A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia  

作　　者：Qi Liu Wanping Chen Yue Zhang Fengyang Hu Xiaoman Jiang Fei Wang Yang Liu Lixin Ma 

机构地区：[1]State Key Laboratory of Biocatalysis and Enzyme Engineering,Hubei Collaborative Innovation Center for Green Transformation of Bioresources,Hubei Key Laboratory of Industrial Biotechnology,School of Life Sciences,Hubei University,Wuhan 430062,China [2]School of Pharmacy,Qingdao University,Qingdao 266071,China [3]Hubei Jiangxia Laboratory,Wuhan 430200,China

出　　处：《Acta Biochimica et Biophysica Sinica》2023年第8期1204-1212,共9页生物化学与生物物理学报（英文版）

基　　金：supported by the grants from the China National Key Research and Development(R&D)Program(No.2021YFC2100100);the Wuhan Science and Technology Bureau Knowledge Innovation Special Dawning Program(No.2022020801020326);the Postdoctoral Innovation Project of Shandong Province(No.SDCX-ZG-202203010);the China Postdoctoral Science Foundation(No.2022M721074).

摘　　要：Argonaute(Ago)proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements.Almost all characterized pAgos prefer to cleave DNA targets.Here,we describe a novel pAgo from Verrucomicrobia bacterium(VbAgo)that can specifically cleave RNA targets rather than DNA targets at 37℃ and function as a multiple-turnover enzyme showing prominent catalytic capacity.VbAgo utilizes DNA guides(gDNAs)to cleave RNA targets at the canonical cleavage site.Meanwhile,the cleavage activity is remarkably strengthened at low concentrations of NaCl.In addition,VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets,and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage.Moreover,VbAgo can efficiently cleave highly structured RNA targets at 37℃.These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.

关 键 词：RNA target specificity Verrucomicrobia bacterium prokaryotic argonaute programmable nucleases 

分 类 号：Q51[生物学—生物化学]