Self-Hybridization Chain Polymerization-Driven Transcriptional Synthesis of Fluorescent Aptamers for One-Step Detection of Cancer-Associated RNA Biomarkers

Aberrant RNA expression is closely linked to cancer development, making its quantification vital for diagnosis and therapy. However, existing RNA assays often face some limitations such as complex workflows, limited sensitivity, involvement of multiple probes, and nonspecific background. To address these challenges, we developed a primer-free method for one-step detection of cancer-associated RNA biomarkers based on self-hybridization chain polymerization-driven transcriptional synthesis of fluorescent aptamers. In this assay, the target RNA-induced conformational switch of the hairpin probe triggers the autonomous polymerization of a long DNA strand containing tandem T7 promoters that subsequently transcribes numerous Mango RNA aptamers. The resultant aptamers can bind to the fluorogenic dye TO1-biotin, generating a strong amplified fluorescence signal. This assay can be completed within 40 min in a homogeneous and isothermal manner, and it possesses excellent single-base mismatch discrimination capability and enables ultrasensitive and label-free detection of thymidine kinase 1 (TK1) mRNA with a limit of detection of as low as 49.09 aM. Moreover, this method can profile endogenous mRNA expression in cancer cells and accurately differentiate breast cancer tissues from healthy ones. Moreover, this method can be readily adapted to detect other RNA biomarkers (e.g., miR-21) by simply reconfiguring the recognition sequence of the hairpin probe, providing a versatile and powerful platform for biomedical research, molecular diagnostics, and point-of-care testing.