Construction of a Structure-Switchable Toehold Dumbbell Probe for Sensitive and Label-Free Measurement of MicroRNA in Cancer Cells and Tissues.

MicroRNAs (miRNAs) play multiple crucial roles in post-transcriptional regulating gene expression, and the abnormal expression may induce various human diseases. Herein, we demonstrate the construction of a structure-switchable toehold dumbbell probe for sensitive and label-free measurement of microRNA in cancer cells and tissues on the basis of integrating exponential-rolling circle amplification (EXP-RCA) with linear-rolling circle amplification (LRCA). We designed a structure-switchable toehold dumbbell probe with annular and symmetric structure whose either side can hybridize with target miRNA to initiate EXP-RCA, greatly improving the detection sensitivity. Moreover, the dumbbell probe is designed with an appropriate standard free energy ( G ), and it cannot be activated by mismatched miRNAs, endowing this assay with good specificity. When target miRNA is present, it interacts with the dumbbell probe to activate EXP-RCA via toehold-mediated stand displacement, generating abundant triggers. The resulting triggers and target miRNA can function as primers to initiate LRCA, producing abundant long tandem repeats that can generate a distinct fluorescence signal using SYBR Gold as the indicator. This assay can be carried out homogeneously and isothermally without the requirement for either sophisticated modification/separation steps or any extra primers. It displays ultrahigh sensitivity with a limit of detection of 8.45 × 10 -17 M and excellent specificity, and it can differentiate let-7a from its homologous analogues. Moreover, this method can accurately quantify let-7a expression at a single-cell level and can even distinguish the let-7a expression between non-small cell lung cancer patient tissues and healthy person tissues.