CN114438084A - Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase - Google Patents

Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase Download PDF

Info

Publication number
CN114438084A
CN114438084A CN202210110835.7A CN202210110835A CN114438084A CN 114438084 A CN114438084 A CN 114438084A CN 202210110835 A CN202210110835 A CN 202210110835A CN 114438084 A CN114438084 A CN 114438084A
Authority
CN
China
Prior art keywords
ala
leu
glu
lys
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210110835.7A
Other languages
Chinese (zh)
Inventor
钱剩金
陈丽
杨侠斐
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Zhaowei Bioengineering Co ltd
Shanghai Hongene Biotech Corp
Original Assignee
Shanghai Zhaowei Bioengineering Co ltd
Shanghai Hongene Biotech Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Zhaowei Bioengineering Co ltd, Shanghai Hongene Biotech Corp filed Critical Shanghai Zhaowei Bioengineering Co ltd
Priority to CN202210110835.7A priority Critical patent/CN114438084A/en
Publication of CN114438084A publication Critical patent/CN114438084A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The application relates to the technical field of genetic engineering, in particular to a transcription reaction solution, a preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase. The transcription reaction solution for preparing the locked nucleic acid modified RNA comprises a DNA template, mutated T7RNA polymerase, LNA-NTPs and a buffer solution; wherein the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7. When the mutant T7RNA polymerase is added into a transcription reaction solution to synthesize the locked nucleic acid modified RNA, the transcription yield of the locked nucleic acid modified RNA can be obviously improved, the operation is simple, and the industrial production is facilitated.

