Size-Discriminative DNA Nanocage Framework Enables Sensitive and High-Fidelity Imaging of Mature MicroRNA in Living Cells

Abstract

Mature microRNAs (miRNAs) are closely associated with cell proliferation and differentiation, stress response, and carcinogenesis, and monitoring intracellular miRNAs can contribute to the studies of their regulatory roles and molecular mechanisms of disease progression. However, accurate and reliable detection of mature miRNAs in complex physiological environments encounters the challenge of undesired detection accuracy ascribed to the coexistence of their precursor microRNAs (pre-miRNAs) and degradation of sensing probes. Here, we demonstrate the synthesis of a new size-discriminative DNA nanocage framework (DNF) for the sensitive monitoring of mature miRNA-21 in living cells with high accuracy via cascaded toehold-mediated strand displacement reaction (TSDR) amplifications. The DNF is prepared by a simple self-assembly of six ssDNAs, and the signal probes are docked inside the DNF. Because of its rigid framework structure, the DNF shows enhanced enzyme stability. Upon entering cells, only the short target mature miRNA-21 sequences instead of the large-sized pre-miRNAs are allowed to be accommodated inside the cavity of the DNF owing to the size-discriminative capability of the DNF. The cascaded TSDR amplifications can thus be activated by the mature miRNA-21 together with endogenous ATP to result in magnified fluorescence for sensitive detection and selective discrimination of miRNA-21 from the interference pre-miRNAs. Our results indicate that the DNF probes can offer robust sensing means for detecting various intracellular mature miRNAs with high accuracy for disease diagnoses and biomedical studies.

Endogenous Enzyme-Driven Amplified DNA Nanocage Probe for Selective and Sensitive Imaging of Mature MicroRNAs in Living Cancer Cells

“…Two cancer cell lines with obviously varied mature miR-21 expression levels, including MCF-7 (higher expression level of mature miR-21) and HeLa cells (lower expression level of mature miR-21), were incubated with Acage and a free probe, respectively. In the case of Acage probe, the fluorescence intensity in HeLa cells is much lower than that in MCF-7 cells (Figure d), indicating the lower expression level of miRNA-21 in HeLa cells than that of MCF-7 cells, which is consistent with the result in previous literature ,, as well as the result of qRT-PCR (Figure S20a). However, in the case of the free probe, the fluorescence intensity of HeLa cells is strong (Figure c).…”

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging

“…Considerable effort has been invested in improving the sensitivity of sensing systems, particularly the introduction of various DNA cascade circuit-based isothermal amplification techniques, such as catalytic hairpin assembly (CHA), entropy-driven catalysis (EDC), and hybridization chain reaction (HCR). − These amplification approaches, performing one-to-many signal output, allow a single target molecule to activate many signal molecules by programmed DNA assembly or a DNA chain-growth polymerization reaction, paving a facial path for nonenzymatic signal amplification using only several artificially programmable DNA sequences. Accordingly, extensive research endeavor has been directed toward utilizing DNA cascade circuits to identify cancer-related biomarkers, such as microRNAs, adenosine triphosphate (ATP), and telomerase. − However, the DNA cascade circuits triggered by a single biomarker are prone to unreliable cellular imaging output due to the lack of gating for specific cancer cells. In other words, even for downregulated biomarkers in normal cells, the DNA cascade circuit can automatically execute an unrestricted cyclic amplification reaction and eventually provide a false-positive image output to negative cells and on-target off-cancer risks, leading to increased sensitivity but compromised specificity for cancer sensing.…”

NIR Photocontrolled Fluorescent Nanosensor under a Six-Branched DNA Nanowheel-Induced Nucleic Acid Confinement Effect for High-Performance Bioimaging

“…As a result, seeking an effective means to confine the reactants into a restrictive contact space is another difficulty. With the rapid extension of ingenious DNA nanostructures, a concept named nucleic acid confinement effect is capable of being utilized to intensively accelerate the intermolecular mass transportation efficiency. − Among the available structure types (e.g., cage, tetrahedron, triangular prism, and wheel), the wheel case containing multiple DNA branches has not only the advantage of loading abundant fluorescence output units but also a more flexible construction.…”