Spatial confinement-based Figure-of-Eight nanoknots accelerated simultaneous detection and imaging of intracellular microRNAs

Developing highly efficient and reliable methods for simultaneous imaging of microRNAs in living cells is often appealed to understanding their synergistic functions and guiding the diagnosis and treatment of human diseases, such as cancers. In this work, we rationally engineered a four-arm shaped nanoprobe that can be stimuli-responsively tied into a Figure-of-Eight nanoknot via spatial confinement-based dual-catalytic hairpin assembly (SPACIAL-CHA) reaction and applied for accelerated simultaneous detection and imaging of different miRNAs in living cells. The four-arm nanoprobe was facilely assembled from a cross-shaped DNA scaffold and two pairs of CHA hairpin probes (21HP-a and 21HP-b for miR-21, while 155HP-a and 155HP-b for miR-155) via the "one-pot" annealing method. The DNA scaffold structurally provided a well-known spatial-confinement effect to improve the localized concentration of CHA probes and shorten their physical distance, resulting in an enhanced intramolecular collision probability and accelerating the enzyme-free reaction. The miRNA-mediated strand displacement reactions can rapidly tie numerous four-arm nanoprobes into Figure-of-Eight nanoknots, yielding remarkably dual-channel fluorescence proportional to the different miRNA expression levels. Moreover, benefiting from the nuclease-resistant DNA structure based on the unique arched DNA protrusions makes the system ideal for operating in complicated intracellular environments. We have demonstrated that the four-arm-shaped nanoprobe is superior to the common catalytic hairpin assembly (COM-CHA) in stability, reaction speed, and amplification sensitivity in vitro and living cells. Final applications in cell imaging have also revealed the capacity of the proposed system for reliable identification of cancer cells (e.g., HeLa and MCF-7) from normal cells. The four-arm nanoprobe shows great potential in molecular biology and biomedical imaging with the above advantages.