, KT-qf, KT-qu, KT-qu7, KT-pt1, and KT-vt 1. When the DNA template shown in SEQ ID NO: 9 is used, the only difference between it and the DNA template shown in SEQ ID NO: 5 is that the T7 promoter sequence is connected to AGG to initiate transcription. Under the same IVT reaction conditions, Figure 4 Middle B shows that the yield of WT and mutants such as KT-pt1 and KT-vt1 decreased significantly, while KT-2q and KT-qf mutants had high yields; Figure 4 Center C shows low dsRNA content in the co-transcriptional capping products of KT-2q and KT-qf mutants. This indicates that KT-2q and KT-qf mutants are suitable for a co-transcriptional capping system, i.e., transcription from the DNA template shown in SEQ ID NO: 9, initiated by a T7 promoter sequence linked to an AGG sequence.

[0160] Example 5: Application of T7 RNAP mutants in group I intron self-cleavage and circularization to prepare circRNA

[0161] In this example, WT T7 RNAP and a linear DNA template (shown in SEQ ID NO: 10) were used to perform an IVT reaction to test the effect of dsRNA on the self-cleavage and circularization of group I introns. SEQ ID NO: 10 is from reference

[10] , and its main sequence components are 3'-intron-E2-EMCV IRES-Gaussia luciferase-E1-5'-intron. Introns and IRES sequences in pre-mRNA contain a large number of secondary structures. The IVT reaction system is shown in Table 2, where 10x buffer I was replaced with 10x buffer II (400mM Tris-HCl, pH 7.5, 100mM MgCl2, 20mM Spermidine, 100mM DTT). The reaction was incubated at 37°C for 3 hours, the DNA template was digested with DNase I, and the RNA was purified by LiCl precipitation. The RNA was dissolved in 50ul of RNase-free ddH2O to complete step one of the two-step circularization reaction. 5ul of RNA product was mixed with 14.5ul of RNase-free ddH2O, incubated at 70°C for 5 minutes, and then placed on ice for 3 minutes. Then, 0.5ul of GTP (100mM) and 5ul of 5x circularization buffer (250mM Tris-HCl, pH 7.5, 50mM MgCl2, 5mM DTT) were added and incubated at 55°C for 20 minutes to complete the second step of the two-step circularization reaction. In addition, by introducing a T7 promoter sequence into the 3' end of the PCR primer, a linear DNA template with T7 promoters at both ends was obtained. That is, both strands of the double-stranded DNA can be transcribed as template strands in the IVT reaction, and the resulting RNA can complement each other (i.e., dsRNA).

[0162] like Figure 5 As shown, two groups of IVT reactions were performed, one group was a normal circularization reaction template (SEQ ID NO: 10), and the other group was a linear DNA template with T7 promoters at both ends. Four different loading amounts of each reaction product were subjected to urea gel electrophoresis ( Figure 5 In the figure, from left to right, the sample loading amounts are 11 ng, 33 ng, 100 ng, and 300 ng, respectively. CircRNA formation can be observed in all four lanes of the normal circularization reaction products. However, in the reaction products of another group of linear DNA templates with T7 promoters at both ends, dsRNA is produced. No circRNA formation is observed in the four lanes, and no clear RNA bands are found. It is possible that the long-chain dsRNA cannot be completely denatured on the urea gel, resulting in uneven migration. Figure 5Experiments have shown that dsRNA cannot be cyclized by ribozymes. Therefore, obtaining high pre-mRNA yield and low dsRNA byproducts in the IVT reaction is beneficial to improving the cyclization efficiency and yield of circRNA.

[0163] During in vitro synthesis of circRNA, various linear RNA impurities are generated, such as uncircularized linear pre-mRNA, nicked RNA (i.e., self-cleaved introns without cyclization), and intron fragments. Distinguishing circRNA from these impurities on nucleic acid agarose gels is difficult. Invitrogen E-gel, a precast urea gel, offers better resolution, but precast gels are expensive. The experiments presented here utilize the following denaturing urea gel preparation, sample loading, and electrophoresis methods, developed by our team, to better characterize circRNA products.

