Argonaute (Ago) proteins mediate target recognition guiding miRNA to bind complementary mRNA primarily in the seed region. However, additional pairing can occur beyond the seed, forming a supplementary duplex that can contribute to the guide-target affinity. In order to shed light on the connection, between protein-RNA interactions and miRNA-mRNA seed and supplementary duplex mobility, we carried out molecular dynamics simulations at the microsecond time-scale using a different approach compared to the ones normally used. Until now, theoretical investigations with classical MD on Ago-RNA complexes have been focused primarily on pure water solvent, which mimics the natural environment of biological molecules. Here, we explored the conformational space of a human Ago2 (hAgo2) bound to the seed + supplementary miRNA-mRNA duplex, using the solvent environment as a molecular probe. MD simulations have been performed in a mixture of water/MeOH at a molar ratio of 70 : 30 as well as in pure water for comparison. Our findings revealed that the mixed solvent promotes protein RNA association, principally enhancing salt-linkages between basic amino acid side-chains and acidic phosphates of the sugar-phosphate backbone. The primary effect registered was the restriction of supplementary duplex flexibility and the stabilization of the miRNA 3' terminus. Interestingly, we observed that the influence of the solvent appears to have almost no impact on the conformation of the seed duplex.