Ultrasensitive detection of mRNA in extracellular vesicles using DNA tetrahedron-based thermophoretic assay

The analysis of messenger RNA (mRNA) in extracellular vesicles (EVs) may provide unprecedented opportunities for non-invasive and rapid diagnosis of cancers. However, clinical utility of EV mRNA is often hampered by the extremely low abundance of mRNA in EVs and the complicated procedures for mRNA detection. Here we address these challenges by developing an ultrasensitive assay for in situ measurement of EV mRNA, which combines FRET-based DNA tetrahedron (FDT) for efficient EV internalization and target mRNA detection, and size-selective thermophoretic accumulation for amplification of FRET signal in EVs. This DNA tetrahedron-based thermophoretic assay (DTTA) achieves a limit of detection (LoD) of 14 aM for detecting PSA mRNA in serum EVs, with resorting to RNA extraction and enzyme amplification. The DTTA showed that EV PSA mRNA outperforms serum PSA protein, the gold standard in PCa screening, for discrimination between prostate cancer and benign prostatic hyperplasia (area under the curve: 0.93 versus 0.74; 42 patients). This technology may greatly expand the applications of DNA nanostructure-enabled liquid biopsy.