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T7-RNA polymerase mutant and application thereof

A technology of T7-RNA, 1.T7-RNA, applied in the field of nucleic acid tool enzymes and nucleic acid biology, can solve the problem of maintaining high-efficiency transcription RNA product heterogeneity

Active Publication Date: 2021-05-25
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these existing technologies also cannot make it maintain high-efficiency transcription while reducing RNA product heterogeneity

Method used

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  • T7-RNA polymerase mutant and application thereof
  • T7-RNA polymerase mutant and application thereof
  • T7-RNA polymerase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: In vitro termination efficiency detection using T7--RNA polymerase mutant

[0026] (1) Expression and purification of T7-RNA polymerase mutant

[0027]The T7-RNA polymerase mutants S43A, S43L, S43K, S43D, S43Y and S43F were respectively constructed by molecular cloning, and then the prokaryotic expression vector pQE82L containing these mutants was transformed into the E.coli BL21 expression strain, and the bacteria were picked and expanded for cultivation , place the bacteria in LB medium containing 100 μg / ml ampicillin and culture on a shaker at 37°C until the OD600 value is close to 1.2, and then add isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.5mM (IPTG) was induced to express on a shaking table at 30°C for 4h, and then centrifuged at 4°C and 5000rpm for 20min to collect the cell pellet, and then the cell was fully resuspended in a solution containing 300mM NaCl, 20mM Tris-HCl (pH=7.5), 0.5mg / ml lysozyme, 0.5mM DTT lysate, frozen at ...

Embodiment 2

[0038] Example 2: Comparison of T7-RNA polymerase mutant S43Y and wild type for class I termination efficiency from different sources

[0039] (1) Acquisition of transcription reaction template

[0040] The sequences of several class I terminators T3 TΦ, thr attenuator, rrnBT1 terminator and rrnC terminator reported in T3 phage and Escherichia coli by molecular cloning (respectively see the sequence table SEQ ID NO.3-6 ) to replace the neck loop structure sequence of the class I terminator T7 TΦ in the vector pETsnrs-1 (see the sequence listing SEQ ID NO.7). Carry out PCR amplification with the general primer of embodiment 1 after the successful construction of vector, then utilize DNA purification kit Clean&Concentrator TM -5 Purify and measure the concentration of the PCR product.

[0041] (2) Detection of transcription termination efficiency in vitro

[0042] The in vitro transcription reaction (10μL) contained 40mM Tris-HCl (pH=8.0), 16mM MgCl 2 , 2mM spermidine, 5mM D...

Embodiment 3

[0043] Example 3: Comparison of T7-RNA polymerase mutant S43Y and wild type on the termination efficiency of class II from different sources

[0044] (1) Acquisition and purification of transcription reaction templates

[0045] By means of molecular cloning, the class II termination signal sequences (respectively see SEQ ID NO. There is a termination sequence. Similarly, the constructed vector was amplified by PCR using the universal primers described in Example 1, and then the PCR product was purified and its concentration was determined using a DNA purification kit.

[0046] (2) Detection of transcription termination efficiency in vitro

[0047] The in vitro transcription reaction components contained 40mM Tris-HCl (pH=8.0), 16mM MgCl 2 , 2mM spermidine, 5mMDTT, 4mM ATP, GTP, CTP, UTP, 0.3μL RNase inhibitor, 0.2μL pyrophosphatase, 50nM RNA polymerase, 14nM PCR template, add DEPC water to 10μL. The transcription reaction conditions were incubation at 37°C for 1 h, followe...

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Abstract

The invention discloses a T7-RNA polymerase mutant. The mutant is obtained by replacing serine at the 43rd site from the N end of an amino acid sequence of wild T7-RNA polymerase with A-type amino acid or B-type amino acid, wherein the A amino acid is tyrosine, phenylalanine, leucine, lysine or aspartic acid, and the B amino acid is tryptophan, isoleucine, arginine, asparagine, glutamine, glutamic acid or proline. The T7-RNA polymerase mutant is suitable for synthesis of RNA internally containing a termination signal and RNA with a hairpin structure formed at the tail end. The method can be widely applied to the aspects of in-vitro transcription, RNA synthesis, gene editing, RNA drug synthesis, in-vivo protein expression or cell-free protein expression in-vitro translation systems, transcription terminator research, RNA-dependent RNA polymerase activity research, biological transcription regulation element synthesis and the like.

Description

technical field [0001] The invention belongs to the field of nucleic acid tool enzyme and nucleic acid biology, in particular to a phage body T7-RNA polymerase mutant and its application. Background technique [0002] RNA (ribonucleic acid), as a class of biological macromolecules for genetic information transmission, widely exists in eukaryotes, prokaryotes, some viruses and viroids, and has many different types and functions. In addition to the three types of RNA initially identified: mRNA, rRNA, and tRNA, several novel types of RNA such as microRNA (miRNA) (Cheng et al., 2005), long non-coding RNA (lncRNA) (Dey et al. ., 2014), Circular RNAs (circRNA) (Memczak et al., 2013), etc. have also become hot fields in RNA research in recent years. In addition, the gradual deepening of RNA-related research reveals that RNA has a very important application value in disease treatment. siRNA and mRNA synthesized in vitro can become important drugs for RNA-targeted therapy (Sahin et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12P19/34C12Q1/686
CPCC12N9/1247C12P19/34C12Q1/686C12Y207/07006C12Q2521/101
Inventor 朱斌吴慧
Owner HUAZHONG UNIV OF SCI & TECH
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