title: A new method for sensitive detection of microphthalmia-associated transcription factor based on "OFF-state" and "ON-state" equilibrium of a well-designed probe and duplex-specific nuclease signal amplification.
abstract: Herein, we report a new method which the "stage change" of DNA1 and the cleavage feature upon recycled recognition by DSN was used to detect target microphthalmia-associated transcription factor (MITF) in cell nuclear extracts. In this method, we employed a well-designed DNA1 as a recognition element which can converting in two states, "ON-state" and "OFF-state". Also, the DNA1 is modified with 2-OMe-RNA on hybridization part in the "OFF-state" to prevent meaningless digestion. By taking advantage of the high amplification efficiency of DSN-aided recycling, high sensitivity of MITF is realized with a detection limit as low as 1.1 pM, which is superior or comparable to that of the reported literature. This method is a fast and easy-to-use one-pot method that was carried out in a tube, while being quantitative and applicable to other proteins in the sample without involving complicated procedures or sophisticated instrumentations. It is very simple and fast, needing only mixing of DNA1, DNA2, MITF and DSN enzyme and incubating within 60min, which is in the homogeneous solution, and not requiring separation and troublesome procedures. Considering the superior sensitivity and specificity, as well as the multiplex and simple-to-implement features, this method holds great promise of becoming a routine tool for simultaneously quantitative analysis of multiple proteins and supplies valuable information for transcription factor-based early stage cancer diagnosis.
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China. Electronic address: zhangkai@jsinm.org.
authors: Kai
authors: K
authors: Zhang
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors: Ke
authors: K
authors: Wang
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors: Xue
authors: X
authors: Zhu
authors: Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China.
authors: Minhao
authors: M
authors: Xie
authors: Department of Neurosurgery, Wuxi People's Hospital affiliated with Nanjing Medical University, Wuxi, Jiangsu 214000, China. Electronic address: doczhangxiaolu@gmail.com.
authors: Xiaolu
authors: X
authors: Zhang
mesh_terms: D000072283:A549 Cells; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000378:metabolism; D015342:DNA Probes / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D051739:Microphthalmia-Associated Transcription Factor / Q000032:analysis* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D015342:DNA Probes; D051739:Microphthalmia-Associated Transcription Factor; D004247:DNA; D004720:Endonucleases
keywords: Cell nuclear extracts; DSN; One-pot; Transcription factor