Fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues

Abstract

Long noncoding RNAs (lncRNAs) act as the essential regulators in various biological processes, and are becoming potential diagnostic and prognostic biomarkers. Herein, we demonstrate the fabrication of on-bead functional nucleic acid nanowires based on cyclic ligation reaction-driven rolling circle amplification for label-free detection of long noncoding RNAs in breast and lung tissues. The presence of lncRNA activates the cyclic ligation reaction, converting low-abundance target to numerous biotinylated ligation products through repetitive denaturation-hybridization-ligation. The resulting ligation products immobilize on the magnetic bead to initiate the downstream rolling circle amplification (RCA), facilitating the synthesis of long G-quadruplex nanowires. The resultant nanowires can light up Thioflavin T (ThT) to generate a high fluorescence signal. This method displays ultrahigh sensitivity with a limit of detection of 9.97 aM, and it possesses excellent single-base mismatch discrimination capability. Moreover, it can accurately quantify cellular Homeobox (HOX) gene antisense intergenic RNA (HOTAIR) at single-cell level and distinguish the HOTAIR expression level in healthy person and lung/breast cancer patient tissues, holding great promise in clinical diagnosis.