Engineering of a size-selective tetrahedral DNA nanocage for high-fidelity imaging of microRNA and precursor microRNA

Mature microRNAs (miRNAs) and their precursors (pre-miRNAs) are closely related to various physiological and pathological processes. The simultaneous monitoring of miRNA and its precursor can discriminate tumor cells from DNA-damaged cells and increase the diagnosis reliability. Herein, we design a size-selective tetrahedral DNA nanocage for high-fidelity imaging of miRNA and its precursor in living cells. In this research, the DNA nanocage framework is self-assembled from six ssDNAs, and hairpin probes (H1 and H2) can bind to two edges of the DNA nanocage for the recognition of pre-miRNA, and entropy-driven reaction substrates are docked inside the DNA nanocage framework for the recognition of miRNA. Due to the size-selectivity of the DNA nanocage, longer pre-miRNAs (60-70 nt) are excluded from the cavity, but shorter miRNAs (19-23 nt) can enter the cavity. Once the DNA nanocage framework is internalized into cells, pre-miRNA-155 initiates catalytic hairpin assembly between H1 and H2 to recover AF647 fluorescence, and miRNA-155 triggers entropy-driven strand displacement to restore AF568 fluorescence. In the presence of intracellular ATP, the fuel strand is released from fuel strand/ATP aptamer duplex to displace miRNA-155 that can initiate new entropy-driven strand displacement to enhance AF568 fluorescence. This nanosensor enables high-fidelity imaging of miRNA-155 and its precursor in living cells. Moreover, it can quantify both miRNA-155 and pre-miRNA-155 at the single-cell level, and distinguish their levels between lung cancer tissues and normal counterparts. Furthermore, this nanosensor enables the differentiation of tumor cells from DNA-damaged cells, offering a powerful tool for precise diagnosis.