A Programmable Automatic Cascade Machinery for Single-Molecule Profiling of Multiple Noncoding RNAs in Breast Tissues

Noncoding RNAs (ncRNAs) are identified as critical regulatory molecules in tumorigenesis and progression. Investigating the expression patterns of multiple ncRNAs in living cells and tissues may facilitate the diagnosis of cancers. Herein, we develop a programmable automatic cascade machinery for single-molecule profiling of multiple ncRNAs. This method involves two successive amplification events that can convert extremely low-abundance target ncRNAs into abundant FAM/Cy5 molecules for the generation of amplified fluorescence signals. The subsequent single-molecule detection can identify piR-36026 with the FAM signal and DSCAM-AS1 with the Cy5 signal. Due to the high efficiency of automatic cascade machinery and the high signal-to-noise ratio of single-molecule imaging, this method can achieve sensitive detection of multiple ncRNAs with a detection limit of 44.67 aM for piR-36026 and 45.71 aM for DSCAM-AS1, and it can measure endogenous piR-36026 and DSCAM-AS1 at the single-cell level. Moreover, the profiling of piR-36026 and DSCAM-AS1 in healthy tissues and breast cancer tissues demonstrates the feasibility of the proposed method in cancer diagnostics. By programming the recognition sequences of dumbbell probes, this method can be extended to measure other cancer-related ncRNAs, with great prospects in clinical applications.