RNA imaging in living mice enabled by an in vivo hybridization chain reaction circuit with a tripartite DNA probe

RNA imaging in living animals helps decipher biology and creates new theranostics for disease treatment. Due to their low delivery efficiency and high background, however, fluorescence probes for in situ RNA imaging in living mice have not been reported. We develop a new cell-targeting fluorescent probe that enables RNA imaging in living mice via an in vivo hybridization chain reaction (HCR). The minimalistic Y-shaped design of the tripartite DNA probe improves its performance in live animal studies and serves as a modular scaffold for three DNA motifs for cell-targeting and the HCR circuit. The tripartite DNA probe allows facile synthesis with a high yield and demonstrates ultrasensitive RNA detection in vitro. The probe also exhibits selective and efficient internalization into folate (FA) receptor-overexpressed cells via a caveolar-mediated endocytosis mechanism and produces fluorescence signals dynamically correlated with intracellular target expressions. Furthermore, the probe exhibits specific delivery into tumor cells and allows high-contrast imaging of miR-21 in living mice. The tripartite DNA design may open the door for intracellular RNA imaging in living animals using DNA-minimal structures and its design strategy can help future development of DNA-based multi-functional molecular probes.