Single-molecule Fluorescence Resonance Energy Transfer spectroscopy Study on the Conformational Interconversion Kinetics of Human Telomere G-quadruplexes Influenced by Local Chemical Environment

• Main Authors: Hsu, Hao-Yi, 許顥頤
• Other Authors: Lee, I-Ren
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/55516823571060235957

碩士 === 國立臺灣師範大學 === 化學系 === 104 === G-quagruplexes, secondary structures formed by guanine-rich DNA sequences in the telomere regions of genome, are highly relevant to cancer due to the inhibition of telomerase, an enzyme that has high activity in tumor cells and causes the proliferation of tumor cell. Recent studies revealed that monovalent metal cations such as potassium, sodium, and ammonium ions stabilize the G-quadruplex structure of in parallel, antiparallel, and their hybrid configurations. In physiological conditions, the abundance of magnesium ion is second to the concentration of potassium ion and it participates in at least 300 biochemical reactions in cell. Previous studies have shown that the addition of magnesium ions to the sodium or potassium solutions does further stabilize certain G-quadruplex. Here, we examined the G-quadruplex formation and stabilization in the presence of magnesium ions only. Single-molecule fluorescence resonance energy transfer spectroscopy(smFRET) was employed to study the transformation between G-quadruplex conformational states and their unfolded counterpart. Surprisingly, our experimental data shows inconsistency with the results in the literatures. In order to address this problem, we varied different experimental conditions such as labeling chemistry and ingredient of oxygen scavenging system solution. We found that the oxygen scavenging systems, frequently used in smFRET experiment for stabilizing the fluorescent dye molecules, and the chemistry of fluorescent dye molecule linking to the oligonucleotides sequence, collaboratively alter the equilibrium between conformational states, hence, form a stable dye-facilitated folded state in certain conditions, especially in the solely presence of magnesium cations. However, the kinetics analysis by excluding the molecules in this specific state showed that the interconversion of G-quadruplex conformational states remain unchanged throughout the conditions we have tested. Finally, we add extra poly-thymine, which does not participate in the formation of G-quadruplexes, to the sequence of interest to increase the distance between the labeling dye pairs and ultimately improve the spatial resolution. We found that two distinct states originated from the previously assigned antiparallel configuration and a fast direct interconversion between these two states is also observed.