Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection

Bioresources and Bioprocessing

Yinhua Li¹  He Fei¹  Kai Li²  Zhonglei Li³  Qian Liu³  Yan Feng³  Yuesheng Chong³  Xiang Guo³  Guanhua Xun³ 

[1] Department of Obstetrics and Gynaecology, The Fifth People’s Hospital of Shanghai Affiliated To Fudan University, 200240, Shanghai, People’s Republic of China;GeneTalks Biotechnology Inc., 410013, Changsha, Hunan, China;State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 200240, Shanghai, People’s Republic of China;

关键词: Thermophiles;    Argonaute;    Nuclease;    Multiplex;    Nucleic acid detection;   

DOI  :  10.1186/s40643-021-00401-6

来源: Springer

【 摘 要 】

Argonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs. We validated that the cleavage products with 5'-phosphorylated ends can be used as a new guide to induce a new round of cleavage. Based on the reprogrammable capacity of Ago’s stepwise activity, we established a rapid and specific platform for unambiguous multiplex gene detection, termed Renewed-gDNA Assisted DNA cleavage by Argonaute (RADAR). Combined with a pre-amplification step, RADAR achieved sensitivity at the femtomolar level and specificity with at least a di-nucleotide resolution. Furthermore, RADAR simultaneously discriminated among multiple target sequences simply by corresponding multiple guides. We successfully distinguished four human papillomavirus serotypes from patient samples in a single reaction. Our technique, based on the unique properties of Ago, provides a versatile and sensitive method for molecular diagnosis.

【 授权许可】

CC BY