ABSTRACT
Highly efficient cellular transfection and intracellular signal amplification is a prerequisite for low-abundant microRNA (miRNA) imaging and biomedical application. Herein, we report a functional cancer cell membrane (CM) vesicle, Au-P/DSN@CM (DSN, double-specific nucleases), which consists of Au nanoparticles modified with three types of fluorescent miRNA detection probes (Au-P) and DSN that simultaneously encapsulate in cancer CM. We find that the Au-P/DSN@CM could specifically target the cancer cell and transfect the cell with higher efficiency than Au nanoparticles. The internalized Au-P/DSN@CM could further specifically recognize the target miRNA and induce DSN-assisted target recycle signal amplification, leading to multiple miRNA simultaneous detection with high sensitivity. It successfully detects oncogenic miRNAs in MCF-7 cells with high sensitivity and is amenable to monitor the dynamic expression change of oncogenic miRNAs in cancer cells. Our study represents a promising gene delivery vector for cancer diagnosis and potential therapy.
Subject(s)
Cell Membrane/chemistry , MicroRNAs/analysis , DNA/chemistry , DNA/genetics , DNA/toxicity , DNA Probes/chemistry , DNA Probes/genetics , DNA Probes/toxicity , Endonucleases/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Gold/chemistry , Gold/toxicity , Humans , MCF-7 Cells , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , MicroRNAs/genetics , MicroRNAs/metabolism , Microscopy, Fluorescence/methods , Nucleic Acid Hybridization , Spectrometry, Fluorescence/methodsABSTRACT
Discriminative identification of homologous miRNAs in miRNA family with high specificity and sensitivity is crucial for accurate classification, diagnosis and prognosis of breast cancer. Herein, we report a reliable, sensitive, and selective assay by coupling fluorescence resonance energy transfer (FRET) with cascade signal amplification. The strategy is developed by designing two programmable DNA probes that can be triggered to shift from "off" to "on" state in a cascade hybridization reaction in the presence of target miRNA let-7a, leading to the generation of an amplified signal. The assay can detect concentrations as low as â¼3.0 pM let-7a and discriminate let-7a from other highly homologous members in the let-7 miRNA family. Moreover, it can also be used to determine let-7a levels at single-cell resolution and evaluate the drug efficacy of let-7a expression among various molecular types of breast cancer cell lines. The advantage of this assay is a combined result of signal generation and amplification triggered by target miRNA, which can satisfy an assay of analogous miRNA in a downregulated manner with high specificity. It has promising potential as a selective assay for homologous miRNAs in precision medicine.
Subject(s)
Breast Neoplasms/metabolism , Fluorescence Resonance Energy Transfer/methods , MicroRNAs/analysis , Antineoplastic Agents/pharmacology , Carbocyanines/chemistry , Carbocyanines/toxicity , Cell Line, Tumor , DNA Probes/chemistry , DNA Probes/genetics , DNA Probes/toxicity , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Humans , Inverted Repeat Sequences , MicroRNAs/genetics , MicroRNAs/metabolism , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Nucleic Acid Hybridization , Paclitaxel/pharmacology , Proof of Concept StudyABSTRACT
DNA fluorescent probes are versatile tools that are widely used for biological detection in tubes. Using DNA probes in living systems, however, represents a significant challenge because of the endogenous nuclease-induced DNA degradation and strong background fluorescence in complex biological environments. Here, we show that assembling DNA probes into core-satellite gold nanoparticle (AuNP) superstructures could unprecedentedly enhance enzymatic stability and reduce background interference. The embedded DNA probes are protected from interaction with nuclease, eliminating the enzymatic degradation. In the meantime, the AuNP superstructures show extremely high quenching efficiency (>98%) toward the embedded DNA probes, whose fluorescence can be instantly turned on by the target binding, resulting in high signal-to-background ratio. To demonstrate these distinct properties, we made use of the assembled nanoprobes to monitor the ATP levels under different stimuli in living cells. The assembly strategy leads to a new opportunity for accurately sensing targets in living systems.
Subject(s)
DNA Probes/chemistry , DNA/chemistry , Metal Nanoparticles/chemistry , Adenosine Triphosphate/analysis , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/toxicity , Carbocyanines/chemistry , Carbocyanines/toxicity , Cell Line, Tumor , DNA/toxicity , DNA Probes/toxicity , Deoxyribonuclease I/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Gold/chemistry , Gold/toxicity , Humans , Metal Nanoparticles/toxicity , Mice , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Proof of Concept StudyABSTRACT
We developed a dual-mode DNA nanoprobe based on covalent linkage for fluorescence imaging and photoacoustic imaging of tumor-related mRNA.
Subject(s)
DNA Probes/chemistry , DNA/chemistry , Fluorescent Dyes/chemistry , Neoplasms/diagnosis , RNA, Messenger/metabolism , Animals , Carbocyanines/chemistry , Carbocyanines/toxicity , Cattle , Cell Line, Tumor , DNA/toxicity , DNA Probes/toxicity , Deoxyribonuclease I/chemistry , Female , Fluorescence , Fluorescent Dyes/toxicity , Graphite/chemistry , Graphite/toxicity , Humans , Mice, Nude , Nucleic Acid Hybridization , Photoacoustic Techniques , RNA, Messenger/genetics , Serum Albumin, Bovine/chemistry , Thymidine Kinase/geneticsABSTRACT
The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G(2) progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2, and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G(2)M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis.
