Construction of a Ligation-Controlled Single-Molecule Biosensor for Simultaneous Measurement of Multiple Cancer-Related circRNAs in Clinical Tissues

Circular RNAs (circRNAs) are noncoding RNAs with covalently closed circular structures that regulate important cellular processes, and their dysregulation is implicated in the pathogenesis and progression of various cancers. Simultaneous and specific detection of multiple circRNAs is of significant importance in the early diagnosis of cancer. Herein, we develop a ligation-controlled single-molecule biosensor for multiplexed measurement of breast cancer-associated circRNAs. This assay integrates the isothermal exponential amplification reaction (EXPAR)-induced generation of multiple DNAzymes with a Au nanoparticle (AuNP)-based spherical nucleic acid nanoprobe. The back-splice junction (BSJ) sequences of circFOXO3 and circMTO1 can serve as the templates to ligate their hairpin probes and helper probes under the catalysis of SplintR ligase, forming complete amplification templates. Afterward, the ligated amplification template can serve as both a primer and a template to initiate the EXPAR, inducing the exponential accumulation of characteristic DNAzyme sequences (i.e., DNAzymes 1 and 2). DNAzymes 1 and 2 can be paired with signal probes 1 and 2 immobilized on the AuNP surface, respectively, inducing cyclic degradation of signal probes to liberate large amounts of Cy5 and Cy3 fluorophores and achieving detection limits of 8.34 aM for circFOXO3 and 9.84 aM for circMTO1. This single-molecule biosensor has been successfully applied for simultaneous analysis of multiple circRNAs in a single cancer cell and differentiation of multiple circRNA levels between breast cancer tissues and healthy para-carcinoma tissues, offering a new paradigm for biomedical research and circRNA-related molecular diagnostics.