[0164] 4% polyacrylamide-urea gels were prepared using the UreaGel system (National Diagnostics) reagents according to Table 4. Electrophoresis was performed using a Shanghai Tianneng VE-180 vertical electrophoresis tank with a gel size of 8.3 x 7.3 cm. Two gels were loaded into the tank, and 2X TBE buffer was used as the running buffer. Before loading, the gels were pre-run at 240V for 30 minutes. CircRNA samples were mixed with 2X Urea-PAGE loading buffer (1 ml 0.5 M EDTA, 9 ml formamide, 0.01% bromophenol blue, 0.01% xylene cyanol, 0.1% SDS), heated at 65°C for 3 minutes, and then placed on ice for 1 minute before loading. The sample volume should exceed half the gel well volume. After the pre-run, the sample wells were flushed with a syringe and the samples were immediately loaded. Electrophoresis was performed at 240V for 90 minutes. The urea gel was stained with safe green for 5 minutes, destained with deionized water for 10 minutes, and photographed using a Biorad gel camera.

[0165] Table 4. 4% polyacrylamide urea glue formula

[0166]

[0167] Using WT T7 RNAP and the DNA template shown in SEQ ID NO: 10, IVT group I intron self-cleavage and cyclization was performed. The product of step 1 in the two-step cyclization reaction was mainly linear RNA, such as Figure 6 Shown in lane 1. Figure 6Lane 2 shows that in step 2, the presence of GTP promotes the ribozyme circularization reaction, resulting in a decrease or disappearance of linear RNA, an increase in the amount of circRNA, and the generation of new RNA bands. Lane 2', in which GTP is not added to the circularization reaction system in step 2, exhibits poor ribozyme circularization, with no significant increase in circRNA observed. However, there is a slight decrease in linear RNA, and the generation of new RNA bands is observed.

[0168] Furthermore, T7RNAP WT and seven mutants KT-m0, KT-pt, KT-prt, KT-u0, KT-u2, KT-u4, and KT-uc were subjected to IVT group I intron self-cleavage and circularization using the DNA template shown in SEQ ID NO: 10. Figure 7 This is a urea gel image of the two-step cyclization products. It can be observed that WT, KT-m0, KT-pt, KT-u0, and KT-uc produce higher amounts of circRNA. WT also produces the most linear RNA impurities, and KT-m0 also has significantly more linear RNAs. The four mutants, KT-u0, KT-u2, KT-u4, and KT-uc, produce the least linear RNA impurities in their circRNA IVT products.

[0169] Furthermore, WT T7 RNAP was used to perform self-cleavage and circularization of the IVT group I intron using the DNA template of SEQ ID NO:11, which is similar to SEQ ID NO:10. The main components of this template are 3'-intron-E2-CVB3 IRES-EGFP-E1-5'-intron. Northern blot analysis was performed to detect the product components of step one (-) and the complete two-step reaction (+) of the IVT two-step group I intron circularization reaction. Figure 8 Figures A and B show that when probe primers were designed at the 3'-intron and 5'-intron, respectively, Northern blot detected only linear RNA. Figure C shows that when probe primers were designed at the EGFP sequence, Northern blot detected both linear RNA and circRNA. Figure D shows that when probe primers were designed at the circular adapter sequence, Northern blot detected only circRNA. This experiment detected two circRNA bands, but the nature and generation mechanism of the weaker circRNA that migrated faster on the urea gel remain unexplained. Figure E shows a 4% polyacrylamide-urea gel. Based on the Northern blot results in Figures A, B, C, and D, the components of the in vitro circRNA synthesis products can be annotated on Figure E.

