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Published on: March 29, 2017
Single-Nucleotide-Specific Lipidic Nanoflares for Precise and Visible Detection of KRAS Mutations via
Chan Li1, Haiyan Jia1, Xiaoling Wei1
1Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China.
Abstract:
Accurate identification of single-nucleotide mutations in circulating tumor DNA (ctDNA) is critical for cancer surveillance and cell biology research. However, achieving precise and sensitive detection of ctDNAs in complex physiological environments remains challenging due to their low expression and interference from numerous homologous species. This study introduces single-nucleotide-specific lipidic nanoflares designed for the precise and visible detection of ctDNA via toehold-initiated self-priming DNA polymerization (TPP). This system can be assembled from only a single cholesterol-conjugated multifunctional molecular beacon (MMB) via hydrophobicity-mediated aggregation. This results in a compact, high-density, and nick-hidden arrangement of MMBs on the surface of lipidic micelles, thereby enhancing their biostability and localized concentrations. The assay commences with the binding of frequently mutated regions of ctDNA to the MMB toehold domain. This domain is the proximal holding point for initiating the TPP-based strand-displacement reaction, which is the key step in enabling the discrimination of single-base mutations. We successfully detected a single-base mutation in ctDNA (KRAS G12D) in its wild-type gene (KRAS WT), which is one of the most frequently mutated ctDNAs. Notably, coexisting homologous species did not interfere with signal transduction, and small differences in these variations can be visualized by fluorescence imaging. The limit of detection was as low as 10 amol, with the system functioning well in physiological media. In particular, this system allowed us to resolve genetic mutations in the KRAS gene in colorectal cancer, suggesting its high potential in clinical diagnosis and personalized medicine.
Insights
This study presents novel lipidic nanoflares for precise detection of circulating tumor DNA (ctDNA) mutations. The system accurately identifies single-nucleotide variations in ctDNA, crucial for cancer diagnostics and research.
Area of Science:
- Biotechnology
- Molecular Biology
- Nanotechnology
Background:
- Accurate detection of circulating tumor DNA (ctDNA) single-nucleotide mutations is vital for cancer surveillance and research.
- Low ctDNA expression and interference from homologous species present significant detection challenges.
Purpose of the Study:
- To develop a single-nucleotide-specific method for precise and visible detection of ctDNA.
- To overcome challenges in ctDNA detection in complex biological environments.
Main Methods:
- Introduction of single-nucleotide-specific lipidic nanoflares assembled from cholesterol-conjugated multifunctional molecular beacons (MMBs).
- Utilizing toehold-initiated self-priming DNA polymerization (TPP) for strand displacement and single-base discrimination.
- Employing hydrophobicity-mediated aggregation for compact MMB arrangement on lipidic micelles.
Main Results:
- Successfully detected KRAS G12D mutations in ctDNA against wild-type KRAS with high specificity.
- Demonstrated robust signal transduction unaffected by coexisting homologous species.
- Achieved a limit of detection as low as 10 amol, with visible fluorescence imaging of mutations.
Conclusions:
- The developed lipidic nanoflare system enables precise, visible, and sensitive detection of ctDNA single-nucleotide mutations.
- The system shows high potential for clinical diagnosis and personalized medicine, particularly in resolving KRAS gene mutations in colorectal cancer.
