DNAzyme-Amplified Cascade Catalytic Hairpin Assembly Nanosystem for Sensitive MicroRNA Imaging in Living Cells

Abstract

Sensitive imaging of microRNAs (miRNAs) in living cells is significant for accurate cancer clinical diagnosis and prognosis research studies, but it is challenged by inefficient intracellular delivery, instability of nucleic acid probes, and limited amplification efficiency. Herein, we engineered a DNAzymeamplified cascade catalytic hairpin assembly (CHA)-based nanosystem (DCC) that overcomes these challenges and improves the imaging sensitivity. This enzyme-free amplification nanosystem is based on the sequential activation of DNAzyme amplification and CHA. MnO 2 nanosheets were used as nanocarriers for the delivery of nucleic acid probes, which can resist the degradation by nucleases and supply Mn 2+ for the DNAzyme reaction. After entering into living cells, the MnO 2 nanosheets can be decomposed by intracellular glutathione (GSH) and release the loaded nucleic acid probes. In the presence of target miRNA, the locking strand (L) was hybridized with target miRNA, and the DNAzyme was released, which then cleaved the substrate hairpin (H 1 ). This cleavage reaction resulted in the formation of a trigger sequence (TS) that can activate CHA and recover the fluorescence readout. Meanwhile, the DNAzyme was released from the cleaved H 1 and bound to other H 1 for new rounds of DNAzyme-based amplification. The TS was also released from CHA and involved in the new cycle of CHA. By this DCC nanosystem, low-abundance target miRNA can activate many DNAzyme and generate numerous TS for CHA, resulting in sensitive and selective analysis of miRNAs with a limit of detection of 5.4 pM, which is 18-fold lower than that of the traditional CHA system. This stable, sensitive, and selective nanosystem holds great potential for miRNA analysis, clinical diagnosis, and other related biomedical applications.

Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing

“…MiR-21 expression is dynamic, and its expression in the same cancer cell will be varied in different stages of tumorigenesis. − As a proof of concept, MCF-7 cells were pretreated with different agents to induce miR-21 expression changes, and the expression fluctuation was monitored by the CRISPR/Cas12a-based biosensor (Figure A). Specifically, the artificially synthesized miR-21 DNA analogue (miR-21 mimic) and anti-miR-21 (miR-21 antisense oligonucleotide with a complementary sequence to miR-21) were transfected into cells to increase or decrease intracellular miR-21, respectively.…”

AND Logic-Gate-Based Dual-Locking Probe System for the Sensitive Detection of microRNA and APE1

“…DNA nanotechnology, as a highly programmable and biocompatible nanomaterial, finds widespread applications in various fields, such as biosensing and biocomputing. DNA circuits represent a distinctive class of DNA nanodevices, enabling accurate and sensitive detection of cancer biomarkers through a series of dynamic reactions based on DNA. − Nucleic acid circuits based on toehold-mediated strand displacement reactions (TMSDRs) form the foundation of dynamic DNA systems. − Some researchers have explored the use of DNA nanostructures for logic detection of APE1 and miRNA, with a focus on ensuring logical reliability, which has remained a core issue in this context. , …”

Endogenous AND Logic DNA Nanomachine for Highly Specific Cancer Cell Imaging

“…Specific imaging of cancer cell based on biomarkers is of high significance to achieve the early cancer diagnosis and the reduction of disease related mortality. − The combination of precise base-pairing, excellent biocompatibility, and the high programmability has rendered DNA circuits excellent devices for biomedical imaging in living cells. − Particularly, DNA circuits that could ingeniously integrate programmable DNA self-assembly and functional modules of signal amplification have been constructed for low-abundance biomarkers imaging. − As a typical isothermal DNA circuit, toehold-mediated strand displacement (TMSD) reaction has been developed for amplification detection of biomarkers of interest. , Besides, spatial-confinement effect limited the substrates into a nanoscale for high local concentration, which significantly enhanced the operating efficiency of those DNA circuits. ,− Based on the principle, some emerging DNA circuits, including localized TMSD circuit, were developed. , Despite the improvements in the sensitivity of these methods, cancer cell imaging specificity is still constrained, mainly owing to the signal leakages from the confined space , and the false positive associated with single-biomarker detection, because most biomarkers overexpressed in cancer cells also exist in normal cells, such as miR-21. , Accordingly, establishing multiple activation DNA circuits with specific control to achieve highly reliable cancer cell imaging is needed.…”