Description

Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase
Technical Field
The application relates to the technical field of genetic engineering, in particular to a transcription reaction solution, a preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase.
Background
Locked Nucleic Acid (LNA) is a modified Nucleic Acid derivative, wherein 2 '-O-and 4' -C-positions on a ribose are bridged by a methylene group to form a 'Locked' structure, and the unique 'Locked' structure reduces the flexibility of a ribose structure and increases the stability of a phosphate skeleton local structure.
RNA is often used as a therapeutic drug to regulate and limit the expression of mRNA of abnormal genes, for example, antisense RNA (antisense RNA), ribozymes (ribozymes), small interfering RNAs (sirna), micro ribonucleic acids (micrornas), and aptamers (aptamers), and after nucleic acid-locked modification, the stability of RNA drugs in vivo is greatly improved.
However, the yield of locked nucleic acid modified RNA is low at present, which results in high preparation cost of locked nucleic acid modified RNA and is not beneficial to industrial production.
Disclosure of Invention
The present application aims to provide a transcription reaction solution for preparing a locked nucleic acid modified RNA, a preparation method of the locked nucleic acid modified RNA, and an application of a variant T7RNA polymerase in preparation of the locked nucleic acid modified RNA, which aim to solve the technical problem of low yield of the existing locked nucleic acid modified RNA.
The first aspect of the application provides a transcription reaction solution for preparing locked nucleic acid modified RNA, which comprises a DNA template, mutated T7RNA polymerase, LNA-NTPs and a buffer solution; wherein the mutated T7RNA polymerase comprises at least one of T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.1, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.2, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.3, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.4, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.5, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.6 and T7RNA polymerase with an amino acid sequence shown as SEQ ID NO. 7.
According to the application, when at least one of the mutant T7RNA polymerases provided by the method is added into a transcription reaction solution to synthesize the locked nucleic acid modified RNA, the transcription yield of the locked nucleic acid modified RNA can be obviously improved, the operation is simple, and the industrial production is facilitated.
In a second aspect of the present application, there is provided a method for producing a locked nucleic acid-modified RNA, comprising subjecting the transcription reaction solution for producing a locked nucleic acid-modified RNA provided in the first aspect to a transcription reaction.
Optionally, the method for preparing locked nucleic acid modified RNA further comprises: adding DNA template degrading enzyme into the transcribed system to obtain a first mixed system, and carrying out digestion reaction; and adding a precipitator into the first mixed system to obtain a second mixed system, and purifying.
Optionally, the DNA template degrading enzyme is selected from DNase I enzyme, the total amount of the DNA template degrading enzyme in the first mixed system is 1-3U.
Optionally, the precipitating agent is selected from ammonium acetate, and the final molar concentration of the ammonium acetate in the second mixed system is 3-6M.
Optionally, the precipitant is selected from lithium chloride, and the final molar concentration of the lithium chloride in the second mixed system is 6-8M.
The transcription yield of the RNA modified by the locked nucleic acid can be obviously improved by carrying out the transcription reaction on the transcription reaction solution of the T7RNA polymerase containing at least one mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 as amino acid sequences; in addition, the digestion reaction and the purification treatment are matched to further improve the yield of the RNA modified by the locked nucleic acid.
In a third aspect of the present application, there is provided a use of a mutant T7RNA polymerase in preparing locked nucleic acid modified RNA, wherein the mutant T7RNA polymerase includes at least one of T7RNA polymerase having an amino acid sequence shown in SEQ ID No.1, T7RNA polymerase having an amino acid sequence shown in SEQ ID No.2, T7RNA polymerase having an amino acid sequence shown in SEQ ID No.3, T7RNA polymerase having an amino acid sequence shown in SEQ ID No.4, T7RNA polymerase having an amino acid sequence shown in SEQ ID No.5, T7RNA polymerase having an amino acid sequence shown in SEQ ID No.6, and T7RNA polymerase having an amino acid sequence shown in SEQ ID No. 7.
The mutant T7RNA polymerase is used for preparing the locked nucleic acid modified RNA, so that the transcription yield of the locked nucleic acid modified RNA can be obviously improved, the operation is simple, and the industrial production is facilitated.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following specifically describes a transcription reaction solution for preparing a locked nucleic acid modified RNA, a preparation method of the locked nucleic acid modified RNA, and an application of the modified T7RNA polymerase in preparing the locked nucleic acid modified RNA, which are provided in the embodiments of the present application.
The application provides a transcription reaction solution for preparing RNA, which comprises a DNA template, mutated T7RNA polymerase, LNA-NTPs and a buffer solution; wherein the mutated T7RNA polymerase comprises at least one of T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.1, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.2, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.3, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.4, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.5, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.6 and T7RNA polymerase with an amino acid sequence shown as SEQ ID NO. 7.
