Nanopores are already used to determine the sequence of nucleic acids, and research in this field now focuses on detecting proteins and determining their amino acid sequences. The method relies on detecting electrical currents and their changes as ions and analytes pass through a nanometre-sized pore inserted into an insulating lipid or polymer membrane. Among the most commonly used biological pores are pore-forming proteins, with FraC being the only actinoporin used for this purpose. As part of the doctoral research, we studied and optimised the pores of an actinoporin-like protein from the Orbicella faveolata (Fav) sea coral for sensing purposes. Despite having a less stable α-helical transmembrane domain compared to β-barrel-forming proteins, the mutant RN1-Fav exhibited stable incorporation into polymeric planar membranes and enabled histone discrimination. Extracellular histones are increasingly mentioned as potential biomarkers, whose rapid and accurate detection in real time with nanopores could replace current antibody and mass spectrometry techniques. The results show that electrostatic interactions between positively charged histones and the negative net charge of the pore lumen are most likely the cause of observed changes in electric currents upon addition of histones H4, H3.1, H3K9ac, H3K23ac, and H3Cit. Additionally, we successfully separated the blocks corresponding to histones in the H4/H3Cit, H4/H3.1, and H4/H3Cit/H3K9ac mixtures using model learning. For the most common serum histones, H3Cit and H4, we showed that their concentration and ratio in the mixture can be determined based on the frequency of events. Despite the inability to detect all five histones simultaneously, the robustness of the pore to changes in the amino acid sequence in the pore lumen provides a good starting point for further optimisation of the pore and detection of histones in much more complex samples.
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