Symmetric exponential amplification reaction-based DNA nanomachine for the fluorescent detection of nucleic acids |
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Authors: | Qi Yan Qiuyue Duan Yuqi Huang Jing Guo Liang Zhong Hong Wang Gang Yi |
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Affiliation: | Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education of China), Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016 P. R. China, Fax: +86 23 68485239, +86 23 68485240 ; Department of Clinical Laboratory, Qingdao Municipal Hospital, 1 Jiaozhou Road, Qingdao 266071 P. R. China |
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Abstract: | By introducing palindromic sequences into the classical exponential amplification reaction (EXPAR), we constructed a new palindromic fragment-incorporated multifunctional hairpin probe (P-HP)-mediated symmetric exponential amplification reaction (S-EXPAR), to significantly reduce the background signal caused by inherent nonspecific amplification. A G-triplex/ThT complex was used as the signal reporter for the proposed label-free DNA nanomachine. The P-HP consists of five functional regions: a C-rich region (C), a target DNA recognition region (T′), two nicking sites (X′) and a palindromic fragment (P). When target DNA (T) hybridizes with P-HP, the palindromic fragment at the 3′ end of P-HP is fully exposed. Then, the P-HP/T duplexes hybridize with each other through the exposed P, and EXPAR occurs automatically and continuously on both sides of P under the synergistic effect of polymerase and nicking endonuclease. This is called the S-EXPAR assay. In this system, one T converts to a large number of G-triplex fragments, which can combine with ThT within a short time. The G-triplex/ThT complexes formed act as the signal reporter in a label-free and environmentally friendly format. In this way, the limit of detection of this method is as low as 10 pM with a dynamic response range of 10 pM to 300 nM. In addition, this method can detect other nucleic acids by simply changing the T′ region of the P-HP. Thus, the proposed DNA nanomachine is a potential alternative method for nucleic acid detection.This label-free and ultra-low background signal DNA nanomachine was based on P-HP mediated S-EXPAR and the G-triplex/ThT complex. |
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