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CN-122012449-A - RNA polymerase variants and uses thereof

CN122012449ACN 122012449 ACN122012449 ACN 122012449ACN-122012449-A

Abstract

The application provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. The present disclosure obtains T7 RNA polymerase variants by engineering wild-type T7 RNA polymerase that can greatly increase the capping rate of RNA products relative to wild-type T7 RNA polymerase.

Inventors

  • XU XIAOYU
  • Jin Qiuheng
  • HE WEI
  • Ji Guiying
  • GONG YUBAO
  • CAO LIN

Assignees

  • 南京诺唯赞生物科技股份有限公司

Dates

Publication Date
20260512
Application Date
20260205

Claims (10)

  1. An rna polymerase variant characterized in that the amino acid sequence of the variant has at least 95% sequence identity compared to SEQ ID No. 1 and comprises a substitution or substitution set selected from any one of k642r+k S, S628P, T630A or G777S relative to SEQ ID No. 1.
  2. 2. The variant according to claim 1, wherein the amino acid sequence of the variant is as shown in any one of SEQ ID NOs 2, 3, 5 and 6.
  3. 3. A biomaterial, characterized in that the biomaterial is selected from one or more of the following: 1) A polynucleotide molecule encoding the RNA polymerase variant of claim 1 or 2; 2) An expression vector comprising the polynucleotide molecule of 1); 3) A host cell comprising a polynucleotide molecule as described in 1), or a host cell comprising an expression vector as described in 2).
  4. 4. A composition comprising the variant of claim 1 or 2.
  5. 5. Kit comprising a variant according to claim 1 or 2.
  6. 6. Use of a variant according to claim 1 or 2, a composition according to claim 4 or a kit according to claim 5 for in vitro transcription for the preparation of RNA.
  7. 7. A method for preparing RNA, comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, with the RNA polymerase variant of claim 1 or 2, and incubating in an in vitro transcription reaction system to obtain a target RNA product.
  8. 8. A method of preparing a capped mRNA comprising contacting a DNA template, a modified or unmodified nucleoside triphosphate, a cap analog with the RNA polymerase variant of claim 1 or 2, and incubating in an in vitro transcription reaction system to obtain a product of interest.
  9. 9. The method of claim 7, further comprising a cap analogue in the in vitro transcription system.
  10. 10. Use of an RNA polymerase variant according to claim 1 or 2 in RNA drug synthesis.

Description

RNA polymerase variants and uses thereof Technical Field The application relates to the field of biotechnology, in particular to an RNA polymerase variant and application thereof in preparation of capped RNA. Background The complete mRNA has mainly five parts, from 5' to 3' including a 5' cap structure, a 5' untranslated region, an open reading frame encoding an antigen, a 3' untranslated region, and a Ploy A tail. In vivo, the 5 'cap structure is formed by the catalysis of RNA triphosphatase, mRNA guanosine transferase, mRNA methyltransferase and mRNA nucleoside 2' -methyltransferase, and is a special structure for forming mature mRNA by post-transcriptional modification of eukaryotes. In Vitro Transcription (IVT) capping can be performed by synthesizing the cap structure through in vivo-like enzyme-linked reaction, also called enzymatic capping, generally using enzymes such as VCE, FCE and the like as capping catalysts, and additionally adding mRNA nucleoside 2' -methyltransferase for capping. Enzymatic capping is affected by its enzyme-linked catalytic efficiency, and capping rate is relatively low, and co-transcription capping by cap analogues is also a currently mainstream method, and the capping rate is far beyond that of enzymatic capping. Nowadays, cap analogues have evolved to the third generation, and the first generation cap analogues are rarely available on the market due to the presence of two free 3' -OH groups, which results in the back incorporation of the cap analogues, and the main second generation ARCA cap analogues (formula I) and the third generation cap analogues (formula II, cleanCap AG) are currently common on the market. I is a kind of II (II) Compared with the second-generation cap analogues, the third-generation cap analogues, such as CLEANCAP AG, have obviously improved capping rate (more than 90% of capping rate), but when the wild-type T7 RNA polymerase is used as a catalyst for preparation, the capping rate is difficult to reach 100% due to steric hindrance and other reasons, and the uncapped RNA products are generated, so that raw materials are wasted, and the uncapped RNA products need to be removed through column purification in the later period, so that the production cost is increased. Therefore, further improvement of the mRNA capping rate in the co-transcription capping process is advantageous for economical production of mRNA. Disclosure of Invention In a first aspect, the present disclosure provides a class of RNA polymerase variants having an amino acid sequence that has at least 95% sequence identity compared to SEQ ID NO. 1 and comprising at least one mutation selected from the group consisting of K642, S628, T630, G777, relative to SEQ ID NO. 1. In a second aspect, the present application provides a class of biological materials selected from one or more of the following: 1) A polynucleotide molecule encoding the variant; 2) An expression vector comprising the polynucleotide molecule of 1); 3) A host cell comprising a polynucleotide molecule as described in 1), or a host cell comprising an expression vector as described in 2). In a third aspect, the present application provides a method for preparing the above RNA polymerase variant. In a fourth aspect, the application provides a composition comprising at least one RNA polymerase variant according to the application. In a fifth aspect, the application provides a kit comprising at least one RNA polymerase variant according to the application. In a sixth aspect, the application provides the use of an RNA polymerase variant as described above for in vitro transcription to produce RNA. In a seventh aspect, the application also provides a method of preparing RNA. Detailed Description RNA polymerase variants The RNA polymerase variant provided by the application has at least 95% sequence identity with the amino acid sequence SEQ ID NO. 1 of the wild-type T7 RNA polymerase, and comprises any one of the following substitutions or substitution groups, namely K642R+K387S, S628P, T A or G777S, relative to the SEQ ID NO. 1. In some embodiments, the variant has an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 1. In some embodiments, the amino acid sequences of the RNA polymerase variants provided herein have at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the sequences set forth in any one of SEQ ID NOs 2, 3, 5, 6. In some embodiments, the amino acid sequence of the variant is as set forth in any one of SEQ ID NOs 2, 3, 5, 6. Biological material The present application provides polynucleotides encoding the RNA polymerase variants of the present application. Due to the degeneracy of the codons or the preference of the codons of the host cell expressing the polypeptide, the polynucleotide sequence is any polynucleotide sequence encoding a variant without altering the amino acid sequence. In some embodiments, the polynucleotide sequence en