Studies Of Human Telomeric DNA Dye And Drug Interactions Of Small Molecules

The binding of small molecules to DNA is related to the replication and transcription of DNA, and can be used to clarify the structure and function of DNA, to understand the action mechanism of some anti-cancer, anti-virus and anti-tumor drugs and to explore the origin of some common diseases.In this paper, the different binding actions of some dyes and drugs molecules to human telomere DNA were investigated by the electrochemistry with simplicity, accuracy, inexpensiveness, sensitivity and selectivity combined with spectrum, the redox-active molecules that have different affinities with single-strand and double-strand human telomere DNA (ssDNA and dsDNA). So, the binding modes and mechanism were discussed and proposed. Combined the electrochemical property of dyes and drugs molecules with good conductive performance in graphene, alizarin/graphene-chitosan (AR/GR-CS/GC) electrode and Hypocrellin A/graphene (HA-GR/GC) electrode were prepared for the first time. The modified electrodes can bind to telomere DNA with high selectivity and high affinity compared with the bare electrode. Also, the modified electrodes with the advantages of relatively easy production, high stability, electrochemical signal obviously have been widely used to develop electrochemical biosensors. Based on the reaction mechanism between HA and DNA, the HA-GR/GCE was successfully applied to determine the telomere DNA in human blood serum and abdominal fluid and the result is satisfactory. The main content and results are listed as following:1. The interaction between pyrocatechol violet (PCV) and telomere DNA was studied in physiological buffer (pH7.4) by spectroscopic and electrochemical methods. These results indicated that PCV could bind strongly to the telomere DNA and the major binding mode was intercalative. Based on the voltammetric titration, the binding constant (β) and binding ration (m) of PCV and telomere DNA were found to be5.3×109mol·L-1and1.7, respectively. The reduction of the peak current of PCV after adding telomere DNA was further used for the quantification of telomere DNA by Differential pulse voltammetry (DPV). The linear range for telomere DNA was in the range of0.2-13.0μmol·L-1with the linear regression equation as△Ipa(μA)=0.075CDNA(μmol·L-1)-0.041, R2=0.9990and the detection limit of0.1μmol·L-1. The determination of telomere DNA was hardly impacted by foreign substances. 2. In this work, the modified glassy carbon electrode of alizarin (AR) intermingled with graphene (GR) and chitosan (CS) was prepared for the first time. The AR/GR-CS electrode exhibited a pair of well-defined reversible redox peaks in0.1M phosphate buffer solution (PBS, pH7.4) with the oxidation peak potential of-0.573V and the reduction peak potential of-0.652V. It exhibited a two-proton and two-electron redox process and a fast heterogeneous electron transfer rate with the rate constant (Ks) of1.69s-1using the cyclic voltammogram. Based on the characteristics of alizarin with the decreased current and negative potential after binding with human telomere DNA, a sensitive, simple, reliable and stable electrochemical method for the detection of alizarin using the AR/GR-CS electrode was developed. The working curve was constructed in the range of8×10-8-1.4×10-5mol·L-1with the linear regression equation as△Ipc(μA)=8.4860+0.5366CDNA(μmol·L-1), R2=0.9990, and the detection limit (S/N) as2×10-8mol·L-1. This method is expected to be used for the determination of actual samples.3. A graphene (GR) and Hypocrellin A (HA) modified glassy carbon electrode was fabricated, which was characterized by transmission electron microscope (TEM) and Fourier transform infrared spectum (FTIR). The electrochemical behavior of human telomere DNA and HA was investigated in pH6.0phosphate buffer solution by cyclic voltammetry and differential pulse voltammetry. The fluorescence results indicated that HA binding with telomere DNA was dynamic quenching. Thermo dynamic study indicated that the binding is mainly van der Waals and hydrogen bond. Based on the fluorescence experiment, the binding ratio (n) of HA and telomere DNA were found to be1.04and0.89at293K and308K, respectively. The electrochemical parameters of human telomere DNA on the modified electrode were calculated with the results of the charge transfer coefficient (a) as0.23and electron transfer rate constant(ks) as1.28×10-2s-1. Under the optimized conditions, the oxidation current of HA was proportional to human telomere DNA concentration in the range from5.91×10-9to7.27×10-7mol·L-1, the linear regression equation as△Ipa(10-5A)=19.67CDNA (10-8mol·L-1)-2.98×10-5and the correlation coefficient of0.9922and the detection limit of1.21×10-10mol·L-1(S/N=3). The proposed method was further applied to determine DNA in blood serum and abdominal fluid samples with satisfactory results. The recovery is99.54%～103.21%.