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.

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.

Exponential and efficient target-catalyst rolling circle amplification for label-free and ultrasensitive fluorescent detection of miR-21 and p53 gene.

MicroRNAs (miRNAs) and p53 gene can serve as valuable biomarkers for the diagnosis of a variety of cancers. Nevertheless, although the development of the DNA nanostructure on the detection of cancer-related biomarkers was initially demonstrated several years ago, the challenges of developing simpler, cheaper, and multi-level detection DNA biosensors persist. Herein, based on the rolling circle amplification (RCA) coupled with the target-triggered skill, we have developed a well-designed detecting platform. In this study, the dumbbell-shaped probes (DPP) could be cyclized and initiated through targets, thus beginning the target-catalyst RCA (tc-RCA) reaction, therefore engendering numerous dumbbell probe amplicons (DPA). Thereafter the probe primers (PP) mutually complementary to the loop of DPA was introduced, leading to the branch strand displacement reaction (B-SDA). SYBR Green I can effectively bind to the amplified double-stranded structures as a fluorescent reporter. Altering the target-binding sequence of the DPP, this biosensor can also be applied to detect different biomarkers. As a consequence, target miR-21 and p53 gene can be detected down to 0.65 fM and 2.04 fM respectively with a wide dynamic range. Moreover, we have also achieved the qualitative detection of interesting targets in cell lysates as well as the complex biological substrates and compared the results with reverse transcription quantitative PCR (RT-qPCR), thereby indicating the potential application in clinical diagnosis and biomedical research.

Reliable Manipulation of Gas Bubble Size on Superaerophilic Cones in Aqueous Media.

Gas bubbles in aqueous media are ubiquitous in a broad range of applications. In most cases, the size of the bubbles must be manipulated precisely. However, it is very difficult to control the size of gas bubbles. The size of gas bubbles is affected by many factors both during and after the generation process. Thus, precise manipulation of gas bubble size still remains a great challenge. The ratchet and conical hairs of the Chinese brush enable it to realize a significant capacity for holding ink and transferring them onto paper continuously and controllably. Inspired by this, a superhydrophobic/superaerophilic cone interface is developed to manipulate gas bubble size in aqueous media. When the resultant force between the Laplace force and the axial component of the buoyancy force approaches zero, the gas bubble is held steadily by the superhydrophobic/superaerophilic copper cones in a unique position (balance position). A new kind of pressure sensor is also designed based on this principle.