Fluorescent sensing of tsRNA by silver nanoclusters with exponential amplification-mediated DNA tetrahedron

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are crucial regulators and potential biomarkers for various cancers, including breast cancer, due to their stability and specific expression in tumor tissues. However, current detection methods face limitations like complex instrumentation and low sensitivity, highlighting the urgent need for the development of rapid and reliable detection platforms for tsRNAs. The EXPAR reaction could amplify the tsRNA by several orders of magnitude into another oligo sequence. The amplified oligos can undergo a toehold-mediated strand displacement reaction (TMDR) with the blocked sequence on one edge of the DNA tetrahedron, exposing a poly C sequence capable of forming a stem-loop structure. During this process, the DNA tetrahedron provides structural stability. The exposed polyC sequence efficiently facilitates the synthesis of silver nanoclusters, enabling the detection of tsRNA through fluorescence signals. Under the optimal condition, the method achieved a detection limit as low as 41.21 fM. Validation using clinical samples from breast cancer patients showed strong correlation with qRT-PCR results, confirming the clinical utility of the platform. The proposed EXPAR-DTH platform addressed the key limitations of current tsRNA detection technologies by offering a highly sensitive, cost-effective, and easy-to-implement method. Its success in clinical sample analysis highlighted its potential for use in cancer diagnostics, particularly in point-of-care settings.