Regulation Of Fragment DNA On Copper-zinc Superoxide Dismutase Aggregation And Its Applications

In recent years,the release of active proteins into target cells to cure the protein mediated diseases has become more and more concerned,gene therapy brings a new dawn for the treatment of DNA damage-related diseases.Cu/Zn superoxide dismutase(SOD1)is one of the most important antioxidant enzymes in the cell,and its main function is to maintain the homeostasis of intracellular H2O2 and superoxide anion(O2).Therefore,to construct biological function SOD 1-nucleic acid aggregation system,which loads to cell by carrier,to achieve active protein and target genes releasing together,may be an effective way to cure the disease.This work studies in the neutral physiological environment,vitamin C damaging conditions,to construct SOD 1-DNA Nano particles aggregation and explore the double functional applications as protein drug therapy and nucleic acid gene vector,the results are as follows:(1)In the physiological environment,the hydrophobic of vitamin C treated SOD1 increased,the secondary structure changed,Surface charge from negative to positive,activity to decrease to some extent,but remains about 75%.(2)Hydrophobic residues of SOD 1 is different from different types of DNA fragments,whicn G-quadruplex DNA is stronger than ss DNA.Guadruplex DNA was significantly stronger than the corresponding ssDNA.(3)RALS experiments show that,the SOD1 aggregation reaction is promoted rapid equilibrium by different types of DNA fragments in the 30 minutes,and the formation of aggregation have significantly different particle size,two sets of columns,which G-quadruplex DNA promotes the formation of SOD1 aggregation level significantly higher than ss DNA.(4)DLS was found that SOD 1 aggregates particle size and surface charge is regulated by the concentration of DNA,when SOD 1:DNA 6:1?4:1?2:1,SOD1 aggregates are formed with the DNA of a certain size,and gathered surface with a certain amount of positive charge.(5)Fluorescent double staining showed that the aggregation containing SOD1 and DNA;TEM study of the morphology of aggregates shows that in acidic conditions,ss DNA promotes SOD1 to form amorphous aggregates,while the G-quadruplex DNA promotes SOD1 to form of fibrous aggregates.Under the neutral pH 7.4,Vc damaged conditions,ss DNA promotes SOD1 to form particle size of about 30nm spherical aggregates,G-quadruplex DNA promotes SOD1 to form similar fibrous aggregates.(6)Using FAM-labeled DNA study different particle size of SOD1-DNA aggregate particles trans-membrane cell experiments.Results show that SOD 1-DNA appear aggregates fluoresce in cells,and is likely to enter the nucleus.