State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
Nucleic Acids Res. 2022 May 20;50(9):5226-5238. doi: 10.1093/nar/gkac315.
Argonaute (Ago) proteins are programmable nucleases found in eukaryotes and prokaryotes. Prokaryotic Agos (pAgos) share a high degree of structural homology with eukaryotic Agos (eAgos), and eAgos originate from pAgos. Although eAgos exclusively cleave RNA targets, most characterized pAgos cleave DNA targets. This study characterized a novel pAgo, MbpAgo, from the psychrotolerant bacterium Mucilaginibacter paludis which prefers to cleave RNA targets rather than DNA targets. Compared to previously studied Agos, MbpAgo can utilize both 5'phosphorylated(5'P) and 5'hydroxylated(5'OH) DNA guides (gDNAs) to efficiently cleave RNA targets at the canonical cleavage site if the guide is between 15 and 17 nt long. Furthermore, MbpAgo is active at a wide range of temperatures (4-65°C) and displays no obvious preference for the 5'-nucleotide of a guide. Single-nucleotide and most dinucleotide mismatches have no or little effects on cleavage efficiency, except for dinucleotide mismatches at positions 11-13 that dramatically reduce target cleavage. MbpAgo can efficiently cleave highly structured RNA targets using both 5'P and 5'OH gDNAs in the presence of Mg2+ or Mn2+. The biochemical characterization of MbpAgo paves the way for its use in RNA manipulations such as nucleic acid detection and clearance of RNA viruses.
Argonaute（AGO）蛋白是真核生物和原核生物中发现的可编程核酸内切酶。原核 AGO（pAgos）与真核 AGO（eAgos）具有高度的结构同源性，而 eAgos 起源于 pAgos。尽管 eAgos 专门切割 RNA 靶标，但大多数已鉴定的 pAgos 切割 DNA 靶标。本研究从嗜冷菌粘液化杆菌中鉴定了一种新型的 pAgo，MbpAgo，它更喜欢切割 RNA 靶标而不是 DNA 靶标。与以前研究过的 AGO 相比，如果引导物长度在 15 到 17 个核苷酸之间，MbpAgo 可以利用 5'磷酸化（5'P）和 5'羟基化（5'OH）DNA 引导物（gDNA）来有效地切割 RNA 靶标在规范的切割位点。此外，MbpAgo 在很宽的温度范围内（4-65°C）都具有活性，并且对引导物的 5'-核苷酸没有明显的偏好。单核苷酸和大多数二核苷酸错配对切割效率没有或几乎没有影响，除了位置 11-13 的二核苷酸错配会显著降低靶标切割。MbpAgo 可以在 Mg2+或 Mn2+存在下使用 5'P 和 5'OH gDNA 有效地切割高度结构化的 RNA 靶标。MbpAgo 的生化特性为其在 RNA 操作中的应用铺平了道路，例如核酸检测和清除 RNA 病毒。
