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Updated: May 21, 2025
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Construction of a Ligation-Controlled Single-Molecule Biosensor for Simultaneous Measurement of Multiple
Qian Zhang1,2, Li-Xue Qiao1, Dong-Ling Li3
1College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China.
Abstract:
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.
Insights
We developed a novel biosensor for detecting multiple circular RNAs (circRNAs) linked to breast cancer. This technology enables highly sensitive, simultaneous measurement of these cancer-associated molecules for early diagnosis.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- Circular RNAs (circRNAs) are crucial regulators of cellular processes, and their aberrant expression is linked to cancer development.
- Accurate and simultaneous detection of multiple circRNAs is vital for early cancer diagnosis and understanding disease progression.
Purpose of the Study:
- To develop a sensitive and specific single-molecule biosensor for the multiplexed detection of breast cancer-associated circRNAs.
- To establish a novel platform for circRNA analysis in clinical samples and single cells.
Main Methods:
- A ligation-controlled single-molecule biosensor integrating isothermal exponential amplification reaction (EXPAR) and Au nanoparticle (AuNP)-based spherical nucleic acid nanoprobe.
- Utilized back-splice junction (BSJ) sequences of circFOXO3 and circMTO1 to trigger EXPAR-induced DNAzyme generation.
- Employed DNAzymes to cyclically degrade signal probes on AuNPs, releasing fluorophores for sensitive detection.
Main Results:
- Achieved ultra-low detection limits of 8.34 aM for circFOXO3 and 9.84 aM for circMTO1.
- Successfully performed simultaneous analysis of multiple circRNAs within a single cancer cell.
- Differentiated circRNA expression levels between breast cancer tissues and healthy adjacent tissues.
Conclusions:
- The developed biosensor offers a powerful tool for simultaneous, highly sensitive detection of multiple circRNAs.
- This technology represents a significant advancement for circRNA-based molecular diagnostics and biomedical research in oncology.
