Live-Cell Imaging of miRNAs with the Combination of CHA and HCR Techniques

The abnormal expression of miRNA-21 is closely related to many cancers, and its sensitive detection plays an important role in the diagnosis and treatment of cancers. In the report, we designed a non-enzymatic amplification sensing system based on the catalytic hairpin assembly (CHA) and palindrome-based hybridization chain reaction (PHCR) technique for the detection and imaging of miRNA in living cells. Based on PHCR technology, two ingeniously designed palindrome-contained fluorescently labeled hairpin probes are sequentially opened upon the stimulation of short oligonucleotide triggers, promoting the autonomous assembly of cross-linked network-like structures (CNSs) and generating fluorescence resonance energy transfer (FRET) signal. Moreover, the PHCR technique can be combined with CHA technology to sufficiently improve amplification efficiency and signal output. By utilizing the CHA-PHCR sensing system, one miRNA-21 activates many triggers through CHA process and in turn initiates the assembly of CNSs by PHCR reaction, efficiently amplifying the FRET signal output. As such, the sensing system can detect target miRNAs down to 10 pm with high detection specificity. Moreover, the CNS exhibits the enhanced intracellular stability, enabling the living cell imaging of miRNA-21. The CHA-PHCR sensing system can also screen the expression level of miRNA-21 in different living cells and differentiate cancer cells from healthy cells, which is consistent with the gold-standard PCR method.