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Multiplexed and amplified chemiluminescence resonance energy transfer (CRET) detection of genes and microRNAs using dye-loaded hemin/G-quadruplex-modified UiO-66 metal-organic framework nanoparticles.

ABSTRACT: Dye-loaded UiO-66 metal-organic framework nanoparticles (NMOFs) modified with catalytic hemin/G-quadruplex DNAzyme labels act as functional hybrid modules for the chemiluminescence resonance energy transfer (CRET) analysis of miRNAs (miRNA-155 or miRNA-21) or genes (p53 or BRCA1). The dye-loaded NMOFs (dye = fluorescein (Fl) or rhodamine 6G (Rh 6G)) are modified with hairpin probes that are engineered to include in their loop domains recognition sequences for the miRNAs or genes, and in their stem regions caged G-quadruplex domains. In the presence of the analytes miRNAs or genes, the hairpin structures are opened, leading, in the presence of hemin, to the self-assembly of hemin/G-quadruplex DNAzyme labels linked to the dye-loaded NMOFs. In the presence of luminol and H₂O₂, the hemin/G-quadruplex DNAzyme labels catalyze the generation of chemiluminescence that provides radiative energy to stimulate the process of CRET to the dye loaded in the NMOFs, resulting in the luminescence of the loaded dye without external excitation. The resulting CRET signals relate to the concentrations of the miRNAs or the genes and allow the sensitive analysis of miRNAs and genes. In addition, the DNA hairpin-functionalized dye-loaded NMOF sensing modules were further applied to develop amplified miRNA or gene CRET-based sensing platforms. The dye-loaded NMOFs were modified with hairpin probes that include in their loop domain the recognition sequences for miRNA-155 or miRNA-21 or the recognition sequences for the p53 or BRCA1 genes. Subjecting the hairpin-modified NMOFs to the respective miRNAs or genes, in the presence of two hairpins H _i and H _j that include in their stem regions caged G-quadruplex subunit domains, results in the analyte-triggered opening of the probe hairpin linked to the NMOFs, and the opened hairpin tethers induce the cross-opening of the hairpins H _i and H _j by the hybridization chain reaction, HCR, resulting in the assembly of G-quadruplex wires tethered to the NMOFs. The binding of hemin to the HCR-generated chains yields hemin/G-quadruplex DNAzyme wires that enhance, in the presence of luminol/H₂O₂, the CRET processes in the hybrid nanostructures. These amplification platforms lead to the amplified sensing of miRNAs and genes. By mixing the Fl- and Rh 6G-loaded hairpin-functionalized UiO NMOFs, the multiplexed CRET detection of miRNA-155, miRNA-21 and the p53 and BRCA1 genes is demonstrated.

SUBMITTER: Zhang P

PROVIDER: S-EPMC8179566 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The use of drug delivery systems in targeting and achieving the targeting of drugs in treating diseases, especially cancer, has attracted the attention of researchers. Letrozole is one of the drugs for the treatment of breast cancer. In this study, the organic-metallic pharmaceutical porous nanostructure based on zirconium UiO-66 loaded letrozole was synthesized. Its cytotoxicity and effect on apoptosis and migration against breast cancer cell line were investigated. In this experimental study, the UiO-66 nanoparticle-loaded letrozole was synthesized using zirconium chloride (ZrCl4), dimethylformamide (DMF), and HCl. Its characteristics were determined by scanning electron microscopy, and its average size was determined by the DLS method. Also, the rate of letrozole drug release from the nanoparticle was investigated in 24, 48, and 72 h. In addition, its cytotoxicity effects were investigated using the MTT colorimetric method at concentrations of 3.125-100 µg/ml against the breast cancer cell line (MCF-7) in the periods of 48 and 72 h. Also, the expression level of apoptotic genes Bax and Bcl2 was investigated by the Real-Time PCR method. Also, the amount of cell migration was done by the migration assay method. The results showed that UiO-66 bound to letrozole had a spherical morphology and an average size of 9.2 ± 160.1. Also, the letrozole drug was loaded by 62.21 ± 1.80% in UiO-66 nanoparticles and had a slower release pattern than free letrozole in the drug release test, so within 72 h, 99.99% of free letrozole was released in If in UiO-66 containing letrozole, 57.55% of the drug has been released. Also, the cytotoxicity results showed that UiO-66 bound to letrozole has more significant cytotoxic effects than free letrozole (p < 0.05). Also, the results of Bax and Bcl2 gene expression showed that the treatment of MCF-7 cells with UiO-66 nanoparticles attached to letrozole increased the expression of Bax and Bcl2 genes compared to the reference gene Beta-actin in MCF-7 cell line, respectively. (p < 0.05) increased by 3.71 ± 0.42 and (p < 0.01) decreased by 0.636 ± 0.034 (p < 0.05). Cell migration results showed that the concentration of 50 µg/ml of UiO-66 bound to letrozole decreased the migration of MCF-7 cells. Generally, the results of this study showed that UiO-66 loaded letrozole can be used as a suitable drug carrier for cellular purposes, as it has increased the effects of cytotoxicity and the rate of apoptosis in breast cancer cell line (MCF-7), so it can be used with more studies used nanocarriers as a drug delivery system.

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Multiplexed and amplified chemiluminescence resonance energy transfer (CRET) detection of genes and microRNAs using dye-loaded hemin/G-quadruplex-modified UiO-66 metal-organic framework nanoparticles.

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