Construction of Dual-Color Probes with Target-Triggered Signal Amplification for <i>In Situ</i> Single-Molecule Imaging of MicroRNA.

Abstract

The <i>in vitro</i> detection of low abundance biomolecules <i>via</i> nonenzymatic signal amplification is an attractive strategy. However, it remains a challenge to monitor targets of interest <i>in situ</i> in living cells by low-background interference and visualized enzyme-free signal amplification strategies. Taking advantage of the single-molecule imaging and dynamic DNA nanotechnologies, we have achieved the target-triggered self-assembly of nanostructure-based dual-color fluorescent probes (NDFPs) by an enzyme-free toehold-mediated strand displacement cascade. NDFPs will facilitate the simple and visualized monitoring of microRNA (miRNA) at the femtomolar level. The recycled miRNA can be considered as the catalyst for the assembly of multiple H1/H2 duplexes. This generated the fluorescence signal of the enhanced target expression, indicating both <i>in vitro</i> and <i>in vivo</i> signal-amplified imaging. Moreover, the NDFPs improved the measurement accuracy by dual-color colocalization imaging to greatly avoid false-positive signals and enabled the successful <i>in situ</i> imaging of miRNA in living cells in real time. This work provides a strategy to visually monitor and study the integration of signal amplification detection and single-molecule imaging. NDFPs may be an important step toward the enzyme-free amplified monitoring and imaging of various biomolecules in living cells at the single-molecule level.