Conditional regulation of HCR for rapid visualization of endogenous miR-21 in cancer cells

MicroRNA-21 (miR-21) is a significant tumor marker for early cancer screening. Hybridization chain reaction (HCR) as an enzyme-free signal amplification method has been used for intracellular miR-21 detection in living cells, but it is limited by unclear reaction details and prolonged detection time. In this study, by effectively regulating the HCR reaction conditions, we achieved rapid visualization of endogenous miR-21 in live cells. A pair of HCR DNA hairpins H₁ and H₂ was designed, and the effects of reaction concentration, the type of HCR products, ionic selectivity, and the reaction order of H₁ and H₂ on the HCR process were investigated. We discovered that the cascade reaction produced by HCR in cells primarily formed six distinct assemblies and was dependent on different concentrations of Na⁺ and Mg²⁺ salts. Importantly, it was found that the addition sequence of the two hairpin DNAs was closely related to the speed of the cascading reaction in living cells. Adding H₁ first and then H₂ resulted in stronger detection signals in a relatively short time. By elucidating these details, we carried out the rapid fluorescence colocalization detection of endogenous miR-21 in living cells.