Argonaute proteins (Agos) are found in all domains of life. Eukaryotic Argonautes (eAgos) are the catalytic core of RNA interference machinery, whereas prokaryotic Argonautes (pAgos) provide innate immunity to exogenic nucleic acids, such as plasmid or bacteriophage DNA. All pAgos share a similar mechanism of action, they use DNA or RNA guides to recognize complementary DNA or RNA targets. Upon target recognition, pAgo either cleaves the target strand or employs effector proteins, which leads to degradation of invading nucleic acids or, in some cases, cell death [3,4]. Knowledge of preferred guides and targets reveals valuable information about how pAgos obtain their guides and recognize invading nucleic acids.
The object of this study is an archaeal Argonaute protein AfAgo from Archaeoglobus fulgidus. Several groups published crystal structures of AfAgo with various nucleic acid substrates [2]. Therefore, it served as a structural Argonaute model in RNA interference studies. Although AfAgo was widely-studied, recently new features of AfAgo were described, such as ability to form homodimers and create DNA loops [1]. Latest crystal structure of AfAgo obtained in our group revealed several base specific contacts with three terminal base pairs of guide-target duplex. In addition to that, we showed that in vivo AfAgo binds RNA guides (gRNA) enriched in 5'-AUU sequence. Studies show that similarly to eAgos, pAgos may exhibit specificity to the guide strand 5' terminal nucleotide and respective complementary target strand nucleotide. However, this is the first example of more extensive specificity towards guide and target sequence [3].
In this study we compare AfAgo affinity towards RNA guides with different 5'-end sequence using electrophoretic mobility shift assay (EMSA). Results provide experimental evidence that AfAgo specifically recognizes RNA guides with 5'-AUU sequence. We also demonstrate that AfAgo:gRNA binary complex preferentially binds complementary DNA targets. These results challenge previous knowledge about AfAgo, since it was thought that AfAgo uses ssDNA guides and recognizes DNA targets, possibly due to the use of nonoptimal 5'-sequence guides in experiments [2].