Autonomously driving multiplexed hierarchical hybridization chain reaction of a DNA cobweb sensor for monitoring intracellular microRNA

Abstract

Here, we present an autonomously driving multiplexed hierarchical hybridization chain reaction (MH-HCR) of a DNA cobweb sensor for monitoring intracellular microRNAs. The amplifier of the sensor consists of an enzyme-free amplification, two-step and multiple round hybridization chain reaction (HCR) circuit, where the target miRNA-triggered output of the first HCR stage (HCR1) serves as input to generate the following HCR stage (HCR2) and then produces n cycles (HCR1-HCR2) amplifying processes to result in a DNA cobweb sensor. By introducing a pair of fluorescence resonance energy transfer (FRET) probes, this MH-HCR-based DNA cobweb sensor enables selective and sensitive detection of miRNAs down to 1 fM. In situ imaging of miRNAs in several living cells was successfully achieved. Compared with other nonlinear HCRs, the initiation sequence is enclosed in the stable multiple capture probe, avoiding interference reaction of the capture probe with a hairpin containing DNA fluorescent agent to generate false signals. This MH-HCR amplifier provides a general and robust method for the highly sensitive and selective detection of multiple miRNAs and has the potential for early cancer diagnosis.