LNA-NTPs (nucleotide triphosphates) are nucleoside triphosphates bridged between the 2 '-O-and 4' -C-positions of ribose via a methylene group, and are used as raw materials for synthesizing nucleic acid-modified RNA. In the present application, LNA-NTPs refers to a base in a molecular structure that may be natural or modified, and LNA-NTPs refers to a triphosphate structure in a molecular structure that may be modified or unmodified.
In this embodiment, the LNA-NTPs may be selected from LNA-ATP, LNA-GTP, LNA-CTP, and LNA-UTP.
The transcription reaction solution can be used for transcription reaction, and in a buffer solution system, a medium template chain of a DNA template is used as a template, LNA-NTPs are used as raw materials, and in-vitro transcription is carried out under the catalysis of mutant T7RNA polymerase to synthesize the RNA modified by locked nucleic acid.
When the mutant T7RNA polymerase with the amino acid sequence shown as at least one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 is added into the transcription reaction solution to synthesize the locked nucleic acid modified RNA, compared with the case of using the natural T7RNA polymerase as the RNA polymerase in the transcription system, the method can obviously improve the transcription yield of the locked nucleic acid modified RNA, is simple to operate and is beneficial to industrial production.
In this example, the DNA template includes the target sequence and a promoter corresponding to a mutated T7RNA polymerase having the amino acid sequence shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or SEQ ID NO. 7.
In this example, the DNA template is selected from linearized plasmid DNA.
The transcription reaction solution for preparing RNA in the present application may include NTPs (i.e., modified or unmodified nucleoside triphosphate) in addition to LNA-NTPs, such as natural ATP, natural GTP, natural CTP, natural UTP, modified ATP, modified GTP, modified CTP, and modified UTP.
In this example, the pH of the buffer is 6.5-7.5, which facilitates better transcription reaction for the synthesis of locked nucleic acid modified RNA. Illustratively, the pH of the buffer may be 6.5, 7.0, 7.2, and 7.5, among others.
In this embodiment, the buffer includes a biological buffer, an activator, a polyamine, a detergent, and a reducing agent. Biological buffers are used to maintain the pH required for the transcription reaction. The activator is used to form a complex with LNA-NTPs and the DNA template to be recognized by the mutated RNA polymerase, thereby allowing the DNA template to be stably transcribed to synthesize locked nucleic acid modified RNA. Polyamines have a promoting effect on the recognition and binding of the DNA template and the mutated RNA polymerase, facilitating easier transcription reactions for the synthesis of locked nucleic acid modified RNA. The detergent functions to increase the stability of the enzyme in the transcription reaction solution. The reducing agent can inhibit dimerization of DNA, and is beneficial to promoting high-efficiency progress of transcription reaction to synthesize RNA modified by locked nucleic acid.
In this example, the biological buffer in the buffer may be selected from Tris (Tris), HEPES (4-hydroxyethylpiperazineethanesulfonic acid), MOPS (3- (N-morpholino) propanesulfonic acid) or PIPES (piperazine-1, 4-diethylsulfonic acid). The above substances are effective in maintaining the pH required for the transcription reaction. In other embodiments of the present application, the biological buffer may not be limited to the above-mentioned substances, and for example, the biological buffer may be BICINE (N, N-dihydroxyethylglycine), CAPS (3- (cyclohexylamine) -1-propanesulfonic acid), TAPS (N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid), EPPS (N- (2-hydroxyethyl) piperazine-N' -3-propanesulfonic acid), MOPSO (3- (N-morpholinyl) -2-hydroxypropanesulfonic acid), or the like.
Further, in this example, the final molar concentration of the biological buffer in the transcription reaction solution is 25-35mM, which can maintain the stability of the transcription reaction solution, and facilitate the transcription reaction to synthesize the locked nucleic acid modified RNA. Illustratively, the final molar concentration of the biological buffer in the transcription reaction solution can be 25mM, 27mM, 30mM, 32mM, 35mM, and so forth.
In this embodiment, the activator may be selected from magnesium ions or sodium ions, which can better form a complex with LNA-NTPs and DNA template to be more easily recognized by the mutated RNA polymerase, facilitating the transcription reaction to synthesize locked nucleic acid modified RNA.
Furthermore, in this example, the final molar concentration of the activator in the transcription reaction solution is 4.5-5.5mM, which not only provides the ion concentration required for the transcription reaction, but also facilitates the complexing of magnesium or sodium ions with LNA-NTPs and DNA templates to form a complex that is easily recognized by the mutated RNA polymerase. Illustratively, the final molar concentration of the activator in the transcription reaction solution may be 4.5mM, 4.8mM, 5.0mM, and 5.5mM, and the like.
In this embodiment, the polyamine may be selected from spermidine, putrescine or spermine, which may better facilitate the recognition of the DNA template and the mutated RNA polymerase, facilitating easier transcription reactions for the synthesis of locked nucleic acid modified RNA. It should be noted that in other embodiments of the present application, the above substances may also be replaced by spermidine salts, such as spermidine phosphate or spermidine hydrochloride.
Furthermore, the polyamine can further promote the recognition and binding of the DNA template and the mutant RNA polymerase by setting the final molar concentration of the polyamine in the transcription reaction solution to 0.8 to 1.2 mM. Illustratively, the final molar concentration of the polyamine in the transcription reaction solution may be 0.8mM, 1.0mM, and 1.2mM, and so forth.