[0170] Furthermore, T7RNAP WT, competitors C1, C2, C3 and six mutants KT-m0, KT-p3, KT-2q, KT-pt, KT-u0 and KT-uc were used to perform IVT group I intron self-cleavage and circularization with the DNA template shown in SEQ ID NO: 11. The circRNAs after in vitro transcription and purification were quantified by nanodrop, and equal amounts of products were electrophoresed. Figure 9 Figure A is a urea gel image of the two-step cyclization product. From the circRNA band intensity on the gel image, the circRNA content in the cyclization products of WT, competitors C2, C3, mutants KT-u0, and KT-uc is relatively high. Figure 9 Middle B shows the results of quantitative detection of dsRNA content in circRNA products by MDA5 ATPase assay. KT-pt, KT-u0, KT-uc and competitor C1 have the lowest dsRNA byproduct content, and WT TRNAP produces the highest amount of dsRNA.

[0171] Next, we tested the transfection expression of these circularized products. HEK293 cells in the logarithmic growth phase were counted using a Countstar instrument, centrifuged, and the cell density adjusted to 2.5E6 cells / ml. The cells were then inoculated into 24-well plates with 0.5ml per well. 1 μg of circRNA purified from WT, different T7 RNAP mutants, and competitors was transfected into the cells with Lipo3000. 24 hours after circRNA transfection, the cells were stained with trypan blue and then counted to obtain cell viability. Figure 9 Middle C shows that the cell viability of KT-u0 and KT-uc circularization products after transfection is higher than that of competitors and other mutants. The cell viability of the circularization product of WT enzyme after transfection is the lowest. EGFP fluorescence signal was then detected by flow cytometry. Untransfected HEK293 cells were used as negative control. The EGFP signal intensity was used to evaluate the expression of EGFP protein encoded by circRNA. Figure 9 Two peaks are observed in the spectrum in Figure D: the first peak represents cells without fluorescence signal, and the second peak represents cells expressing EGFP and showing fluorescence signal. The circRNA products of KT-u0, KT-uc, and KT-pt express stronger EGFP fluorescence signals than the competitor and WT.

[0172] These results indicate that mutants KT-u0, KT-uc, and KT-pt are suitable for in vitro transcription preparation of group I intron self-cleavage circular RNA.

[0173] Example 6: Application of T7 RNAP mutants in saRNA in vitro transcription

[0174] Self-amplifying RNA (saRNA) is an emerging technology for RNA drugs and vaccines. Its characteristic is that saRNA has the ability to self-replicate and amplify after entering the cell, achieving the same level of immune response, and its immune dose is significantly lower than that of traditional mRNA. saRNA is longer than traditional mRNA, reaching 9-12kb. This is because in addition to containing the same framework structure parts as traditional mRNA, such as 5'Cap, 5'UTR, 3'UTR and poly (A), saRNA also carries a sequence that can express RNA-dependent RNA polymerase (RdRP), exerting its self-amplification function.

[0175] The DNA template used to transcribe saRNA in this embodiment is 8.5 kb in length, as shown in SEQ ID NO: 12, and its composition includes 5'-UTR, nsP1-4, EGFP ORF (underlined part), 3'-UTR and 3' poly (A) tail. Except for the EGFP ORF sequence, the other element sequences are from the literature

[16] . The IVT reaction system for transcribing saRNA is shown in Table 5. T7 RNAP WT, competitor enzymes C1, C2, C3 and 6 mutants KT-m0, KT-p3, KT-2q, KT-uc, KT-pt, KT-u0 were used for IVT reaction. After purification, the IVT reaction product was analyzed by Q-sep1 (Guangding Biotechnology) capillary electrophoresis, using N3 high-sensitivity large fragment cartridge. The electrophoresis conditions are shown in Table 6, and the electronic marker information is shown in Table 7. Figure 10 The capillary electrophoresis gel shows the distribution of product molecules of varying lengths within the IVT reaction. The apparent molecular sizes of the RNA products on the capillary electrophoresis graph, obtained by comparison with electronic markers, deviate somewhat from the theoretical RNA molecular size. T7 RNAP WT, KT-2q, KT-p3, KT-pt, and KT-m0 are observed to be within the theoretical 8.5 kb range. The amount of saRNA is very low, with a major electrophoretic band observed at approximately 4500 bp. RNA products of approximately 4000 bp or shorter are primarily observed in C1, C2, and C3, with a small amount of saRNA product with an apparent size of approximately 7600 bp also observed. The apparent molecular weights of the IVT products of the mutants KT-uc and KT-u0 of the present invention on the capillary electrophoresis graph are primarily saRNAs larger than 8000 bp.

