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

被引:13
作者
Zhang, Kai [1 ]
Wang, Ke [1 ]
Zhu, Xue [1 ]
Xie, Minhao [1 ]
Zhang, Xiaolu [2 ]
机构
[1] Jiangsu Inst Nucl Med, Key Lab Nucl Med, Jiangsu Key Lab Mol Nucl Med, Minist Hlth, Wuxi 214063, Jiangsu, Peoples R China
[2] Nanjing Med Univ, Wuxi Peoples Hosp, Dept Neurosurg, Wuxi 214000, Jiangsu, Peoples R China
关键词
Transcription factor; One-pot; DSN; Cell nuclear extracts; MOLECULAR BEACON PROBES; MULTIPLEX DETECTION; ASSAY; BINDING; QUANTIFICATION; INFECTIONS; MICRORNAS; BIOSENSOR; VIRUS;
D O I
10.1016/j.bios.2016.08.070
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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 60 min, 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. (C) 2016 Elsevier B.V. All rights reserved.
引用
收藏
页码:299 / 304
页数:6
相关论文
共 25 条
[1]   MAMMALIAN DNA NUCLEOTIDE EXCISION-REPAIR RECONSTITUTED WITH PURIFIED PROTEIN-COMPONENTS [J].
ABOUSSEKHRA, A ;
BIGGERSTAFF, M ;
SHIVJI, MKK ;
VILPO, JA ;
MONCOLLIN, V ;
PODUST, VN ;
PROTIC, M ;
HUBSCHER, U ;
EGLY, JM ;
WOOD, RD .
CELL, 1995, 80 (06) :859-868
[2]   A general approach to the design of allosteric, transcription factor-regulated DNAzymes [J].
Adornetto, G. ;
Porchetta, A. ;
Palleschi, G. ;
Plaxco, K. W. ;
Ricci, F. .
CHEMICAL SCIENCE, 2015, 6 (07) :3692-3696
[3]   Development of a nucleic acid sequence-based amplification assay that uses gag-based molecular beacons to distinguish between human immunodeficiency virus type 1 subtype C and C′ infections in Ethiopia [J].
Ayele, W ;
Pollakis, G ;
Abebe, A ;
Fisseha, B ;
Tegbaru, B ;
Tesfaye, G ;
Mengistu, Y ;
Wolday, D ;
van Gemen, B ;
Goudsmit, J ;
Dorigo-Zetsma, W ;
de Baar, MP .
JOURNAL OF CLINICAL MICROBIOLOGY, 2004, 42 (04) :1534-1541
[4]   Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor [J].
Bonham, Andrew J. ;
Hsieh, Kuangwen ;
Ferguson, B. Scott ;
Vallee-Belisle, Alexis ;
Ricci, Francesco ;
Soh, H. Tom ;
Plaxco, Kevin W. .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2012, 134 (07) :3346-3348
[5]   Validation of an assay for the determination of levoglucosan and associated monosaccharide anhydrides for the quantification of wood smoke in atmospheric aerosol [J].
Cordell, Rebecca L. ;
White, Iain R. ;
Monks, Paul S. .
ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 2014, 406 (22) :5283-5292
[6]   Lab in a Tube: Ultrasensitive Detection of MicroRNAs at the Single-Cell Level and in Breast Cancer Patients Using Quadratic Isothermal Amplification [J].
Duan, Ruixue ;
Zuo, Xiaolei ;
Wang, Shutao ;
Quan, Xiyun ;
Chen, Dongliang ;
Chen, Zhifei ;
Jiang, Lei ;
Fan, Chunhai ;
Xia, Fan .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2013, 135 (12) :4604-4607
[8]   Label free analysis of transcription factors using microcantilever arrays [J].
Huber, F ;
Hegner, M ;
Gerber, C ;
Güntherodt, HJ ;
Lang, HP .
BIOSENSORS & BIOELECTRONICS, 2006, 21 (08) :1599-1605
[9]   Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA [J].
Leone, G ;
van Schijndel, H ;
van Gemen, B ;
Kramer, FR ;
Schoen, CD .
NUCLEIC ACIDS RESEARCH, 1998, 26 (09) :2150-2155
[10]   Multiplex detection of single-nucleotide variations using molecular beacons [J].
Marras, SAE ;
Kramer, FR ;
Tyagi, S .
GENETIC ANALYSIS-BIOMOLECULAR ENGINEERING, 1999, 14 (5-6) :151-156