In this example, the detergent may be selected from Triton X-100 (polyethylene glycol octylphenyl ether), which may better stabilize the mutant T7RNA polymerase action. Further, the detergent has a mass fraction of 0.01 to 0.03% in the transcription reaction solution, and the effect of stabilizing the mutated T7RNA polymerase can be further achieved. Illustratively, the mass fraction of the detergent in the transcription reaction solution may be 0.01%, 0.02%, 0.03%, and the like.
In this embodiment, the reducing agent may be selected from DTT (dithiothreitol) or β -mercaptoethanol, which is a reducing agent that better inhibits dimerization of DNA.
Further, dimerization of DNA can be further inhibited by adjusting the final molar concentration of the reducing agent in the transcription reaction solution to 2.5 to 3.5 mM. Illustratively, the final molar concentration of the reducing agent in the transcription reaction solution may be 2.5mM, 3.0mM, and 3.5mM, and the like.
In addition, in this embodiment, the buffer solution further includes inorganic pyrophosphatase and a nuclease inhibitor. The inorganic pyrophosphatase can promote the transcription reaction and is favorable for improving the yield of the RNA modified by the locked nucleic acid prepared by transcription. Nuclease inhibitors have the effect of inhibiting rnases.
Further, the final concentration of inorganic pyrophosphatase in the transcription reaction solution is 0.01-1U/. mu.l, which can further promote the transcription reaction to synthesize the RNA modified by locked nucleic acid; as an example, the final concentration of inorganic pyrophosphatase in the transcription reaction solution may be 0.01U/. mu.l, 0.02U/. mu.l, 0.05U/. mu.l, 1U/. mu.l, and the like. The final concentration of the nuclease inhibitor in the transcription reaction solution is 1-5U/mul, and the action of inhibiting the RNase can be further realized; illustratively, the final concentration of nuclease inhibitor in the transcription reaction solution may be 1U/. mu.l, 2U/. mu.l, 4U/. mu.l, 5U/. mu.l, and the like.
The application also provides a preparation method of the RNA modified by the locked nucleic acid, which comprises the step of carrying out transcription reaction on the transcription reaction liquid for preparing the RNA modified by the locked nucleic acid.
The transcription yield of the RNA modified by the locked nucleic acid can be obviously improved by carrying out the transcription reaction on the transcription reaction solution of the T7RNA polymerase containing at least one mutation shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 in amino acid sequence.
In this example, the transcription reaction for preparing the locked nucleic acid modified RNA is carried out at a temperature of 30-40 ℃ for 10-18 hours. Further, the temperature of the transcription reaction was 37 ℃ for 16 hours.
In this embodiment, after the transcription reaction, adding a DNA template degrading enzyme to the transcribed system to obtain a first mixed system, and performing a digestion reaction; can digest the original DNA template, is beneficial to subsequent purification reaction and is beneficial to improving the purity of the locked nucleic acid modified RNA in a system after in vitro transcription.
In some embodiments of the present application, the DNA template degrading enzyme is selected from DNase I enzyme (Chinese name: DNase I, an endonuclease capable of digesting single-stranded or double-stranded DNA to generate single-stranded or double-stranded oligodeoxynucleotides), which can digest the original DNA template well and is beneficial for subsequent purification reactions.
In some embodiments of the present application, the total amount of DNA template degrading enzyme in the first mixed system is 1-3U, which may further facilitate digestion of the original DNA template. Illustratively, the total amount of the DNA template degrading enzyme in the first mixed system may be 1U, 2U, and 3U, and so on.
In this embodiment, after the digestion reaction, a second mixed system is obtained by adding a precipitant to the first mixed system after the digestion reaction, and a purification treatment is performed: so that the reaction product nucleic acid-locked modified RNA can be precipitated, and other impurities do not precipitate, thereby realizing purification.
In some embodiments of the present application, the precipitating agent may be selected from ammonium acetate or lithium chloride, which may be better to allow the reaction product to precipitate locked nucleic acid modified RNA, while other impurities do not precipitate.
Further, the purification treatment comprises adding a precipitant into the first mixed system after the digestion reaction, fully mixing to obtain a second mixed system, carrying out ice bath and centrifugation, and removing the supernatant to obtain a precipitate.
In the embodiment, after precipitating the reaction product of the locked nucleic acid modified RNA by using the precipitating agent, the method further comprises washing the precipitate to obtain the final locked nucleic acid modified RNA. Further, in this example, the precipitate was washed with ethanol at a mass fraction of 70%.
The preparation method of the locked nucleic acid modified RNA provided by the application at least has the following advantages:
the transcription yield of the RNA modified by the locked nucleic acid can be obviously improved by carrying out the transcription reaction on the transcription reaction solution of the T7RNA polymerase containing at least one mutant T7RNA polymerase shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 as amino acid sequences; in addition, the digestion reaction and the purification treatment are matched to further improve the yield of the RNA modified by the locked nucleic acid.
The application also provides an application of the mutant T7RNA polymerase in preparing locked nucleic acid modified RNA; wherein the mutated T7RNA polymerase comprises at least one of T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.1, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.2, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.3, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.4, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.5, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.6 and T7RNA polymerase with an amino acid sequence shown as SEQ ID NO. 7.
