Hubei University

Wanping Chen

Lixin Ma

Fei Wang

Longyu Wang

Xiaochen Xie

Yang Liu

Jiakai Cui

Qi Liu

Miaomiao Chen

Fuyong Huang

An efficient synthesis and assembly method for the large fragment DNA based on the programmable nuclease Argonaute, specifically includes: constructing and treating antibiotic resistance gene reconstructed vectors with linearization, dividing a target DNA into multiple small DNA fragments and then synthesizing the small DNA fragments, followed by loading the synthesized small DNA fragments to the antibiotic resistance gene reconstructed vectors; and the SLIC and resistance gene reconstruction are used to achieve assembly of the target DNA. The method combines the SLIC with a resistance gene reconstruction strategy, allowing for the assembly of 5-6 small fragments in a single resistance gene reconstruction, which is more efficient and time-saving. Moreover, the number of the resistance gene reconstructions can be flexibly chosen according to the length of the DNA fragments. Mutations are not introduced caused by PCR, and the reconstructed large fragment do not need a second sequencing, saving time and costs.

Efficient synthesis and assembly method for large fragment DNA based on programmable nuclease Argonaute

AArgonaute (PfAgo) mutant protein with mesophilic target nucleic acid cleavage activity and an application thereof are provided. The PfAgo mutant protein has an amino acid mutation on at least one of a 617position and a 618position relative to the sequence shown in SEQ ID NO: 1, specifically, K617E/G and/or L618Y/F/W/G. Compared with a wild-type PfAgo protein, the provided PfAgo mutant protein has activity in the temperature range of 30-95 Celsius degree (° C.), effectively expanding application range of prokaryotic Argonaute (pAgo) protein.

PfAgo mutant protein mesophilic target nucleic acid cleavage activity and application thereof

An application of a prokaryotic Argonaute protein with only a cleavage activity of a target ribonucleic acid (RNA) in RNA editing is provided. The Argonaute protein is derived from a mesophilic prokaryotes, and its amino acid sequence is shown in SEQ ID NO: 1 or a protein with high similarity to SEQ ID NO: 1 and the same function. This protein has binding activity to a single-stranded guide DNA and nuclease activity only to the target RNA complementarily paired with the single-stranded guide DNA. Therefore, the protein can be utilized for in vitro and in vivo targeted RNA editing, providing a new powerful tool for RNA editing.

Application of prokaryotic Argonaute protein with only RNA target cleavage activity in RNA editing

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Wuhan, China

Wanping Chen