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

The in vitro detection of low abundance biomolecules via nonenzymatic signal amplification is an attractive strategy. However, it remains a challenge to monitor targets of interest in situ 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 in vitro and in vivo 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 in situ 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.