Excessive Existence of Positively Charged Amino Acids Caused Off-Target Recognition in the Seed Region of Clostridium butyricum Argonaute

<i>Clostridium butyricum</i> Argonaute (<i>Cb</i>Ago) can achieve DNA-guided DNA recognition and cleavage at physiological temperatures (~37 °C), making it a promising tool for gene editing. However, its significant off-target effects, particularly associated with the seed region (sites 2-8), pose challenges for precise gene therapy. This study focuses on enhancing the specificity of the seed region recognition to mitigate these off-target effects. We investigated the molecular recognition process between the <i>Cb</i>Ago-gDNA complex and the seed region of the target DNA using molecular dynamics simulations and automated path searching. Our findings reveal that positively charged residues located in an α-helix domain at the DNA-protein interface (R279, H285, K287, K288, K291, K298) facilitate rapid binding to the DNA phosphate backbone. Such interaction enhances the pre-formation of the DNA double helix, reducing the reliance on base complementarity during duplex pairing. Further simulations showed that alanine replacement of these positively charged residues led to significantly improved sequence specificity for the target DNA seed region. Collectively, these results offered critical insights into the origin of off-target recognition by <i>Cb</i>Ago in its seed region, shedding lights on its fidelity enhancement.