Symmetric exponential amplification reaction-based DNA nanomachine for the fluorescent detection of nucleic acids

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.