3D DNA Framework-Encoded Multicolor DNA Amplifiers Permit Dual-Modal Simultaneous Imaging of Intracellular Multiplexed mRNAs

Abstract High-sensitivity, simultaneous visualization of multiple low-abundance intracellular mRNAs is appealed to elucidating their synergistic functions and guiding cancer diagnosis and treatment. Engineered nanoprobes integrating anti-enzymatic degradation, efficient signal amplification, and multimodal output are highly desirable for sensitive and accurate in situ imaging in living cells. Herein, we design a 3D tetrahedral DNA-encoded four-color entropy-driven amplifiers (TFEA) for simultaneous fluorescence/surface-enhanced Raman scattering (FL-SERS) dual-modal imaging of four intracellular mRNAs. Target mRNA binding activates the TFEA to simultaneously trigger a multi-path entropy-driven DNA circuit, resulting in the disassociation of multicolor fluorophore-tagged DNAs and the aggregation of gold nanobipyramids (Au NBPs) mediated by tetrahedral DNA (TDN) nanostructures, thus producing strong FL and SERS signals that directly report on the location and concentration of the mRNAs. The TDN assembles equal amounts of different entropy-driven amplifier probes on a single nanostructure, confers potent resistance to nuclease degradation, maintains structural integrity in complex biological systems, and facilitates amplifier probe delivery. Impressively, the multiple Au NBPs assembled by TDN promote the formation of dense plasmonic hotspots in TDN-confined spaces, thereby eliciting a significant boost in the SERS signal. The amplifiers are endowed with potent delivery, high sensitivity, and desirable stability and can simultaneously monitor the four types of cellular mRNAs with FL-SERS dual-modal imaging, thus providing accurate information on localization and concentration of targets. Notably, it can distinguish the expression signatures of multiplex mRNAs in different cells and in tissues from healthy controls and liver cancer patients, offering a promising platform for clinical diagnosis and biomedical research.