Synchronous 3D-DNA Walking-Driven Dual-Color RNA Aptamers Lighting-Up for Label-Free and Attomolar Profiling of Multiple circRNAs in Breast Cancer

Circular RNAs (circRNAs) are covalently closed, long-lived, single-stranded RNAs with key roles in tumorigenesis and progression through diverse molecular mechanisms. Nevertheless, the accurate detection of multiple circRNAs poses a major challenge due to their interspecies diversity and high degree of sequence homology. Herein, we demonstrate synchronous 3D-DNA walking-driven dual-color RNA aptamer lighting-up for label-free and attomolar measurement of multiple circRNAs in breast cancer. This nanodevice comprises two pairs of capture probes and padlock probes. In the presence of target circRNAs (circMTO1 and circCDYL), they hybridize specifically with the back-splice junction (BSJ)-binding domains of capture probes anchored on magnetic beads to obtain the RNA/DNA heteroduplexes, initiating duplex-specific nuclease-powered 3D-DNA walking and subsequent T7/T3 RNA polymerase-catalyzed rolling circle transcription (RCT) to produce long-repeat ssRNAs (i.e., Mango and MG aptamers). Then, TO1-biotin and MG selectively bind with Mango and MG aptamers to obtain the TO1-biotin/MG nanostructures for the generation of amplified dual-color fluorescence signals. This nanodevice achieves excellent specificity and attomolar sensitivity with a limit of detection of 10.96 aM for circMTO1 and 17.78 aM for circCDYL. Moreover, it can accurately quantify circRNAs in diverse cell types at the single-cell level, reliably distinguish breast cancer cells from normal cells, and effectively discriminate circRNA expressions in breast cancer tissues and healthy counterparts. Importantly, it exhibits high accuracy in diagnosing breast cancer across the clinical spectrum from early stage I-II (91.1%) to advanced stage III-IV (99.4%), providing a valuable platform for biomedical research and clinical diagnostics.