Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens

Abstract

Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo functions as a programmable DNA endonuclease by utilizing both short 5′-phosphorylated and 5′-hydroxylated single-stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double-stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago-based DNA detection toolbox.

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo

“…In summary, similar to previously characterized pAgos from thermophilic prokaryotes such as TtAgo, PfAgo, MjAgo, and FpAgo (Swarts et al, 2014a;Swarts et al, 2015;Willkomm et al, 2017;Zander et al, 2017;Guo et al, 2021), TtdAgo prefers to use DNA guides to cleave DNA targets. This result is similar to a simultaneous study of TtrAgo (TtdAgo) reported earlier by Fang et al (2022). However, in contrast to most eAgos and pAgos including hAgo2, IbAgo, KmAgo, and MbpAgo (Rivas et al, 2005;Kropocheva et al, 2021;Li W. et al, 2022), whose target cleavage resulted in a shift of the cleavage site mediated by 5′-OH guides compared to that using 5′-P-gDNA as shown by Fang et al (2022), we did not observe the cleavage position shift for TtdAgo with 5′-P-gDNA compared to that with 5′-OH-gDNA.…”

“…This result is similar to a simultaneous study of TtrAgo (TtdAgo) reported earlier by Fang et al (2022). However, in contrast to most eAgos and pAgos including hAgo2, IbAgo, KmAgo, and MbpAgo (Rivas et al, 2005;Kropocheva et al, 2021;Li W. et al, 2022), whose target cleavage resulted in a shift of the cleavage site mediated by 5′-OH guides compared to that using 5′-P-gDNA as shown by Fang et al (2022), we did not observe the cleavage position shift for TtdAgo with 5′-P-gDNA compared to that with 5′-OH-gDNA. This result may be due to the different targets and reaction conditions such as the concentration of divalent ion and the temperature both being different.…”

“…Our study thus demonstrated that TtdAgo cleaved DNA at temperatures ranging from 30 °C to 95 °C and had good DNA cleavage activity at 70-80 °C, which is consistent with the previous study (Fang et al, 2022). Furthermore, Fang et al (2022) also found that TtdAgo was active at Mn 2+ concentrations ≥ 0.1 mM, which is consistent with our results.…”

“…For TtdAgo, a minimum of 16 nt gDNA is required. Furthermore, TtdAgo is most active with a length of 17-19 nt for 5′-P-gDNA and 16-19 nt for 5′-OH-gDNA, with a lower efficiency observed with longer or shorter guides, which is consistent with the previous study (Fang et al, 2022).…”

“…The presence of excess Co 2+ is likely to inhibit the activity of TtdAgo. In contrast, Fang et al (2022) did not observe the cleavage product under the presence of Mg 2+ , which may be due to the 0.5 mM Mg 2+ they used being lower than that required for TtdAgo to be actively functional as we demonstrated that TtdAgo functioned at Mg 2+ concentrations ≥ 2.5 mM (Figure 2D). These studies thus demonstrated that it is crucial to obtain accurate concentrations of divalent cations for the functionality of pAgos.…”