Compared with the use of natural T7RNA polymerase as RNA polymerase in a transcription system, the mutant T7RNA polymerase is used for preparing locked nucleic acid modified RNA, can obviously improve the transcription yield of the locked nucleic acid modified RNA, is simple to operate and is beneficial to industrial production.
The features and properties of the present application are described in further detail below with reference to examples.
Example 1
The present embodiment provides a transcription reaction solution for preparing a locked nucleic acid modified RNA and a method for preparing a locked nucleic acid modified RNA, including the following steps:
(1) preparation of transcription reaction solution:
mu.l of 10 Xbuffer, 2.5. mu.l of 20mM LNA-ATP, 2.5. mu.l of 20mM GTP, 2.5. mu.l of 20mM CTP, 2.5. mu.l of 20mM LNA-UTP, 2. mu.l of the enzyme mixture, and 2. mu. l X. mu.g/. mu.l of the DNA template were mixed, and nuclease-free water was added thereto to give a total volume of 20. mu.l, thereby preparing a transcription reaction solution.
Wherein, the preparation of 10 × buffer solution: mixing HEPES-sodium hydroxide (pH7.0@25 ℃), magnesium acetate, spermidine, Triton X-100 and DTT to obtain 10 × buffer solution; HEPES-sodium hydroxide, magnesium acetate, spermidine, Triton X-100 and DTT were present in 10 Xbuffer at concentrations of 300mM, 50mM, 10mM, 0.02% (mass fraction) and 30mM, respectively.
Preparation of mixed enzyme solution: mixing mutated T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.1, inorganic pyrophosphatase and nuclease inhibitor to prepare a mixed enzyme system; the concentrations of the mutated T7RNA polymerase, the inorganic pyrophosphatase and the nuclease inhibitor, the amino acid sequences of which are shown in SEQ ID NO.1, in the mixed enzyme solution are 200U/. mu.l, 0.5U/. mu.l and 20U/. mu.l, respectively.
The sequence of the DNA template is as follows 1:
Figure BDA0003495039320000101
Figure BDA0003495039320000102
(Note: in the sequence of the above DNA template, the underlined part is a primer, and the bold part is a promoter).
(2) The preparation method of the locked nucleic acid modified RNA comprises the following steps:
after the transcription reaction solution provided above was subjected to transcription reaction at 37 ℃ for 16 hours, 1U of DNase I enzyme was added to digest the original DNA template at 37 ℃ for 30 min. Then 20. mu.l of 5M ammonium acetate was added thereto, mixed well, ice-cooled for 30min, centrifuged at 4 ℃ for 15min, the supernatant was removed, and the precipitate was retained. Eluting and precipitating twice by using ethanol with the mass fraction of 70 percent to obtain the RNA modified by the locked nucleic acid.
Example 2
Example 2 differs from example 1 in that: HEPES-sodium hydroxide was replaced with Tris-HCl at a concentration of 350mM in 10 Xbuffer.
Example 3
Example 3 differs from example 1 in that: magnesium acetate was replaced by sodium acetate, 55mM in 10 Xbuffer.
Example 4
Example 4 differs from example 1 in that: spermidine was replaced with putrescine, the concentration of putrescine in 10 × buffer being 12 mM.
Example 5
Example 5 differs from example 1 in that: DTT was replaced by beta-mercaptoethanol, which was present in 10 Xbuffer at a concentration of 35 mM.
Example 6
Example 6 differs from example 1 in that: the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO. 2.
Example 7
Example 7 differs from example 1 in that: the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO. 3.
Example 8
Example 8 differs from example 1 in that: the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO. 4.
Example 9
Example 9 differs from example 1 in that: the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO. 5.
Example 10
Example 10 differs from example 1 in that: the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO. 6.
Example 11
Example 11 differs from example 1 in that: the amino acid sequence of the mutant T7RNA polymerase is shown as SEQ ID NO. 7.
Comparative example 1
Comparative example 1 differs from example 1 in that: the RNA polymerase is natural T7RNA polymerase.
Comparative example 2
Comparative example 2 differs from example 1 in that: the concentration of HEPES-sodium hydroxide in 10 Xbuffer was 100 mM.
Comparative example 3
Comparative example 3 differs from example 1 in that: magnesium acetate was replaced by sodium acetate, the concentration of sodium acetate in 10 Xbuffer was 30 mM.
Comparative example 4
Comparative example 4 differs from example 1 in that: spermidine was replaced with putrescine, the concentration of putrescine in 10 × buffer was 5 mM.
Comparative example 5
Comparative example 5 differs from example 1 in that: DTT was replaced by beta-mercaptoethanol, which was present in 10mM buffer at a concentration of 10 mM.
Test examples
The transcription reaction solutions for preparing the locked nucleic acid modified RNAs and the methods for preparing the locked nucleic acid modified RNAs provided in examples 1 to 5 and comparative examples 1 to 5 were subjected to yield measurement. The locked nucleic acid-modified RNA precipitates obtained in examples 1 to 5 and comparative examples 1 to 5 were dried in the sun, the precipitates were dissolved in nuclease-free water, and OD was measured260And calculating the yield of nucleic acid-modified RNA (RNA yield-M dilution fold x 0.04 μ g/μ l volume of system, wherein 0.04 μ g/μ l is OD2601 is the corresponding RNA concentration value), the results are shown in Table 1.
TABLE 1
Yield (ug) Yield (ug) Yield (ug)
Example 1 18 Example 7 11 Comparative example 1 4
Example 2 15 Example 8 10 Comparative example 2 10
Example 3 10 Example 9 8 Comparative example 3 6
Example 4 14 Example 10 6 Comparative example 4 9
Example 5 12 Example 11 6 Comparative example 5 8
Example 6 10
