Toehold-mediated ligation-free rolling circle amplification enables sensitive and rapid imaging of messenger RNAs in situ in cells.

In situ analysis of tumor-related messenger RNAs (mRNAs) is significant in identifying cancer cells at the genetic level in the early stage. Rolling circle amplification (RCA)-based methods are primary tools for in situ mRNA assay, however, the necessary ligation reaction not only shows low ligation efficiency, but also greatly prolongs the assay time that increases the risk of cells losing and mRNAs leakage. In this work, we propose a novel toehold-mediated ligation-free RCA (TMLFRCA) on a designed structure-switchable dumbbell-shaped probe (SDP). Target mRNA can specifically activate SDP from its circular form by toehold strand displacement, thereby initiates in situ RCA for mRNA imaging with the help of a short DNA primer. For the proof-of-concept demonstration, the TK1 mRNA was sensitively detected by TMLFRCA in less than 3.5 h with a limit of detection (LOD) of 0.39 fM (corresponds to 2.39×10copiesL), and significantly improved specificity capable for distinguishing single base difference. The sensitivity of the TMLFRCA for TK1 mRNA in situ assay is ∼29-fold and ∼7-fold higher than that of FISH and ligase-assisted RCA method, respectively, which enables the TMLFRCA method capability of highly sensitive and specific distinction mRNA expression levels between cancer cells and normal cells. We believe this TMLFRCA strategy would be of great value in both basic research and clinical diagnosis.