Modular engineering of a hierarchical hybridization-reconfiguration-replication circuit for rapid detection of long noncoding RNA with attomolar sensitivity

Long non-coding RNAs (lncRNAs) are important regulators that affect gene expression at multiple levels, and their dysregulation are positively correlated with the pathogenesis and progression of various cancers. Herein, we engineer a hierarchical hybridization-reconfiguration-replication circuit for simple, rapid, and sensitive detection of lncRNA in cancer cells and tissues. We rationally design a structure-switchable dumbbell probe. The presence of target lncRNA can specifically compel the dumbbell probe to transform into an activated configuration through toehold-mediated strand migration, thereby initiating the hierarchical priming co-directed amplification reaction wherein each amplicon can be recycled to promote the signal gain without wasting species. This all-in-amplification and circuiting-to-feedback pattern result in the exponential accumulation of numerous G-quadruplex-containing motifs that can be lighted up by thioflavin T (ThT) to produce a significantly enhanced fluorescence signal. Notably, the self-primer within the dumbbell probe can prominently speed up the responsiveness of the whole amplification process due to the spatial confinement effect. This method enables one-probe, one-step, and rapid (within 30 min) detection of lncRNA HOTAIR with a detection limit of 6.78 aM. Moreover, it can be used to quantify cellular HOTAIR expression at single-cell level, and discriminate HOTAIR expression between breast cancer tissues and corresponding healthy para-carcinoma tissues.