College of Science, National University of Defence Technology , Changsha, 410073, People's Republic of China.
Anal Chem. 2017 Feb 7;89(3):1892-1899. doi: 10.1021/acs.analchem.6b04360. Epub 2017 Jan 20.
Fluorogenic probes targeting G-quadruplex structures have emerged as the promising toolkit for functional research of G-quadruplex and biosensor development. However, their biosensing applications are still largely limited in in-tube detection. Herein, we proposed a fluorescent bioimaging method based on enzyme-generated G-quadruplexes for detecting apoptotic cells at the cell and tissue level, namely, terminal deoxynucleotidyl transferase (TdT)-activated de novo G-quadruplex synthesis (TAGS) assay. The detection target is genomic DNA fragmentation, a biochemical hallmark of apoptosis. The TAGS assay can efficiently "tag" DNA fragments via using their DNA double-strand breaks (DSBs) to initiate the de novo synthesis of G-quadruplexes by TdT with an unmodified G-rich dNTP pool, followed by a rapid fluorescent readout upon the binding of thioflavin T (ThT), a fluorogenic dye highly specific for G-quadruplex. The feasibility of the TAGS assay was proved by in situ sensitive detection of individual apoptotic cells in both cultured cells and tissue sections. The TAGS assay has notable advantages, including being label-free and having quick detection, high sensitivity and contrast, mix-and-read operation without tedious washing, and low cost. This method not only shows the feasibility of G-quadruplex in tissue bioanalysis but also provides a promising tool for basic research of apoptosis and drug evaluation for antitumor therapy.
荧光探针靶向 G-四链体结构已成为研究 G-四链体功能和开发生物传感器的有前途的工具。然而，它们的生物传感应用在很大程度上仍然局限于管内检测。在此，我们提出了一种基于酶促 G-四链体的荧光生物成像方法，用于在细胞和组织水平检测凋亡细胞，即末端脱氧核苷酸转移酶（TdT）激活的从头 G-四链体合成（TAGS）检测法。检测目标是基因组 DNA 片段化，这是细胞凋亡的生物化学标志。TAGS 检测法可以通过利用 DNA 双链断裂（DSB）来有效地“标记”DNA 片段，从而由 TdT 用未修饰的富含 G 的 dNTP 池起始从头 G-四链体合成，随后通过硫代黄素 T（ThT）结合进行快速荧光读出，ThT 是一种高度特异性结合 G-四链体的荧光染料。TAGS 检测法通过在培养细胞和组织切片中对单个凋亡细胞进行原位灵敏检测，证明了其可行性。TAGS 检测法具有显著的优点，包括无标记、快速检测、高灵敏度和对比度、无需繁琐洗涤的混合读取操作以及低成本。该方法不仅展示了 G-四链体在组织生物分析中的可行性，还为细胞凋亡的基础研究和抗肿瘤治疗的药物评价提供了一种有前途的工具。
