Framework Nucleic Acid-Based Spatial-Confinement Amplifier for miRNA Imaging in Living Cells

Abstract

Real-time in situ monitoring of miRNAs in living cells is often appealed to signal amplifiers to tackle their low abundance challenges. However, the poor kinetics of amplifiers and potential interferences from the complex intracellular environment hamper its widespread applications in vivo. Herein, we report a framework nucleic acid (FNA)-based nonenzymatic spatial-confinement amplifier for rapid and reliable intracellular miRNA imaging. The amplifier consists of a localized catalytic hairpin assembly (L-CHA) reactor encapsulated in the inner cavity of an FNA (a 20 bp cube). The L-CHA reactor is certainly confined to the internal frame by integrating two probes (H1 and H2) of the L-CHA within a DNA strand and harnessing it to the opposite angles of the cube. We find that the stability of the amplifier is remarkably improved due to the protection of the FNA. More importantly, the spatial-confinement effect of the FNA can endow the confined L-CHA amplifier with enhanced local concentrations of reagents (5000-fold), thereby accelerating the reaction rate and improving the dynamic performance (up to 14.34-fold). With these advantages, the proposed amplifier can enable accurate and effective monitoring of miRNA expression levels in living cells and poses great potential in medical diagnostics and biomedical research.

Discrimination between Cancer Cells and DNA-Damaged Cells: Pre-miRNA Region Recognition Based on Hyperbranched Hybrid Chain Reaction Amplification for Simultaneous Sensitive Detection and Imaging of miRNA and Pre-miRNA

“…MicroRNAs (miRNAs) are a series of small regulatory RNAs whose abnormal expression is associated with a variety of malignancies, so they have been suggested as a diagnostic method. − However, miRNAs are not only aberrantly expressed in tumor cells but also in some cells in the stage of DNA damage, such as those after radiopharmaceutical treatment. − Thus, researchers use only miRNA biomarkers to diagnose cellular disease types that may risk a false discovery rate result in overmedicalization. − Therefore, it remains a huge challenge in tumor diagnosis and prognosis to distinguish tumor cells from DNA-damaged cells. Medical studies have shown that tumor cells witnessed the anomalous expression of mature microRNA (miRNA) and the normal expression of precursor microRNA (Pre-miRNA), while both were expressed abnormally in DNA-damaged cells. − Accordingly, when both Pre-miRNA and miRNA can be simultaneously and accurately detected, it will be possible to distinguish tumor cells from DNA-damaged cells.…”

Advances and prospects of dynamic DNA nanostructures in biomedical applications

“…Effective and sensitive detection of cancer-associated miRNAs is strongly needed in cancer diagnosis and related biological studies. Compared to other imaging methods which need preprocessing to extract miRNA from cell lysates, 58 DDNs allow in situ bioimaging of miRNA expression in living cells with better cell specicity and temporal accuracy. able to measure survivin mRNA, which is highly associated with malignant tumors, in live cancer cells with accuracy and sensitivity, but also can track survivin mRNA dynamically.…”

“…Effective and sensitive detection of cancer-associated miRNAs is strongly needed in cancer diagnosis and related biological studies. Compared to other imaging methods which need pre-processing to extract miRNA from cell lysates, 58 DDNs allow in situ bioimaging of miRNA expression in living cells with better cell specificity and temporal accuracy.…”

Target-mediated self-assembly of DNA networks for sensitive detection and intracellular imaging of APE1 in living cells

“…Nevertheless, traditional CHA reactions relied on random collisions of reactants in a homogeneous solution with low collision efficiency and long reaction time, resulting in a low reaction rate and nonspecic background leakage, which limit further exploration and application of CHA. [30][31][32][33] Thus, how to improve the CHA reaction kinetics is the direction of our continuous efforts. For the past few years, tetrahedral DNA nanostructures (TDNs), as the classical frame nucleic acid, have attracted great attention [33][34][35][36] owing to their pre-engineered sizes, structural stability, excellent biocompatibility and cell permeability.…”