As can be seen from table 1: the yield of the locked nucleic acid modified RNA of example 1 and examples 6 to 11 is higher than that of the locked nucleic acid modified RNA of comparative example 1, indicating that the mutant T7RNA polymerase having the amino acid sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 can effectively improve the yield of the locked nucleic acid modified RNA relative to the natural T7RNA polymerase; meanwhile, the yield of the locked nucleic acid modified RNA of example 1 and example 2 is higher than that of the locked nucleic acid modified RNA of comparative example 2, the yield of the locked nucleic acid modified RNA of example 1 and example 3 is higher than that of the locked nucleic acid modified RNA of comparative example 3, the yield of the locked nucleic acid modified RNA of example 1 and example 4 is higher than that of comparative example 4, and the yield of the locked nucleic acid modified RNA of example 1 and example 5 is higher than that of comparative example 5, which indicates that the concentration of substances in the buffer of the present application is advantageous and the yield of the locked nucleic acid modified RNA can be further improved.
In addition, the mutant T7RNA polymerase used in the transcription reaction solution for preparing the locked nucleic acid modified RNA and the preparation method of the locked nucleic acid modified RNA provided in example 1 is a polymerase having an amino acid sequence shown as SEQ ID NO.1, and the yield of the locked nucleic acid modified RNA can be up to 18 μ g by matching with a specific substance and concentration of a buffer solution.
Therefore, the transcription reaction solution for preparing the locked nucleic acid modified RNA and the preparation method of the locked nucleic acid modified RNA provided by the application can effectively improve the yield of the locked nucleic acid modified RNA, are simple to operate and are beneficial to industrial production.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shanghai megadimensional science and technology development Co., Ltd
SHANGHAI ZHAOWEI BIOENGINEERING Co.,Ltd.
<120> a transcription reaction solution, a method for preparing locked nucleic acid-modified RNA, and a method for producing mutant T7RNA polymerase
By using
<130> 2022.01.28
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 883
<212> PRT
<213> Artificial sequence
<400> 1
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 2
<211> 883
<212> PRT
<213> Artificial sequence
<400> 2
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 3
<211> 883
<212> PRT
<213> Artificial sequence
<400> 3
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 4
<211> 883
<212> PRT
<213> Artificial sequence
<400> 4
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 5
<211> 883
<212> PRT
<213> Artificial sequence
<400> 5
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 6
<211> 883
<212> PRT
<213> Artificial sequence
<400> 6
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val Ala
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala
<210> 7
<211> 883
<212> PRT
<213> Artificial sequence
<400> 7
Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu
1 5 10 15
Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu
20 25 30
Ala Arg Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu
35 40 45
Ala Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val
50 55 60
Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys
65 70 75 80
Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg
85 90 95
Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu
100 105 110
Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125
Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140
Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala Lys
145 150 155 160
His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys Arg Val Gly His
165 170 175
Val Tyr Lys Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser
180 185 190
Lys Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp
195 200 205
Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220
Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp
225 230 235 240
Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255
Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270
Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr Trp Ala
275 280 285
Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala
290 295 300
Leu Met Arg Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile
305 310 315 320
Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335
Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile
340 345 350
Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365
Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380
Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe
385 390 395 400
Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe
405 410 415
Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430
Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys
435 440 445
Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460
Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys
465 470 475 480
Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495
Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510
Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr
515 520 525
Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln
530 535 540
His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn
545 550 555 560
Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575
Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn
580 585 590
Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605
Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620
Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Tyr Gly
625 630 635 640
Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln
645 650 655
Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670
Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr
675 680 685
Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700
Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg
705 710 715 720
Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735
Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750
Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu
755 760 765
Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His
770 775 780
Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu
785 790 795 800
Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815
Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met
820 825 830
Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845
Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860
Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe
865 870 875 880
Ala Phe Ala

