人類端粒序列為一段富含鳥嘌呤的序列，可形成DNA二級結構，鳥嘌呤四股結構。先前的研究指出金屬陽離子對鳥嘌呤四股結構具有穩定其結構的效果，在生理條件下，鉀、鈉與銨等陽離子分別能穩定鳥嘌呤四股結構，形成平行、混和型以及反平行構形，在整體實驗和單分子實驗均已經得到證實。在人體細胞中，鎂離子的含量僅次於鉀離子而且參與300多種生化反應，因此我們想要知道單純鎂離子是否能幫助人類端粒序列形成特定的鳥嘌呤四聚體或穩定其結構，對於已被證實存在人體的鳥嘌呤四聚體結構應用在相關疾病的治療有所幫助。
然而，在實驗的過程中實驗控制組與文獻出現形成構形的比例不一致的情況，為了找出實驗條件中的差異分別針對(1)酵素型除氧系統的穩定性 (2)寡核苷酸上標記螢光染料分子的核苷酸修飾方式 (3)增加實驗的空間解析度進行改善。
在單分子螢光共振能量轉移實驗中所使用的酵素型除氧系統為葡萄糖氧化酶與過氧化氫酶系統，可移除實驗中氧分子的存在與減緩光漂白的現象能有效的增加螢光穩定性與提升時間解析度延長實驗錄製軌跡圖的時間。但是，葡萄糖氧化酶與過氧化氫酶的反應生成物葡萄糖酸，會造成酸鹼值快速下降影響對pH值敏感的實驗，藉由改變酵素型除氧系統希望能找到適合的實驗條件。此外，單分子螢光實驗所使用的寡核苷酸，依實驗需求標記有螢光染料分子在特定位置的核苷酸上可能會造成實驗結果穩定在特定鳥嘌呤四股結構構形，但是對於不同構形之間轉換的動力學實驗結果並無顯著影響。另外，單分子螢光實驗中常用的DNA架構中由互補的雙股DNA作為基底可能會影響人類端粒序列在摺疊成鳥嘌呤四股結構時傾向形成特定的構形而影響實驗結果，對此我們在寡核苷酸序列中額外加入不會參與摺疊的胸腺嘧啶，以增加人類端粒序列可以進行摺疊的空間，同時也改善了螢光共振能量轉移的空間解析度。綜合以上可能會影響實驗結果的因素分別進行討論，希望能對利用單分子螢光共振能量轉移的研究提供最接近真實的生理條件且穩定的實驗環境。

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.

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