Study On The Interaction Mechanism Of Pyrene And Dibenzo[a,h]Anthracene With DNA In Vitro

In this paper,pyrene(PYR)and dibenzo [a,h] anthracene(DBA)were selected as two kinds of typical polycyclic aromatic hydrocarbons(PAHs)for this study.Firstly,the interaction mode of two PAHs with DNA in vitro were studied through a serious of experiments,and then the binding strength were further calculated by various formulas.By comparison,it was found that the interaction between DBA and DNA was stronger.Therefore,DBA was selected as the research object,and the influence of various environmental factors on the interaction between DBA and DNA were determined.The results showed that:(1)Both PYR and DBA could interact with DNA in vitro,the addition of PYR and DBA could increase the melting point temperature of DNA by about 10 °C and 12 °C,respectively.In the fluorescence spectra experiments,DNA had fluorescence quenching effect on both PYR and DBA.Circular dichroism experiments showed that the addition of PYR and DBA could change the secondary conformation of DNA.All these phenomena were consistent with the intercalation characteristics of small molecules with DNA.Finally,using ethidium bromide(EB)as the control group,magnetic beads adsorption combined with fluorescence microscopy was used to visually confirmed the intercalation binding mode of two PAHs with DNA.(2)The quenching mechanism of DNA for the two PAHs were static quenching through formation of the PAH-DNA complex.The binding constants of DNA interact with PYR and DBA were 1.3890 × 105 L/mol and 2.0394 × 105 L/mol at 25 °C,respectively.Thermodynamic results showed that the process of formation PYR-DNA and DBA-DNA complexes were spontaneous,and the main driven forces were hydrogen bond and van der Waals force.At room temperature,the DNA binding saturation values of PYR and DBA were 0.70 and 0.81,respectively.Based on the above results,it can be seen that the intercalation binding strength of DBA with DNA was stronger in vitro.(3)In resonance light scattering experiments,the optimal temperature and p H conditions for the DBA-DNA interaction system were 35 °C and p H 7.4,respectively,and the DNA binding saturation value of DBA was 0.97.Under the optimal conditions,adding different concentrations of environmental coexistence to the reaction system,in addition to sodium chloride and calcium chloride could reduce the resonance light scattering signal intensity of DBA-DNA system,other environmental coexistence(leucine,lysine,sodium lauryl sulfonate,glucose,vitamin C and urea)could enhance the resonance light scattering signal intensity of the reaction system,and increase the DNA binding saturation values.(4)In the experiment of magnetic beads adsorption combined with high performance liquid chromatography,when the dosage of DNA was 0.74μg,the removal rate of DBA at the initial concentration of 2.76 × 10-5 mol/L was 91.82%.Except the presence of sodium chloride and calcium chloride were not conducive to the removal of DBA by DNA,other environmental coexisting substances could promote the removal of DBA by DNA.The repeatability experiments on magnetic beads showed that the magnetic beads used in the experiment could be reused at least three times.In conclusion,both PYR and DBA could intercalate with DNA in vitro,but the binding strength of DBA to DNA was stronger.Optimizing the reaction conditions could further increase the adsorption of DNA to DBA.This study will help to understand the interaction mechanism between PAHs and DNA in vitro,and can lay the foundation for the establishment of a new method to remove PAHs from the environment based on PAHs-DNA intercalation.