<span class="paragraphSection"><div class="boxTitle">Abstract</div>Argonautes are an evolutionarily conserved family of proteins that use guide oligonucleotides for specific recognition of nucleic acid targets. While eukaryotic Argonautes share a conserved structure and act in RNA interference, prokaryotic Argonautes (pAgos) display remarkable structural and functional variations, including various guide and target specificities. Most studied catalytically active pAgos use 5′-phosphorylated DNA guides to recognize and cleave DNA targets. Here, we describe a new group of pAgos from mesophilic bacteria that use RNA guides to cleave DNA targets and are active at physiological temperatures. In contrast to most characterized pAgo nucleases, these proteins can utilize guides of varying lengths containing either a 5′-phosphate or a 5′-hydroxyl group with similar efficiencies. Measurements of the kinetics of target DNA binding show that the annealing of the guide–target duplex in pAgo occurs in an ordered way and that pAgo increases the rate of guide–target interaction in the seed region. We show that the analyzed pAgos can produce alternative DNA products depending on the structure of guide RNA and the cleavage conditions. The results demonstrate that RNA-guided pAgo nucleases are more widespread than previously anticipated and that these pAgos have a relaxed specificity for target DNA cleavage.</span>