Self-assembly of protein-DNA hybrids dedicated to an accelerated and self-primed strand displacement amplification for reinforced serum microRNA probing.

BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.

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.

Construction of a 3D rigidified DNA nanodevice for anti-interference and reinforced biosensing by turning nuclease into a catalyst.

The practical application of DNA biosensors is impeded by numerous limitations in complicated physiological environments, particularly the susceptibility of common DNA components to nuclease degradation, which has been recognized as a major barrier in DNA nanotechnology. In contrast, the present study presents an anti-interference and reinforced biosensing strategy based on a 3D DNA-rigidified nanodevice (3D RND) by converting a nuclease into a catalyst. 3D RND is a well-known tetrahedral DNA scaffold containing four faces, four vertices, and six double-stranded edges. The scaffold was rebuilt to serve as a biosensor by embedding a recognition region and two palindromic tails on one edge. In the absence of a target, the rigidified nanodevice exhibited enhanced nuclease resistance, resulting in a low false-positive signal. 3D RNDs have been proven to be compatible with 10% serum for at least 8 h. Once exposed to the target miRNA, the system can be unlocked and converted into common DNAs from a high-defense state, followed by polymerase- and nuclease-co-driven conformational downgrading to achieve amplified and reinforced biosensing. The signal response can be improved by approximately 700% within 2 h at room temperature, and the limit of detection (LOD) is approximately 10-fold lower under biomimetic conditions. The final application to serum miRNA-mediated clinical diagnosis of colorectal cancer (CRC) patients revealed that 3D RND is a reliable approach to collecting clinical information for differentiating patients from healthy individuals. This study provides novel insights into the development of anti-interference and reinforced DNA biosensors.

Microencapsulation of tomato seed oil using phlorotannins-adducted pea protein isolate-chitosan and pea protein isolate-chitosan complex coacervates.

Pea protein isolates (PPI)/phlorotannins (PT)/chitosan (CS) ternary complex and PPI/CS binary complex were synthesized to prepare tomato seed oil (TSO) microcapsules. The concentration of PT was determined to be 0.025% (w/w) based on the solubility, emulsification, and UV-visible spectrum of PPI-PT complex. Subsequently, the optimal pHs associated with the formation of PPI/CS and PPI-PT/CS complex coacervates were determined to be pH 6.6 and 6.1, while the optimal ratios were 9:1 and 6:1, respectively. The coacervate microcapsules were successfully produced by freeze-dried method and those formulated with PPI-PT/CS displayed significantly lower surface oil content (14.57 ± 0.22%), higher encapsulation efficiency (70.54 ± 0.13%), lower particle size (5.97 ± 0.16 µm), and PDI (0.25 ± 0.02) than PPI/CS. The microcapsules were characterized by scanning electron microscopy and Fourier Transform infrared spectroscopy. Furthermore, the encapsulated TSO exhibited enhanced thermal and oxidative stability than that of free oil, along with microcapsules fabricated with PPI-PT/CS ternary complex showed better protection than that of free PT. Overall, PPI-PT/CS complex as an effective wall material in delivery system presented great potential.

Preparation and physicochemical stability of tomato seed oil microemulsions.

In this study, microemulsions were fabricated using tomato seed oil, water, Tween 80 and citric acid, and then the physicochemical characteristics and the influence of environmental stress were investigated. The physicochemical properties of the microemulsions were evaluated by transmission electron microscopy (TEM), mean particle diameter, polydispersity index (PDI) and conductivity. The phase diagrams of tomato seed oil/Tween 80/citric acid/water microemulsions were constructed under different pHs and ionic strengths. Storage stability of the systems was investigated at 4, 37 and 65°C, and changes in turbidity and lipid oxidation products were monitored. Nano-size zeta potential analyzer results demonstrated that the mean particle diameter and polydispersity index of tomato seed oil microemulsions were 14 nm and 0.014. The transition from W/O to O/W could be detected from electrical conductivity and viscosity data with the increasing of water content. The results showed that the microemulsion areas decreased with increasing pH and NaCl concentrations. What is more, the study proved that tomato seed oil microemulsions exhibited a good storage stability. PRACTICAL APPLICATION: In this study, the preparation of tomato seed oil microemulsion can not only make full use of the nutritional value of tomato seed oil, but also ensure the effective protection of the nutrients contained in it, and improve the problem of adding difficult. By using microemulsion as delivery carrier of tomato seed oil, the application of tomato seed oil in food, cosmetics and other fields could be enhanced. Therefore, the preparation of tomato seed oil microemulsion provides a theoretical basis for production practice.