Enzyme-Activated G-Quadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis.
Zhuoliang LiuXingyu LuoZhu LiYan HuangZhou NieHong-Hui WangShouzhuo YaoPublished in: Analytical chemistry (2017)
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.
Keyphrases
- label free
- cell cycle arrest
- cell death
- induced apoptosis
- sensitive detection
- endoplasmic reticulum stress
- quantum dots
- loop mediated isothermal amplification
- single molecule
- oxidative stress
- high throughput
- circulating tumor
- living cells
- pi k akt
- cell free
- magnetic resonance
- stem cells
- small molecule
- gene expression
- high resolution
- nucleic acid
- emergency department
- magnetic resonance imaging
- computed tomography
- signaling pathway
- anti inflammatory
- bone marrow
- mesenchymal stem cells
- gold nanoparticles
- drug delivery
- real time pcr
- photodynamic therapy
- cancer therapy