Study On The Structure Of Long Telomeric DNA

Telomeres are protective terminal of linear chromosomes in eukaryotic cells, which are closely related to the life span of cells.The number of telomere repeats is reduced during mitosis, and cell become aging when Hayflick limit is reached. If telomerase are activated in cells,telomere length can be maintained and cells could divide endlessly and become immortal, which is the mechanism of the most tumor cells' proliferation.The G-quadruplexes structures formed by 3' ss DNA overhangs at the telomere terminal are potential anti-cancer targets because their formation can inhibit the activity of telomerase. According to these knowledge, many small molecule ligands that can stabilize G-quadruplex structure were designed as anticancer drugs to inhibit tumor growth.During early stages many researches started with single G-quadruplex model system. As research continues, more attentions were focused on long telomeric DNA, which are more consistent with the real system. A variety of complex conformations may form in long telomeric sequences and G-quadruplexes may also interact with each other in different ways.Synthesis difficulty and complexity of conformations have always been the major challenges in the study of long telomeric DNA. In this study, RCA(Rolling Circle Replication) was used for the preparation of long telomeric DNA. The structure and properties of those long telomeric DNA were studied:In chapter 1, the origin, evolutionary sense and physiological functions of the telomere and telomerase are briefly introduced. In addition the relationship between the telomere and the cancer formation as well as aging mechanism are reviewed from macrocosmic to microcosmic.Besides, the current understanding on G-quadruplex structures are also reviewed. Finally, in the end of this chapter, the principle of atomic force microscopy(AFM)-based imaging and single molecular force spectroscopy as well as the and the applications of such technique in relevant fields, such as modern biology are introduced in detail.In the chapter 2, the preparation of long telomeric DNA sequences is firstly introduced. Then the products were characterized by AFM imaging,UV-melting, circular dichroism spectra(CD) etc.. Our results showed the formation of high order structures in long telomeric DNA: G-quadruplexes folded side by side forming the "beads-on-a-string" structure. CD spectrum indicates that the conformations of G-quadruplexes in long telomeric DNA are different from the single isolated one. We speculate that the freedom of two terminals of every G4 unit in long sequence may be restricted by neighbouring sequence leading to the conformation change.By using functionalized primer, RCA reaction has been carried out on solid substrates. And the covalently immobilized long telomeric DNA products can be used for the further research. Besides, we also developed a new method for the preparation of double-stranded DNA with repeating sequence.This method can overcome the shortcomings, such as diffused strip, low yield, and mess base pairing after annealing, of previous methods.In chapter 3,the incomplete folding model of G4 DNA in long telomeric DNA has been proposed based on the experimental results. This model could explain the lower thermo stability of long telomeric DNA. This model can interpret the conflict between lower Tm of long telomeric DNA and the stable higher order structure formed by QQI(Quadruplex- Quadruplex Interaction). The UV-melting and CD results in our researches support the partly folded model. By comparing CD signals obtained on different mutations of telomeric DNA, we speculate that the outermost four guanineson terminal of each G4 unit can hardly fold into G-quadruplex structure,which may be caused by the restriction from the neighbouring G4 unit in the long telomeric DNA as well as the thermal fluctuations. Different from the G-triplex, the disruptions caused by the thermo fluctuation are symmetrical on both sides of the quadurplex structure. This new model deepened our understanding on structure of the long telomeric DNA, which provide new pathway for the design of G4-based small molecule anticancer drugs.In the chapter 4, the mechanical stability of long telomeric single stranded DNA(ss DNA) was investigated by AFM-based single molecule force spectroscopy(SMFS). Our results show that the 55 p N plateau always represent higher order structure formed by QQI. During stretching-relaxing cycle, hysteresis was observed on the force curves of telomeric sequence, which indicate the formation and destruction of high order structure. The area of hysteretic region ?E was found to increase linearly with the increase of contour length ?L. The slope,achieved by linear fitting, can be a measure of the stability of high order structures formed by telomeric DNA. For example, the slope is higher in40 % PEG than in water in good agreement with previous study on that the presence of PEG can stabilize the G4 structure. This single molecule method can also be used to measure many other factors affecting the stability of G-quadruplex such as p H, ion environment, ligand etc..Systematic SMFS investigations on long telomeric DNA show that the hysteresis, observed in telomeric DNA, get disappeared when the repeat units of telomeric DNA are separated from one another by random sequence,which suggest that the formation of G-quadruplexes can induce/speed up the folding of its neighboring telomeric sequence. However, the presence of separation sequence greatly weakened the inductive effect.