A mesophilic Argonaute from Clostridium formicaceticum with efficient DNA cleavage activity guided by small DNA

Jianrui Li¹, Meixia Yu¹, Zhijia Yang¹, Yueheng Zhou¹, Yunpeng Teng¹, Zijian Wang¹, Jian Chen², Jinsheng Lai³, Beibei Xin⁴

Affiliations

¹ State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
² State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China; Key Laboratory of Genome Editing Research and Application, Ministry of Agriculture and Rural Affairs, Beijing 100193, China.
³ State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China; Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing 100193, China; Sanya Institute of China Agricultural University, Sanya 572025, China; Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China; Key Laboratory of Genome Editing Research and Application, Ministry of Agriculture and Rural Affairs, Beijing 100193, China. Electronic address: jlai@cau.edu.cn.
⁴ State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. Electronic address: bxin@cau.edu.cn.

PMID: 40157363
DOI: 10.1016/j.str.2025.03.002

A mesophilic Argonaute from Clostridium formicaceticum with efficient DNA cleavage activity guided by small DNA

Jianrui Li et al. Structure. 2025.

. 2025 Jun 5;33(6):1101-1111.e3.

doi: 10.1016/j.str.2025.03.002. Epub 2025 Mar 28.

Authors

Jianrui Li¹, Meixia Yu¹, Zhijia Yang¹, Yueheng Zhou¹, Yunpeng Teng¹, Zijian Wang¹, Jian Chen², Jinsheng Lai³, Beibei Xin⁴

Affiliations

¹ State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China.
² State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China; Key Laboratory of Genome Editing Research and Application, Ministry of Agriculture and Rural Affairs, Beijing 100193, China.
³ State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China; Center for Crop Functional Genomics and Molecular Breeding, China Agricultural University, Beijing 100193, China; Sanya Institute of China Agricultural University, Sanya 572025, China; Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China; Key Laboratory of Genome Editing Research and Application, Ministry of Agriculture and Rural Affairs, Beijing 100193, China. Electronic address: jlai@cau.edu.cn.
⁴ State Key Laboratory of Maize Bio-breeding, National Maize Improvement Center, Frontiers Science Center for Molecular Design Breeding, Department of Plant Genetics and Breeding, China Agricultural University, Beijing 100193, China. Electronic address: bxin@cau.edu.cn.

PMID: 40157363
DOI: 10.1016/j.str.2025.03.002

Abstract

We characterized a new Argonaute protein (pAgo), CfAgo, from the mesophilic bacterium Clostridium formicaceticum. CfAgo possesses DNA-guided DNA endonuclease activity and cleaves DNA targets at the canonical site. It is active from 28°C to 75°C and prefers DNA guides with a 5'-phosphate group and thymidine as the first nucleotide. Cleavage activity is reduced by single-nucleotide mismatches in the seed, central, and 3'-supplementary regions of guides, with stronger mismatch discrimination observed for 5'hydroxylated (5'OH) guides compared to 5'phosphorylated (5'P) guides. Moreover, structural analysis suggests that the MID domain of CfAgo is crucial for recognizing the 5' guide and it influences the binding specificity. CfAgo catalyzes programmable cleavage of double-stranded DNA in AT-rich regions in the presence of Mn²⁺ and Mg²⁺ ions at appropriate salt concentrations. These properties could make CfAgo a promising tool for DNA manipulation such as nucleic acid detection and cleavage.

Keywords: Clostridium formicaceticum; argonaute; endonuclease; mesophilic bacterium.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

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A mesophilic Argonaute from Clostridium formicaceticum with efficient DNA cleavage activity guided by small DNA

Affiliations

A mesophilic Argonaute from Clostridium formicaceticum with efficient DNA cleavage activity guided by small DNA

Authors

Affiliations

Abstract

Conflict of interest statement

MeSH terms

Substances

LinkOut - more resources

Full Text Sources