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.

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.