[0176] Table 5 saRNA IVT transcription reaction system (20 μl)

[0177]

[0178] Table 6 Q-sep1 capillary electrophoresis conditions

[0179] No Action High voltage Duration Position 1 Purge / 60 Park 2 Wash step / 1 Wash buffer 3 Marker injection 2.00 3 UserDefine 4 Sample injection 4.00 20 S2 5 Separation&Detection 2.00 1500 Seperation Buffer

[0180] Table 7 Electronic marker information of Q-sep1 capillary electrophoresis

[0181]

[0182] Note: Template sequences used in the examples

[0183] T7 promoter GGG (SEQ ID NO: 4)

[0184] TAATACGACTCACTATA GGG

[0185] IVT reaction DNA template SEQ ID NO: 5

[0186] TAATACGACTCACTATAGGGT TCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCA TTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACG AACGATAGCatgggctggtcctgcatcatcttgttcttggtggctactgccactggagtacacagcGAGATTGTCC TTACACAGTCACCAGGCACCCTTTCCTTGTCTCCTGGGGAACGAGCCACCCTCAGTTGTCGGTCAAGCAAGAATCT GCTGCATTCCAATGGGATCACATACCTGTATTGGTACCAGCAGAAGCCTGGACAGGCACCCAGACTGCTCATCTAC CAGATGTCCAATCTGGCCTCAGGCATTCCTGACAGGTTTTCCGGCAGCGGGTCTGGCACCGATTTCACCCTGACCA TATCCAGGCTCGAACCAGAGGATTTTGCCGTGTATTACTGCGCACAGAATCTGGAGATTCCCCGCACTTTTGGCCA AGGGACTAAACTGGAGATCAAGCGGggaggaggcggctctggaggtggcggcagtggtggaggcgggtctggcggt ggcggatctggaggtggtgggagcCAGGTCCAGCTTGTTCAGTCAGGCGCAGAGGTGAAGAAACCCGGAGCTAGCG TGAAAGTCTCCTGCAAAGCGTCAGGGTACACCTTCACCAACTATGGGATGAACTGGGTACGTCAAGCCCCAGGGCA AAGACTCGAATGGATGGGTTGGATCAACACGTACACAGGGGAACCGACTTATGGCGAGGACTTCAAGGGGAGAGTG ACCATAACACTGGACACATCCGCTAGCACAGCGTATATGGAGCTGAGCAGTCTGAGGAGCGAAGATACGGCTGTTT ACTATTGTGCCCGCTTTGGTAACTACGTGGACTATTGGGGTCAGGGAACTCTGGTGACGGTTTCTAGCagtggcgg cggaggatccCAGGTGCAGCTGCAGCAGTCTGGCGCTGAGCTGGCTAGACCTGGCGCCTCCGTGAAGATGTCCTGC AAGACCTCCGGCTACACCTTCACCCGGTACACCATGCACTGGGTCAAGCAGAGGCCTGGACAGGGCCTGGAATGGA TCGGCTACATCAACCCCTCCCGGGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACAACCGA CAAGTCCTCCTCCACCGCCTACATGCAGCTGTCCTCCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGG TACTACGACGACCACTACTCCCTGGACTACTGGGGCCAGGGCACCACACTGACAGTGTCTAGCggaggcggaggat ctggtggtggcggatctggcggcggtggaagtggcggaggtggtagcCAGATCGTGCTGACCCAGTCTCCCGCCAT CATGTCTGCTAGCCCTGGCGAGAAAGTGACAATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATGAACTGGTAT CAGCAGAAGTCCGGCACCTCCCCCAAGCGGTGGATCTACGACACCTCCAAGGTGGCCTCTGGCGTGCCCTACAGAT TCTCCGGCTCTGGCTCTGGCACCTCCTACAGCCTGACCATCTCCAGCATGGAAGCCGAGGATGCCGCCACCTACTA CTGCCAGCAGTGGTCCTCCAACCCCCTGACCTTTGGCGCTGGCACCAAGCTGGAACTGAAGGGCGGCTCTcaccac caccatcaccacTGATGAGCTGCAGAATTCGTCGACGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCC CCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACT CACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCC CACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCA ATTTCGTGCCAGCCACACCCTCGAGCTAGCaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0187] The underlined sequence (SEQ ID NO: 6) is from the EpCAM×CD3 DNA sequence in reference

