Conditional Regulation of HCR for Rapid Visualization of Endogenous miR-21 in Cancer Cells

Abstract

MicroRNA-21 (miR-21) is a significant tumor marker for early cancer screening. Hybrid chain reaction (HCR) as a 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, types of HCR product, ionic selectivity, and 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 adding order 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 detecting signals in a relative short time. By elucidating these details, we carried out the rapid fluorescence colocalization detection of endogenous miR-21 in living cells.