Hairpin self-assembly powered by exonuclease III for highly sensitive and cross-validated miRNA-155 detection

Abstract

Cancer is one of the most important causes of human death and poses a serious threat to human health. As a cancer biomarker, microRNA-155 (miRNA-155) is highly expressed in various types of cancer tissues and is involved in the proliferation of tumor cells. Therefore, developing a miRNA-155 detection technology with high specificity and sensitivity is of great significance for the early detection, accurate treatment and prognostic evaluation of tumors. Here, we developed a fluorescence detection method using exonuclease III-assisted target cycling and catalytic hairpin assembly (CHA) as a signal amplification technique. This study developed a biosensor for the detection of miRNA-155, utilizing a DNA hairpin (Hp) for target recognition and generating double-stranded DNA (dual-Hp-T). The 3′ flat end of the double-stranded DNA can be cleaved by exonuclease III to achieve the target cycle, and a large amount of single-stranded DNA (fuel) can trigger CHA to achieve signal amplification. Simultaneously, the fluorescence resonance energy transfer (FRET) of signal probes with different fluorescence labels on H1 and H2 ends occurs with the CHA reaction. The two fluorescence signals obtained can be used to cross-validate the experimental results. The biosensor exhibits excellent performance of high recovery, high sensitivity and high operability, which can achieve the specific detection of miRNA-155 with a detection limit as low as 8.3 pM. Additionally, the detection efficacy in a human serum environment is also highly satisfactory. This technology provides strong technical support for the development of nucleic acid probes and the diagnosis and treatment of cancer, demonstrating significant practical application value.