A nuclease-associated short pAgo system from Pleomorphomonas koreensis with dual DNA and RNA collateral activity.

Argonaute (Ago) proteins play pivotal roles in nucleic acid targeting across eukaryotes and prokaryotes. While eukaryotic Ago (eAgo) proteins utilize RNA guides to cleave complementary RNA, prokaryotic Ago (pAgo) proteins often employ DNA guides for DNA targeting. This study focuses on the short pAgo system from Pleomorphomonas koreensis (Pko-SPARDA), which consists of a DUF4365-APAZ protein (PkoRE) and an Ago protein (PkoAgo). Upon RNA-guided target DNA recognition, Pko-SPARDA exhibits nonspecific cleavage activity toward ssDNA, dsDNA, and RNA, demonstrating potential for nucleic acid detection. Biochemical characterization revealed that Pko-SPARDA prefers Mn2+ or Mg2+ as cofactors, operates optimally at 40-45 °C, and is sensitive to mismatches in the central and 3'-supplementary regions of the guide RNA. Notably, the system accommodates a range of guide lengths (14-40 nt) and shows higher activity with 5'-phosphorylated guides. Additionally, guide RNA 5'-phosphorylation universally enhances Pko-SPARDA trans-cleavage activity, and this principle extends to microRNA-guided activation, advancing the understanding of short pAgos and highlighting Pko-SPARDA's utility in molecular diagnostics by bridging a gap left by CRISPR systems due to its lack of sequence constraints like PAM motifs.