Onconase is a distinctive RNase due to a prominent feature that enables this protein to exhibit selective toxicity against cancer cells. Therefore, in this study, we aimed to modify Onconase and then assess engineered Onconase characteristics such as cellular uptake, cytotoxicity, the ability to evade RNase inhibitor, and protein functionality. For this purpose, six models of Onconase enzyme were designed based on protein docking and Molecular Dynamic simulation results. For this purpose, fve mutations (K31A, K55A, S72C, R73A and K76A) were selected, because MD results showed that this combination was efective for the goal of this study. Then, the engineering Onconase was synthesized and cloned into the pET22b (+). Recombinant protein was produced in E. coli BL21 (DE3) and after protein purifcation, enzyme properties and cytotoxicity were investigated. Modeling results revealed that replacement of lysine with alanine could lead to an increase in surface charge and cellular uptake. Furthermore, these changes had no infuence on the structural stability of Onconase and the enzyme could still evade RI. Experimental results revealed that the activity of the recombinant Onconase produced in this study was 213±19 U/mol, which is 3.5 times lower than RNase A (746±13 U/nmol); however, this enzyme is resistant to RI. Also, our recombinant Onconase was able to induce apoptosis in HeLa cancer cells at 1.99 µg/uL Onconase, whereas RNase A had no cytotoxic efects on these cells under similar conditions. Our fndings suggested that an in silico study could be highly useful for enzyme engineering and drug discovery.

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