Claims (10)

1. A transcription reaction solution for preparing locked nucleic acid modified RNA is characterized by comprising a DNA template, mutated T7RNA polymerase, LNA-NTPs and a buffer solution;
wherein the mutated T7RNA polymerase comprises at least one of T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.1, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.2, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.3, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.4, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.5, T7RNA polymerase with an amino acid sequence shown as SEQ ID NO.6 and T7RNA polymerase with an amino acid sequence shown as SEQ ID NO. 7.
2. The transcription reaction solution for preparing locked nucleic acid according to claim 1, wherein the buffer solution comprises a biological buffer, an activator, a polyamine, a detergent, and a reducing agent.
3. The transcription reaction solution according to claim 2, wherein the biological buffer is selected from Tris, HEPES, MOPS or PIPES;
the final molar concentration of the biological buffer in the transcription reaction liquid is 25-35 mM;
optionally, the buffer has a pH of 6.5 to 7.5.
4. The transcription reaction solution according to claim 2, wherein the activator is selected from magnesium ions or sodium ions;
the final molar concentration of the activator in the transcription reaction solution is 4.5-5.5 mM.
5. The transcription reaction solution according to claim 2, wherein the polyamine is selected from spermidine, putrescine, or spermine;
the polyamine is present in the transcription reaction solution at a final molar concentration of 0.8 to 1.2 mM.
6. The transcription reaction solution according to claim 2, wherein the detergent is selected from the group consisting of Triton X-100;
the mass fraction of the detergent in the transcription reaction solution is 0.01-0.03%.
7. The transcription reaction solution according to claim 2, wherein the reducing agent is selected from DTT or β -mercaptoethanol;
the final molar concentration of the reducing agent in the transcription reaction solution is 2.5-3.5 mM.
8. The transcription reaction solution according to claim 2, wherein the buffer solution further comprises an inorganic pyrophosphatase and a nuclease inhibitor;
the final concentration of the inorganic pyrophosphatase in the transcription reaction solution is 0.01-1U/microliter;
the nuclease inhibitor is in the transcription reaction solution at a final concentration of 1-5U/. mu.l.
9. A method for producing a locked nucleic acid-modified RNA, which comprises subjecting the transcription reaction solution for producing a locked nucleic acid-modified RNA according to any one of claims 1 to 8 to a transcription reaction;
optionally, the method for preparing locked nucleic acid modified RNA further comprises: adding DNA template degrading enzyme into the transcribed system to obtain a first mixed system, and carrying out digestion reaction; adding a precipitator into the first mixed system to obtain a second mixed system, and purifying;
optionally, the DNA template degrading enzyme is selected from DNase I enzyme, the total amount of the DNA template degrading enzyme in the first mixed system is 1-3U.
10. The application of the mutant T7RNA polymerase in preparing locked nucleic acid modified RNA is characterized in that the mutant T7RNA polymerase comprises at least one of T7RNA polymerase with an amino acid sequence shown as SEQ ID No.1, T7RNA polymerase with an amino acid sequence shown as SEQ ID No.2, T7RNA polymerase with an amino acid sequence shown as SEQ ID No.3, T7RNA polymerase with an amino acid sequence shown as SEQ ID No.4, T7RNA polymerase with an amino acid sequence shown as SEQ ID No.5, T7RNA polymerase with an amino acid sequence shown as SEQ ID No.6 and T7RNA polymerase with an amino acid sequence shown as SEQ ID No. 7.
CN202210110835.7A 2022-01-29 2022-01-29 Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase Pending CN114438084A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210110835.7A CN114438084A (en) 2022-01-29 2022-01-29 Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210110835.7A CN114438084A (en) 2022-01-29 2022-01-29 Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase

