In contrast to eukaryotic Argonaute proteins that act on RNA targets, prokaryotic Argonautes (pAgos) can target DNA, using either small RNA or small DNA guides for its recognition. Since pAgos can recognize only a single strand of DNA and lack a helicase activity, it remains unknown how double-stranded DNA can be bound both in vitro and in vivo. Here, using in vitro reconstitution and footprinting assays we analyze formation of specific complexes with target DNA by a catalytically inactive pAgo, RsAgo from Rhodobacter sphaeroides programmed with small guide RNAs. We showed that RsAgo can recognize a specific site in double-stranded DNA after stepwise reconstitution of the complex from individual oligonucleotides or after prior melting of the DNA target. When bound, RsAgo stabilizes an open DNA bubble corresponding to the length of the guide molecule and protects the target DNA from nuclease cleavage. The results suggest that RsAgo and, possibly, other RNA-guided pAgos cannot directly attack double-stranded DNA and likely require DNA opening by other cellular processes for their action.

与作用于 RNA 靶标的真核 Argonaute 蛋白相反，原核 Argonaute (pAgos) 可以靶向 DNA，使用小 RNA 或小 DNA 引导进行识别。由于 pAgos 只能识别单链 DNA 并且缺乏解旋酶活性，因此目前尚不清楚如何在体外和体内结合双链 DNA。在这里，我们使用体外重建和足迹分析分析了来自球形红杆菌的催化失活 pAgo（RsAgo）与目标 DNA 形成的特定复合物，该 pAgo 是由小引导 RNA 编程的。我们证明，在从单个寡核苷酸逐步重建复合物后或在 DNA 靶标预先解链后，RsAgo 可以识别双链 DNA 中的特定位点。结合后，RsAgo 会稳定与引导分子长度相对应的开放 DNA 气泡，并保护目标 DNA 免受核酸酶切割。结果表明，RsAgo 以及可能的其他 RNA 引导的 pAgos 不能直接攻击双链 DNA，并且可能需要其他细胞过程打开 DNA 才能发挥作用。