[14] , and its components are listed as follows:

[0188] 5'UTR(TEV)-secretion signal-VL(anti-EpCAM)-linker-VH(anti-EpCAM)-linker-VH(anti-CD3)-linker-VL(anti-CD3)-6×His tag-spacer-3'UTR(FI)-spacer-poly(A)tail(A30)-poly(A)tail linker-poly(A)tail(A70)

[0189] T7pro-512B(SEQ ID NO:7)

[0190] TAATACGACTCACTATA GGGAGAAGCTCTCTTACACCTGATTCATTTCCATTGTTTTCTGCAGCAGCAATCCGGTTTCTGTCTTCAATTGTCAAGTTCCTCCTCCATGCACTTATCCAAGACGTCTCTAACTAGAAGCTTGTC CACCAGAGTGGGCTGAAGGAGGTTCAGCAGTTGGAGATATTCATCATGAGCGTTCTCAAACGATGGAGAGGGCAAGTCCGTGAGCTCAGGGTTCATGTAGCGGCGGCCAGAGGCTGCCGGTTCTCC GGAGGGCCTCCACGAATTCCCGAGTCCAACCAAGGTGCCAGACTCCCTTCTCCAAGGTGCTCAGCAGCAGTTCAACTGCCTGCATGTTCCCGGAGGTGGCGACTGTCCTCTGAATCTGCTCCTTCACC TCTGCAGGCAGAAAGGTCAGGTAGTCCAGCAGGCTCCACCTGGATGTACATTTTCCACCCTGGCCCTGAAGCACGAGATGAGATAGCGGAAATTCTCGTCTGTGGAATACCCATTCGACATTCTCCC

[0191] The T7pro-512B (T7 Pro-512B) is a fully functional model[8].

[0192] T7 promoter-AGG(SEQ ID NO:8)

[0193] TAATACGACTCACTATA AGG

[0194] IVT reaction DNA template SEQ ID NO: 9 (SEQ ID NO: 8 joined with SEQ ID NO: 6)

