Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory ‘pancreatic-type’ RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.

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