Visually predicting microRNA-regulated tumor metastasis by intracellularly 3D counting of fluorescent spots based on in situ growth of DNA flares.

INTRODUCTION: MicroRNAs (miRNAs) have been revealed to be critical genetic regulators in various physiological processes and thus quantitative information on the expression level of critical miRNAs has important implications for the initiation and development of human diseases, including cancers. OBJECTIVES: We herein develop three-dimensionally (3D) counting of intracellular fluorescent spots for accurately evaluating microRNA-21 (miRNA-21) expression in individual HeLa cells based on stimuli-activated in situ growth of optical DNA flares, grid-patterned DNA-protein hybrids (GDPHs). METHODS: Target miRNA is sequence-specifically detected down to 10 pM owing to efficient signal amplification. Within living cells, GDPH flares are nuclease resistant and discrete objects with retarded mobility, enabling the screening of intracellular location and distribution of miRNAs and realizing in situ counting of target species with a high accuracy. RESULTS: The quantitative results of intracellular miRNAs by 3D fluorescence counts are consistent with qPCR gold standard assay, exhibiting the superiority over 2D counts. By screening the expression of intracellular miR-21 that can down-regulate the programmed cell death 4 (PDCD4) protein, the proliferation and migration of HeLa cells, including artificially-regulated ones, were well estimated, thus enabling the prediction of cancer metastasis in murine tumor models. CONCLUSION: The experiments in vitro, ex vivo and in vivo demonstrate that GDPH-based 3D fluorescence counts at the single cell level provide a valuable molecular tool for understanding biological function of miRNAs and especially for recognizing aggressive CTCs, offering a design blueprint for further expansion of DNA structural nanotechnology in predicting distant metastasis and prevention of tumor recurrence after primary resection.

Stimuli-Responsive Autonomous-Motion Molecular Machine for Sensitive Simultaneous Fluorescence Imaging of Intracellular MicroRNAs.

DNAzymes with enzymatic activity identified from random DNA pools by in vitro selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.

Programmably tiling rigidified DNA brick on gold nanoparticle as multi-functional shell for cancer-targeted delivery of siRNAs.

Small interfering RNA (siRNA) is an effective therapeutic to regulate the expression of target genes in vitro and in vivo. Constructing a siRNA delivery system with high serum stability, especially responsive to endogenous stimuli, remains technically challenging. Herein we develop anti-degradation Y-shaped backbone-rigidified triangular DNA bricks with sticky ends (sticky-YTDBs) and tile them onto a siRNA-packaged gold nanoparticle in a programmed fashion, forming a multi-functional three-dimensional (3D) DNA shell. After aptamers are arranged on the exterior surface, a biocompatible siRNA-encapsulated core/shell nanoparticle, siRNA/Ap-CS, is achieved. SiRNAs are internally encapsulated in a 3D DNA shell and are thus protected from enzymatic degradation by the outermost layer of YTDB. The siRNAs can be released by endogenous miRNA and execute gene silencing within tumor cells, causing cell apoptosis higher than Lipo3000/siRNA formulation. In vivo treatment shows that tumor growth is completely (100%) inhibited, demonstrating unique opportunities for next-generation anticancer-drug carriers for targeted cancer therapies.

Periodically Ordered, Nuclease-Resistant DNA Nanowires Decorated with Cell-Specific Aptamers as Selective Theranostic Agents.

DNA nanostructures have shown potential in cancer therapy. However, their clinical application is hampered by the difficulty to deliver them into cancer cells and susceptibility to nuclease degradation. To overcome these limitations, we report herein a periodically ordered nick-hidden DNA nanowire (NW) with high serum stability and active targeting functionality. The inner core is made of multiple connected DNA double helices, and the outer shell is composed of regularly arranged standing-up hairpin aptamers. All termini of the components are hidden from nuclease attack, whereas the target-binding sites are exposed to allow delivery to the cancer target. The DNA NW remained intact during incubation for 24 h in serum solution. Animal imaging and cell apoptosis showed that NWs loaded with an anticancer drug displayed long blood-circulation time and high specificity in inducing cancer-cell apoptosis, thus validating this approach for the targeted imaging and therapy of cancers.