Thermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria, Paenibacillus borealis (PbAgo) and Brevibacillus laterosporus (BlAgo), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

嗜热 Argonaute 蛋白 (Agos) 已被证明可以利用小 DNA 引导物在体外切割互补 DNA，这在核酸检测方面显示出巨大的潜力。在本研究中，我们探索了嗜温 Agos 通过与变构转录因子 (aTF) 配合来检测小分子。来自嗜温细菌的两种 Ago，即北极类芽孢杆菌( Pb Ago) 和侧孢短芽孢杆菌( Bl Ago)，通过使用 5'-磷酸化和 5'-羟基化 DNA 向导，在中等温度 (37 °C) 下显示出单链 DNA 的核酸酶活性。两种 Ago 均可对双链 DNA 进行可编程切割，尤其是在质粒中富含 AT 的区域。此外，我们基于Agos的DNA引导DNA切割和HosA的变构效应，开发了一种简单且低成本的对羟基苯甲酸检测方法，拓展了Agos在小分子检测方面的潜在应用。