Studies On The Interactions Of Some Hazardous Materials With DNA By Spectrometric And Electrochemical Approaches

This work studied the interaction of DNA and some small molecules, i.e. isoprenaline, brodifacoum, leucomalachite green and two environmental hormone materials with the spectrometric, electrochemical and circular dichroism techniques combined with chemometrics methods. The work advanced the interpretation of the biological process coupled with the study of the pathogenesis at the genetic level, which greatly facilitated the design of the new and low-toxic medicines.The thesis is divided into five Parts. Part one reviews the importance of DNA in life process, the structural characteristics of DNA and the different binding modes between DNA and some small molecules. In addition, the review gives recent advances of the analysis of nucleic acids by using some methods such as fluorescence method, UV-vis method, electrochemical method and so on, and discuss the possibility of the application of chemometrics in the field. Finally, we look into the trends on the application of chemometrics methods for the investigation of the interaction beween small molecules and DNA. Part two is using spectral and electrochemical techniques to study on the interaction of isoprenaline (ISO) with calf thymus DNA. The characteristics of ISO was investigated at a glassy carbon electrode by using electrochemical techniques such as cyclic voltammetry (CV) and differential pulse stripping voltammetry (DPSV), and it was found that an irreversible oxidation peak of ISO appeared in the electrochemical process. The binding constant, K, and the stoichometric coefficient, m, of the interaction between ISO and DNA were evaluated. Moreover, with the addition of DNA, hyperchromicity of the UV absorption spectra of ISO was observed, while the fluorescence intensity greatly decreased under the same conditions. Subsequently, multivariate curve resolution-alternating least squares method (MCR-ALS) was applied to resolve the augmented data matrices from UV-vis and fluorescence spectroscopy, and the pure spectra of ISO, DNA and the ISO-DNA complex, and their concentration profiles were successfully obtained. The results implied that the ISO intercalated into the base-pairs of DNA. Part three investigates the interaction of brodifacoum (BDF) with DNA in Tris-HCl buffer (pH7.4) using neutral red (NR) dye as a probe with the use of fluorescence and UV-vis spectroscopy techniques. The binding constant and the stoichometric coefficient of the interaction of BDF with DNA were caculated to be (6.40±0.5)×104M-1and1.0, respectively, by using multivariate curve resolution-alternating least squares (MCR-ALS). Moreover, parallel factor analysis (PARAFAC)was used to resolve the three-way fluorescence data from the experiment of Vitamin K1/BDF-DNA. It was found that when Vitamin K1was added into the BDF-DNA system, a ternary complex BDF-DNA-VK1was formed, suggesting that Vitamin K1relieved the damage of BDF to DNA. Part four is using fluorescence, UV-vis spectroscopy and circular dichroism (CD) experiments to study the interaction of leucomalachite green(LMG)with DNA. All these results suggested that LMG intercalated into DNA. CD experiments indicated that LMG induced conformational changes of DNA. Furthermore, multivariate curve resolution-alternating least squares (MCR-ALS) method was applied to resolve the augmented data matrices from the absorption and fluorescence spectra. The binding constant and the stoichometric coefficient of the interaction between LMG and DNA were calculated. Beside the fluorescence enhancement of [LMG] induceed by different ratio of CTAB:DNA was investigated. Part five is using fluorescence, UV-vis spectroscopy, voltammetry and circular dichroism (CD) techniques to explore the binding of calf thymus DNA (ctDNA) with two environmental hormone materials such as4-octylphenol (4-OP) and4-nonylphenol(4-NP). The binding constant of4-NP and DNA was calculated by the fluorescence quenching interaction, and showed a larger value compared to that of4-OP and DNA. Similar result was confirmed by the cyclic voltammetry measurement. At the same time, it was found that the interaction of the two environmental hormone and DNA were intercalation.This work found that most of the hazard materials interact with DNA in the intercalation mode. The surface of intercalative molecule surface sandwiched between the aromatic heterocyclic base pairs of DNA which may further affect the expression and replication of the genetic information. The novelty of the study include:1. the interaction of the hazard materials and DNA were processed by many techniques and the results were similar.2. The chemometrics were applied to resolve the spectrometric data matrices, and the pure spectra of each responding component were recovered, which can not be obtained by conventional methods.3. The valuable information extracted from the data facilitated the study of the interaction between the molecules and DNA, further the design of the new and low-toxic drugs.