title: G-quadruplex-based fluorescent assay of S1 nuclease activity and K+.
abstract: Endonuclease plays an important role in many biological processes, and an assay of endonuclease activity is of great significance. However, traditional methods for the assay of endonuclease activity have undesirable limitations, such as high cost, DNA-consuming and laboriousness. In the present work, a G-quadruplex-based, fluorescent assay of endonuclease activity has been developed with protoporphyrin IX (PPIX) as a signal reporter. S1 nuclease, a single strand DNA (ssDNA)-specific endonuclease, is employed as model system. In the "on" state, G-quadruplex DNA can greatly enhance the fluorescence of PPIX. However, if S1 nuclease could cleave G-quadruplex DNA into small fragments, there would be no formation of G-quadruplexes, accompanied by low emission response of PPIX. This fluorescent discrimination before or after digestion by nuclease can be used to monitor the activity of S1 nuclease. This assay is simple in design and offers a convenient protocol for homogeneous, rapid and high-throughput detection. In addition, the proposed strategy avoids complicated covalent modifications or chemical labeling, and thus offers advantages of simplicity and cost efficiency. More importantly, K(+) is found to well inhibit the activity of S1 nuclease when using certain G-quadruplex DNA as substrate, and thus this system is further used for turn-on detection of K(+). S1 nuclease is critical in the detection of K(+) since it helps to reduce the background signal.
authors: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, PR China.
authors: Zhixue
authors: Z
authors: Zhou
authors: Jinbo
authors: J
authors: Zhu
authors: Libing
authors: L
authors: Zhang
authors: Yan
authors: Y
authors: Du
authors: Shaojun
authors: S
authors: Dong
authors: Erkang
authors: E
authors: Wang
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D004720:Endonucleases / Q000032:analysis* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D054856:G-Quadruplexes; D007477:Ions / Q000737:chemistry; D011188:Potassium / Q000032:analysis*; D011524:Protoporphyrins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D007477:Ions; D011524:Protoporphyrins; C028025:protoporphyrin IX; D004720:Endonucleases; D011188:Potassium