Double-target responsive FRET sensor based on DNA tetrahedron for robust detection of liver fibrosis-related mRNAs in activated hepatic stellate cells.

Early diagnosis and graded evaluation of liver fibrosis remained challenging due to the limited sensitivity and accuracy of current noninvasive diagnostic tools. The imaging strategy of intracellular disease-related biomarker based on DNA nanoparticles had been used for accurate diagnosis and prognosis studies. However, their sensitivity and accuracy were often limited by off-target signal leakage associated with a single-biomarker sensing strategy. Herein, a system for simultaneous detection and imaging of two mRNA targets was constructed by using a double-target responsive FRET sensor (DTFS) that consisted of a DNA tetrahedron nanostructure (DTN), an aptamer and two probes for selective recognition and ratiometric imaging of activated hepatic stellate cells (aHSCs). When the DTFS was internalized into aHSCs through the aptamer, the overexpressed α-smooth muscle actin (α-SMA) and type I collagen α1 chain (COL1A1) could specifically activate the DTFS via toehold-mediated strand displacement (TMSD) reactions, generating significant fluorescence resonance energy transfer (FRET) signal. In this strategy, endogenous α-SMA and COL1A1 acted as two "keys" to activate the DTFS in aHSC cells, thereby reducing off-target signal leakage. Further results supported the intracellular delivery behavior, inhibitor specificity, and extended cellular specificity of DTFS. In a mouse fibrosis model using a redesigned probe with mouse-specific sequences, stage-associated ratiometric fluorescence changes were also observed in vivo. These results suggest that DTFS is expected to provide a useful platform for specific imaging of activated hepatic stellate cells and the graded evaluation of liver fibrosis.