[0195] TAATACGACTCACTATA AGGT TCTCAACACAACATATACAAAACAAACGAATCTCAAGCAATCAAGCA TTCTACTTCTATTGCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACG AACGATAGCatgggctggtcctgcatcatcttgttcttggtggctactgccactggagtacacagcGAGATTGTCC TTACACAGTCACCAGGCACCCTTTCCTTGTCTCCTGGGGAACGAGCCACCCTCAGTTGTCGGTCAAGCAAGAATCT GCTGCATTCCAATGGGATCACATACCTGTATTGGTACCAGCAGAAGCCTGGACAGGCACCCAGACTGCTCATCTAC CAGATGTCCAATCTGGCCTCAGGCATTCCTGACAGGTTTTCCGGCAGCGGGTCTGGCACCGATTTCACCCTGACCA TATCCAGGCTCGAACCAGAGGATTTTGCCGTGTATTACTGCGCACAGAATCTGGAGATTCCCCGCACTTTTGGCCA AGGGACTAAACTGGAGATCAAGCGGggaggaggcggctctggaggtggcggcagtggtggaggcgggtctggcggt ggcggatctggaggtggtgggagcCAGGTCCAGCTTGTTCAGTCAGGCGCAGAGGTGAAGAAACCCGGAGCTAGCG TGAAAGTCTCCTGCAAAGCGTCAGGGTACACCTTCACCAACTATGGGATGAACTGGGTACGTCAAGCCCCAGGGCA AAGACTCGAATGGATGGGTTGGATCAACACGTACACAGGGGAACCGACTTATGGCGAGGACTTCAAGGGGAGAGTG ACCATAACACTGGACACATCCGCTAGCACAGCGTATATGGAGCTGAGCAGTCTGAGGAGCGAAGATACGGCTGTTT ACTATTGTGCCCGCTTTGGTAACTACGTGGACTATTGGGGTCAGGGAACTCTGGTGACGGTTTCTAGCagtggcgg cggaggatccCAGGTGCAGCTGCAGCAGTCTGGCGCTGAGCTGGCTAGACCTGGCGCCTCCGTGAAGATGTCCTGC AAGACCTCCGGCTACACCTTCACCCGGTACACCATGCACTGGGTCAAGCAGAGGCCTGGACAGGGCCTGGAATGGA TCGGCTACATCAACCCCTCCCGGGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACAACCGA CAAGTCCTCCTCCACCGCCTACATGCAGCTGTCCTCCCTGACCTCCGAGGACTCCGCCGTGTACTACTGCGCCCGG TACTACGACGACCACTACTCCCTGGACTACTGGGGCCAGGGCACCACACTGACAGTGTCTAGCggaggcggaggat ctggtggtggcggatctggcggcggtggaagtggcggaggtggtagcCAGATCGTGCTGACCCAGTCTCCCGCCAT CATGTCTGCTAGCCCTGGCGAGAAAGTGACAATGACCTGCCGGGCCTCCTCCTCCGTGTCCTACATGAACTGGTAT CAGCAGAAGTCCGGCACCTCCCCCAAGCGGTGGATCTACGACACCTCCAAGGTGGCCTCTGGCGTGCCCTACAGAT TCTCCGGCTCTGGCTCTGGCACCTCCTACAGCCTGACCATCTCCAGCATGGAAGCCGAGGATGCCGCCACCTACTA CTGCCAGCAGTGGTCCTCCAACCCCCTGACCTTTGGCGCTGGCACCAAGCTGGAACTGAAGGGCGGCTCTcaccac caccatcaccacTGATGAGCTGCAGAATTCGTCGACGGATCCGATCTGGTACTGCATGCACGCAATGCTAGCTGCC CCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACT CACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCC CACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCA ATTTCGTGCCAGCCACACCCTCGAGCTAGCaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

[0196] IVT reaction DNA template SEQ ID NO: 10

[0197] TAATACGACTCACTATA

[0198] IVT reaction DNA template SEQ ID NO: 11

[0199]

[0200] IVT reaction template SEQ ID NO: 12

[0201] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGC TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCT TCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGA GCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCA GCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA CCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAG ctggaattaattcgagctcggtaccttgattctcgaggaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttattttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagc

[0202] References

[0203] 1. Martin C T., et al. Processivity in early stages of transcription by T7 RNA polymerase. Biochemistry. 1988 May 31; 27(11): 3966 - 74.

[0204] 2. Temiakov D., et al. Structural Basis for Substrate Selection by T7 RNA Polymerase. Cell, 116, 381–391(2004)

[0205] 3. Schenborn E T., et al. A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure. Nucleic Acids Research, 13, 6223–6236(1985)

[0206] 4. Krupp G. RNA synthesis: strategies for the use of bacteriophage RNA polymerases. Gene. 72(1 - 2): 75 - 89(1988).