Publications (1)

Publication Number Publication Date
CN114438084A true CN114438084A (en) 2022-05-06

Family

ID=81371927

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210110835.7A Pending CN114438084A (en) 2022-01-29 2022-01-29 Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase

Country Status (1)

Country Link
CN (1) CN114438084A (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495504A (en) * 2005-06-30 2009-07-29 阿切埃米克斯有限公司 Materials and methods for the generation of fully 2'-modified nucleic acid transcripts
CN111893128A (en) * 2020-06-24 2020-11-06 苏州市泽悦生物技术有限公司 Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof
CN112714795A (en) * 2019-08-23 2021-04-27 新英格兰生物实验室公司 Enzymatic RNA capping method
CN112831484A (en) * 2021-01-13 2021-05-25 华中科技大学 T7-RNA polymerase mutant and application thereof
CN112921014A (en) * 2019-12-05 2021-06-08 左炽健 T7RNA polymerase mutant, mRNA, gene, expression vector and cell
CN113166737A (en) * 2018-10-04 2021-07-23 新英格兰生物实验室公司 Methods and compositions for increasing the efficiency of capping of transcribed RNA
US20220056425A1 (en) * 2018-09-12 2022-02-24 Guardian Therapeutics, Llc Rna polymerase for synthesis of modified rna

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101495504A (en) * 2005-06-30 2009-07-29 阿切埃米克斯有限公司 Materials and methods for the generation of fully 2'-modified nucleic acid transcripts
US20220056425A1 (en) * 2018-09-12 2022-02-24 Guardian Therapeutics, Llc Rna polymerase for synthesis of modified rna
CN113166737A (en) * 2018-10-04 2021-07-23 新英格兰生物实验室公司 Methods and compositions for increasing the efficiency of capping of transcribed RNA
CN112714795A (en) * 2019-08-23 2021-04-27 新英格兰生物实验室公司 Enzymatic RNA capping method
CN112921014A (en) * 2019-12-05 2021-06-08 左炽健 T7RNA polymerase mutant, mRNA, gene, expression vector and cell
CN111893128A (en) * 2020-06-24 2020-11-06 苏州市泽悦生物技术有限公司 Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof
CN112831484A (en) * 2021-01-13 2021-05-25 华中科技大学 T7-RNA polymerase mutant and application thereof

Similar Documents

Publication Publication Date Title
US20230250439A1 (en) Polynucleotide secondary structure
US8574864B2 (en) Methods and kits for 3&#39;-end-tagging of RNA
JP2023134670A (en) CRISPR-based compositions and methods of use
JPH05503423A (en) Nucleic acid enzyme for DNA cutting
CN113943764B (en) Method for preparing double-stranded RNA
CN113957108A (en) Synthesis method of capped RNA and capped RNA transcription reaction solution
WO2023132885A1 (en) Methods of purifying dna for gene synthesis
EP4337331A1 (en) Modified mrna, modified non-coding rna, and uses thereof
CN107338245B (en) Method for preparing single-stranded DNA in large scale
WO2023081311A1 (en) Methods of purifying dna for gene synthesis
AU2021217143A1 (en) Chaotropic agents for reducing formation of double-stranded RNA
Shirokikh et al. Chemical and enzymatic probing of spatial structure of the omega leader of tobacco mosaic virus RNA
CN116875658A (en) Deoxyribozyme and method for detecting mRNA capping rate
CN114438084A (en) Transcription reaction solution, preparation method of locked nucleic acid modified RNA and application of mutant T7RNA polymerase
EP4137582A1 (en) Rolling circle reverse transcription of circular rna
CN115807048A (en) Method for preparing double-stranded RNA
van Dijk et al. Distinct RNA structural domains cooperate to maintain a specific cleavage site in the 3′-UTR of IGF-II mRNAs
CN117247932A (en) Method for improving utilization rate of cap analogues in-vitro transcription
US11898186B1 (en) Compositions and methods for preparing capped mRNA
WO2023222114A1 (en) Methods of making circular rna
Dock-Bregeon et al. Nucleic acids and their complexes
Rozhdestvensky et al. Isolation and posttranscriptional modification analysis of native BC1 RNA from mouse brain
WO2023023487A1 (en) Screening codon-optimized nucleotide sequences
EP4388090A1 (en) Screening codon-optimized nucleotide sequences
WO2024044759A2 (en) Methods of using exonucleases to make tiny rnas on argonaute proteins

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination