A triggered DNA nanomachine with enzyme-free for the rapid detection of telomerase activity in a one-step method.

BACKGROUND: Telomerase is considered a biomarker for the early diagnosis and clinical treatment of cancer. The rapid and sensitive detection of telomerase activity is crucial to biological research, clinical diagnosis, and drug development. However, the main obstacles facing the current telomerase activity assay are the cumbersome and time-consuming procedure, the easy degradation of the telomerase RNA template and the need for additional proteases. Therefore, it is necessary to construct a new method for the detection of telomerase activity with easy steps, efficient reaction and strong anti-interference ability. RESULTS: Herein, an efficient, enzyme-free, economical, sensitive, fluorometric detection method for telomerase activity in one-step, named triggered-DNA (T-DNA) nanomachine, was created based on target-triggered DNAzyme-cleavage activity and catalytic molecular beacon (CMB). Telomerase served as a switch and extended few numbers of (TTAGGG)n repeat sequences to initiate the signal amplification in the T-DNA nanomachine, resulting in a strong fluorescent signal. The reaction was a one-step method with a shortened time of 1 h and a constant temperature of 37 °C, without the addition of any protease. It also sensitively distinguished telomerase activity in various cell lines. The T-DNA nanomachine offered a detection limit of 12 HeLa cells µL-1, 9 SK-Hep-1 cells µL-1 and 3 HuH-7 cells µL-1 with a linear correlation detection range of 0.39 × 102-6.25 × 102 HeLa cells µL-1 for telomerase activity. SIGNIFICANCE: In conclusion, our study demonstrated that the triggered-DNA nanomachine fulfills the requirements for rapid detection of telomerase activity in one-step under isothermal and enzyme-free conditions with excellent specificity, and its simple and stable structure makes it ideal for complex systems. These findings indicated the application prospect of DNA nanomachines in clinical diagnostics and provided new insights into the field of DNA nanomachine-based bioanalysis.

Protonation-mediated DNA tile self-assembly with nuclease resistance characteristic for signal-amplified detection of microRNAs.

DNA nanotechnology, developing rapidly in recent years, has unprecedented superiorities in biological application-oriented research including high programmability, convenient functionalization, reconfigurable structure, and intrinsic biocompatibility. However, the susceptibility to nucleases in the physiological environment has been an obstacle to applying DNA nanostructures in biological science research. In this study, a new DNA self-assembly strategy, mediated by double-protonated small molecules instead of classical metal ions, is developed to enhance the nuclease resistance of DNA nanostructures while retaining their integrality and functionality, and the relative application has been launched in the detection of microRNAs (miRNAs). Faced with low-abundance miRNAs, we integrate hybrid chain reaction (HCR) with DNA self-assembly in the presence of double-protonated small molecules to construct a chemiluminescence detection platform with nuclease resistance, which utilizes the significant difference of molecular weight between DNA arrays and false-positive products to effectively separate of reaction products and remove the detection background. This strategy attaches importance to the nucleic acid stability during the assay process via improving nuclease resistance while rendering the detection results for miRNAs more authentic and reliable, opening our eyes to more possibilities for the multiple applications of customized DNA nanostructures in biology, including bioassay, bioimaging, drug delivery, and cell modulation.

A specific and low background nucleic acids sensing strategy based on rolling circle amplification coupled with a magnetic DNA machine.

We propose a universal fluorescence method for detection of nucleic acids based on rolling circle amplification (RCA) combined with a magnetic DNA machine and using dengue virus nucleic acids as an example target. RCA specifically amplifies the target and yields a large number of initiators employing heat-labile double-stranded DNase. The magnetic DNA machine produces a fluorescence signal and eliminates background noise. This method achieved a wide linear range, promising recovery and ultrahigh recognition specificity for one-base mismatches, and indicates the potential application of this sensing strategy in the clinical diagnosis of nucleic acids of pathogens.

Size-Controlled DNA Tile Self-Assembly Nanostructures Through Caveolae-Mediated Endocytosis for Signal-Amplified Imaging of MicroRNAs in Living Cells.

Signal-amplified imaging of microRNAs (miRNAs) is a promising strategy at the single-cell level because liquid biopsy fails to reflect real-time dynamic miRNA levels. However, the internalization pathways for available conventional vectors predominantly involve endo-lysosomes, showing nonideal cytoplasmic delivery efficiency. In this study, size-controlled 9-tile nanoarrays are designed and constructed by integrating catalytic hairpin assembly (CHA) with DNA tile self-assembly technology to achieve caveolae-mediated endocytosis for the amplified imaging of miRNAs in a complex intracellular environment. Compared with classical CHA, the 9-tile nanoarrays possess high sensitivity and specificity for miRNAs, achieve excellent internalization efficiency by caveolar endocytosis, bypassing lysosomal traps, and exhibit more powerful signal-amplified imaging of intracellular miRNAs. Because of their excellent safety, physiological stability, and highly efficient cytoplasmic delivery, the 9-tile nanoarrays can realize real-time amplified monitoring of miRNAs in various tumor and identical cells of different periods, and imaging effects are consistent with the actual expression levels of miRNAs, ultimately demonstrating their feasibility and capacity. This strategy provides a high-potential delivery pathway for cell imaging and targeted delivery, simultaneously offering a meaningful reference for the application of DNA tile self-assembly technology in relevant fundamental research and medical diagnostics.

Shape dependency of gold nanorods through TMB2+-mediated etching for the visual detection of NT-proBNP.

Heart failure (HF) is a major public health problem triggered by heart circulation disorders. Early detection and diagnosis are conducive to the prevention and treatment of HF. Hence, it is necessary to establish a simple and sensitive method to monitor the diagnostic biomarkers of HF. The N-terminal B-type natriuretic peptide precursor (NT-proBNP) is acknowledged as a sensitive biomarker. In this study, a visual detection method for NT-proBNP was developed based on the oxidized 3,3',5,5'-tetramethylbenzidine (TMB2+)-mediated etching of gold nanorods (AuNRs) and double-antibody-sandwich ELISA. The etching color for different amounts of NT-proBNP was obvious and significant differences could be ascertained based on the blue-shift of the longitudinal localized surface plasmon resonance (LLSPR) of the AuNRs. The results could be observed by the naked eye. The constructed system showed a concentration range from 6 to 100 ng mL-1 and a low detection limit of 6 ng mL-1. This method exhibited negligible cross-reactivity toward other proteins, and the recoveries of the samples ranged from 79.99% to 88.99%. These results demonstrated that the established method is suitable for the simple and convenient detection of NT-proBNP.

Kinetics-accelerated one-step detection of MicroRNA through spatially localized reactions based on DNA tile self-assembly.

The localization of isothermal amplification systems has elicited extensive attention due to the enhanced reaction kinetics when detecting ultra-trace small-molecule nucleic acids. Therefore, the seek for an appropriate localization cargo of spatially confined reactions is urgent. Herein, we have developed a novel approach to localize the catalytic hairpin assembly (CHA) system into the DNA tile self-assembly nanostructure. Thanks to the precise programming and robust probe loading capacity, this strategy achieved a 2.3 × 105-fold higher local reaction concentration than a classical CHA system with enhanced reaction kinetics in theory. From the experimental results, this strategy could reach the reaction plateau faster and get access to a magnified effect of 1.57-6.99 times higher in the linear range of microRNA (miRNA) than the simple CHA system. Meanwhile, this strategy satisfied the demand for the one-step detection of miRNA in cell lysates at room temperature with good sensitivity and specificity. These features indicated its excellent potential for ultra-trace molecule detection in clinical diagnosis and provided new insights into the field of bioassays based on DNA tile self-assembly nanotechnology.