title: Enzyme-Activated G-Quadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis.
abstract: 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.
authors.affiliation: College of Science, National University of Defence Technology , Changsha, 410073, People's Republic of China.
authors.forename: Zhuoliang
authors.initials: Z
authors.lastname: Liu
authors.forename: Xingyu
authors.initials: X
authors.lastname: Luo
authors.forename: Zhu
authors.initials: Z
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Zhou
authors.identifier: 0000-0001-9864-2965
authors.initials: Z
authors.lastname: Nie
authors.forename: Hong-Hui
authors.initials: HH
authors.lastname: Wang
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
mesh_terms: D017209:Apoptosis; D015374:Biosensing Techniques; D053903:DNA Breaks, Double-Stranded; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D054856:G-Quadruplexes; D006367:HeLa Cells; D006801:Humans; D057230:Limit of Detection; D057054:Molecular Imaging / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes; D004253:DNA Nucleotidylexotransferase