A Resurfaced Fluorescent Protein H₃₉GFP As A Smart Vector For Nucleic Acid Delivery

Gene therapy is the the therapeutic delivery of nucleic acid polymers into a patient's cells as a drug to treat disease, which is highly dependent on the recent research results of modern medicine and molecular biology technologies. Nowadays, the only way to achieve gene therapy is the "gene supplement". In this way, the abnormal gene doesn't be removed, but the exogenous normal gene will be introduced into cells, and the expression product can compensate for the defective gen e function. Providing effective and safe DNA carriers remains a major challenge for gene delivery so far.Protein resurfacing is a useful technique in endowing protein with novel characteristics, which consists of rationally designing a protein's surface while retaining the overall fold, core domain and boundary regions of the intact protein. Supercharged green fluorescent proteins?Sc GFP? were developed as a new class of resurfaced proteins with high surface net charge. Sc GFP can simply form complexes with negatively charged nucleic acids by electrostatic interaction and then deliver them into mammalian cells. Sc GFPs have several advantages over conventional delivery vectors, including low cytotoxicity, high versatility. As a DNA delivery vector, Sc GFP can transfer various cell types, including normal cells and cancer cells, and is not selective. This prompted us to desire a smart DNA carrier which can respond to the external environment and selectively transfer DNA into target cells. Recently, a new kind of Sc GFPs, containing 39 histidine residues on the surface?H 39GFP?, was developed by further resurfacing of Sc GFP. In this work, we expect to developH₃₉GFP as a smart vector for nucleic acid delivery. The main results are as follows:?1? Construction of a prokaryotic expression plasmid containing the gene of H₃₉GFP, expression and purification of H₃₉GFP. The gene sequence encoding H₃₉GFP?h39gfp? was reversely translated from the amino acid sequence of H 39 GFP and optimized according to E. coli codon usage. The full length gene inserted in plasmid p UC19, was obtained through whole gene synthesis by company. The recombinant plasmid p ET28-h39 gfp was constructed by using “one pot” method and transformed into E. coli for the expression of H₃₉GFP. The purification of H₃₉GFP was performed on an AKTA purifier system by using Ni-NTA agarose column and desalinating column. H₃₉GFP with high purity was characterized by SDS-PAGE, UV and fluorescence spectrophotometries.?2? Characteriazation of surface charge of H₃₉GFP and the electrostatic interaction between H₃₉GFP and DNA. It was found that the surface charge of H₃₉GFP could be controlled by pH and Ni²⁺, characterized by Zeta potential. As a result, slightly acidic environment or the addition of Ni²⁺ made H₃₉GFP positively charge. Its formation was demonstrated by atomic force microscopy. We found that the H₃₉GFP/DNA formed nano-complex under the condition of H₃₉GFP positively charge. Additionally, the fluorescence anisotropy of H₃₉GFP decreased significantly after being encapsulated in nano-complex, which could be reasoned by the homo-FRET among H₃₉GFP molecules in H₃₉GFP/DNA complex. As the ss DNA was labeled with a quencher, the fluorescence of H₃₉GFP in the nanocomplex was efficiently quenched.?3? A series of novel logic gates were constructed based on the electrostatic interaction between H₃₉GFP and DNA. The pH value of the solution, the addition of Ni²⁺ or the addition of DNA were defined as inputs of the logic gates, and the fluorescence change of H₃₉GFP was used as the output of the logic gates to achieve “OR”, “INHIBIT” and “AND” gates.?4? H₃₉GFP worked as a smart nucleic acid delivery vector. In order to reflect the ability of H₃₉GFP to transport DNA into Hela cells, we used confocal laser scanning microscopy to observe the green fluorescence from H₃₉GFP and red fluorescence from Cy5-labeled DNA under different conditions. We found that only in the presence of acidic environment or the addition of Ni²⁺, H₃₉GFP can effectively deliver nucleic acid into cells. As a nucleic acid carrier, H₃₉GFP can achieve the purpose of smart vector. And cellular uptake efficiency was semiquantitatively estimated by flow cytometry.