# A Mesophilic Argonaute from Cohnella algarum Mediates Programmable DNA/RNA Cleavage with Distinctive Guide Specificity

## TL;DR

The study explores CalAgo, a protein from Cohnella algarum, which can cut DNA and RNA using specific short guides at normal temperatures.

## Contribution

CalAgo's unique guide specificity and biochemical properties are characterized, offering new insights into mesophilic prokaryotic Argonaute proteins.

## Key findings

- CalAgo cleaves DNA and RNA using 15–21 nucleotide guides at physiological temperatures.
- CalAgo shows sensitivity to guide–target mismatches, especially at positions 12 and 15.
- Structural modeling links CalAgo's guide specificity to its PAZ domain's 3′-guide binding features.

## Abstract

Argonaute (Ago) proteins are ubiquitous across all domains of life. Some prokaryotic Agos (pAgos) function as endonucleases that utilize short nucleic acid guides to recognize and cleave complementary targets. Yet, considerable diversity within pAgos leaves many of their biochemical and functional features insufficiently understood. This study characterizes CalAgo, an pAgo from the mesophilic bacterium Cohnella algarum, which demonstrates DNA-guided DNA endonuclease and RNA endonuclease activities at physiological temperatures. CalAgo’s cleavage activity depends on Mn2+ and Mg2+ ions and remains effective across a wide range of temperatures and pH levels. CalAgo utilizes only short guides ranging from 15 to 21 nucleotides (nt) in length, in contrast to other reported pAgos that target both DNA and RNA, which often exhibit broad guide selectivity. CalAgo preferentially loads 5′-phosphorylated guides and shows no significant preference among guides with different 5′-end nucleotides. CalAgo is sensitive to guide–target mismatches, and introducing a single mismatch at positions 12 or 15 of the guide strand abolished detectable activity. Structural modeling suggests that this unique guide specificity may originate from structural features in its PAZ domain involved in 3′-guide binding. In summary, this study deepens insight into mesophilic pAgos and supports their potential utility in nucleic acid-based applications.