Single-Nucleotide-Specific Lipidic Nanoflares for Precise and Visible Detection of KRAS Mutations via Toehold-Initiated Self-Priming DNA Polymerization.

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.

Lighting up Lipidic Nanoflares with Self-Powered and Multivalent 3D DNA Rolling Motors for High-Efficiency MicroRNA Sensing in Serum and Living Cells.

Intelligent DNA nanomachines are powerful and versatile molecular tools for bioimaging and biodiagnostic applications; however, they are generally constrained by complicated synthetic processes and poor reaction efficiencies. In this study, we developed a simple and efficient molecular machine by coupling a self-powered rolling motor with a lipidic nanoflare (termed RMNF), enabling high-contrast, robust, and rapid probing of cancer-associated microRNA (miRNA) in serum and living cells. The lipidic nanoflare is a cholesterol-based lipidic micelle decorated with hairpin-shaped tracks that can be facilely synthesized by stirring in buffered solution, whereas the 3D rolling motor (3D RM) is a rigidified tetrahedral DNA scaffold equipped with four single-stranded "legs" each silenced by a locking strand. Once exposed to the target miRNA, the 3D RM can be activated, followed by self-powered precession based on catalyzed hairpin assembly (CHA) and lighting up of the lipidic nanoflare. Notably, the multivalent 3D RM that moves using four DNA legs, which allows the motor to continuously and acceleratedly interreact with DNA tracks rather than dissociate from the surface of the nanoflare, yielded a limit of detection (LOD) of 500 fM at 37 °C within 1.5 h. Through the nick-hidden and rigidified structure design, RMNF exhibits high biostability and a low false-positive signal under complex physiological settings. The final application of RMNF for miRNA detection in clinical samples and living cells demonstrates its considerable potential for biomedical imaging and clinical diagnosis.