To confirm that NgAgo uses ssDNA gu

To confirm that NgAgo uses ssDNA guides, we analyzed the nucleic acids purified from NgAgo expressed in Escherichia coli, as those bacteria produce both 5′-phosphorylated ssRNAs and ssDNAs, and the nucleic acids of proper length (13–25 nt) naturally bind to prokaryotic Argonautes11,12. Nuclease digestion confirmed that DNA but not RNA binds NgAgo (Fig. 1a). To test whether the NgAgo expressed by E. coli can cleave target DNA at 37 °C in vitro, we performed an in vitro plasmid cleavage assay. We designed three 5′ phosphorylated 24-nucleotide (nt) ssDNA guides, among which two guides were complementary to each other (‘FW’ and ‘RV’ guides) and corresponded to a target site in plasmid pACYCDuet-eGFP, which had no homologous sequence to the NgAgo-encoding plasmid pGEX6P-1 (Fig. 1b). The other guide had a random sequence without overlap with pACYCDuet-eGFP (noncomplementary (‘NC’) guide). We also designed a pair of 5′-phosphorylated RNA guides corresponding
to the same target site. Before the cleavage assay, we replaced the native nucleic acids bound to the purified) NgAgo with our designed guides by incubating NgAgo with the guides at 55 °C for 1 h.
NgAgo could not catalyze cleavage without guide or with the NC guide (Fig. 1b). When supplied with either of the complementary FW or RV guides, NgAgo could nick the negatively supercoiled plasmid at 37 °C; when supplied with both FW and RV guides, NgAgo linearized the plasmid. However, neither ssDNA guides without 5′ phosphorylation nor 5′-phosphorylated ssRNA guides led to plasmid cleavage (Fig. 1c). Thus, NgAgo can cleave DNA double helix targets at
37 °C with 5′ phosphorylated ssDNA guide.

結果 (中文) 1:

To confirm that NgAgo uses ssDNA guides, we analyzed the nucleic acids purified from NgAgo expressed in Escherichia coli, as those bacteria produce both 5′-phosphorylated ssRNAs and ssDNAs, and the nucleic acids of proper length (13–25 nt) naturally bind to prokaryotic Argonautes11,12. Nuclease digestion confirmed that DNA but not RNA binds NgAgo (Fig. 1a). To test whether the NgAgo expressed by E. coli can cleave target DNA at 37 °C in vitro, we performed an in vitro plasmid cleavage assay. We designed three 5′ phosphorylated 24-nucleotide (nt) ssDNA guides, among which two guides were complementary to each other (‘FW’ and ‘RV’ guides) and corresponded to a target site in plasmid pACYCDuet-eGFP, which had no homologous sequence to the NgAgo-encoding plasmid pGEX6P-1 (Fig. 1b). The other guide had a random sequence without overlap with pACYCDuet-eGFP (noncomplementary (‘NC’) guide). We also designed a pair of 5′-phosphorylated RNA guides correspondingto the same target site. Before the cleavage assay, we replaced the native nucleic acids bound to the purified) NgAgo with our designed guides by incubating NgAgo with the guides at 55 °C for 1 h.NgAgo 不能催化裂解无导轨或与数控指南 (图 1b)。如果提供两个互补的 FW 或 RV 指南均，NgAgo 可以尼克负超螺旋质粒在 37 ° C;当提供 FW 和 RV 指南，NgAgo 线性化质粒。然而，既不 ssDNA 指导没有 5 ′ 磷酸和 5 ′-磷酸化异性指南导致质粒乳沟 （图 1 c）。因此，NgAgo 可以劈开在 DNA 双螺旋结构目标37 ° C，5 ′ 磷酸化 ssDNA 指南。

結果 (中文) 3:

确认ngago采用单链DNA的指南，我们分析了在大肠杆菌中表达的核酸纯化ngago，这些细菌能产生5′-磷酸化ssrnas和单链DNA，和适当长度的核酸（13–25台币）自然结合原核argonautes11,12。酶切证实DNA而不是RNA结合ngago（图1A）。为了测试是否为大肠杆菌表达可以切割靶DNA在37°C在体外ngago，我们进行了在体外质粒裂解法。我们设计了三个5′磷酸24核苷酸（NT）单链DNA的指南，其中2的指导是相辅相成的（"fw "和" RV指南）和对应的pacycduet EGFP质粒靶位点，没有同源序列编码质粒pgex6p-1的ngago（图1B）。其他指南有一个随机序列没有pacycduet EGFP重叠（互补（"nc”）指南）。我们还设计了一对5个磷酸核糖核酸引导相应的到同一个目标站点。在切割试验，我们取代天然核酸结合的纯化）与我们的设计指南的孵化ngago随着导游在55°C 1小时ngagongago不能催化裂解无指导或与数控导轨（图1B）。当提供的互补FW或RV指南，ngago尼克的负超螺旋质粒能在37°C；当提供FW和RV指南，ngago线性化的质粒。然而，无论是单链DNA的导游没有5′磷酸化和磷酸化5′- ssRNA导游带队质粒切割（图1c）。因此，ngago能切割DNA双螺旋的目标37°C磷酸化5′ssDNA指南。