[0207] 5.Gholamalipour Y.,et al.3'end additions by T7 RNA polymerase are RNAself-templated,distributive and diverse in character-RNA-Seq analyses.NucleicAcids Res.46(18),9253-9263(2018)

[0208] 6.Lyakhov D L.,et al.Mutant bacteriophage T7 RNA polymerases withaltered termination properties.J Mol Biol 269,28-40(1997)

[0209] 7.Macdonald L E.,et al.Termination and slippage by bacteriophage T7RNA polymerase.J.Mol.Biol.232,1030–1047(1993)

[0210] 8.Mu X.,et al.An origin of the immunogenicity of in vitro transcribedRNA.Nucleic Acids Research,46(10),5239–5249(2018)

[0211] 9.Mu X.,et al.Immunogenicity of In Vitro-Transcribed RNA.Acc ChemRes.54(21),4012–4023(2021)

[0212] 10.Wesselhoeft R A.,et al.Engineering circular RNA for potent andstable translation in eukaryotic cells.Nature Communications.9:2629(2018)

[0213] 11.Dousis A.,et al.An engineered T7 RNA polymerase that produces mRNAfree of immunostimulatory byproducts.Nat.Biotechnol.6,1525(2022)

[0214] 12.Lyon,S.,et al.A T7 RNA polymerase mutant enhances the yield of 5'-thienoguanosine-initiated RNAs.Chembiochem.19(2):142–146(2018)

[0215] 13.Fisher,D K.,et al.A sensitive,high-Volume,colorimetric assay forprotein phosphatases.Pharmaceutical Research.11(5):759-763(1994)

[0216] 14.Stadler,C R.,et al.Elimination of large tumors in mice by mRNA-encoded bispecific antibodies.Nature medicine,23(7),815,(2017)

[0217] 15.Henderson,J M.,et al.Cap1 messenger RNA synthesis with co-transcriptional CleanCap analog by in vitro transcription.Curr Protoc1(2):e39,(2021)

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[0219] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An isolated bacteriophage T7 RNA polymerase (T7 RNAP) mutant, characterized in that The T7 RNAP mutant has an amino acid residue mutation at position 744 corresponding to the wild-type T7 RNA polymerase, and comprises an amino acid residue mutation at at least one position selected from the group consisting of: amino acid residues corresponding to positions 62, 68, 72, 152, 173, 535, 538, 539, 633, 723, 767, 881, and 883 of wild-type T7 RNA polymerase; The amino acid numbering is based on the wild-type bacteriophage T7 RNA polymerase sequence shown in SEQ ID NO:

1.

2. The T7 RNAP mutant according to claim 1, wherein The T7 RNAP mutant further comprises an amino acid residue mutation at at least one position selected from the group consisting of: Corresponding to amino acid residues 43 and 880 of wild-type T7 RNA polymerase.

3. An isolated polynucleotide encoding the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1 or 2. A vector comprising the polynucleotide according to claim 3 .

5. A genetically engineered host cell comprising the vector according to claim 4, or a host cell in which the polynucleotide according to claim 3 is integrated into its genome.

6. Use of the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1 or 2 for in vitro transcription reaction (IVT), or for preparing a reagent or kit for in vitro transcription (IVT) reaction.

7. A reagent for in vitro transcription (IVT) reaction, comprising the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1 or 2. A kit comprising the reagent according to claim 7 .

9. A method for performing an in vitro transcription (IVT) reaction, comprising the step of performing a reaction using the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1 or 2.

10. A method for preparing circular RNA (circRNA), comprising the following steps: (i) performing an in vitro transcription (IVT) reaction using the bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1 or 2 to prepare a reaction product comprising a precursor mRNA (pre-mRNA); (ii) performing an in vitro cyclization reaction on the reaction product containing the pre-mRNA obtained in step (i), thereby obtaining CircRNA.

11. A method for preparing self-amplifying RNA (saRNA), comprising the following steps: The bacteriophage T7 RNA polymerase (T7 RNAP) mutant according to claim 1 or 2 is used to perform an in vitro transcription (IVT) reaction to obtain a long-chain saRNA as the target product.

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