Detection Of Intracellular microRNA Research Articles

Triple-hairpin mediated feedback system based on rolling circle amplification for enhanced imaging and detection of intracellular microRNA

It remains challenging to develop highly sensitive biosensing methods for the detection of short, non-coding RNA, such as microRNAs (miRNAs) that were expressed at low levels in biological environments. Here we report a triple-hairpin mediated feedback system based on rolling circle amplification (THF-RCA) for enhanced imaging and detection of intracellular miRNA. THF-RCA contains two RCA-based tandem reactions, Cycle I and Cycle II. Cycle I is initiated by a target-primed RCA reaction to produce the RCA product that can successively open HP1, HP2, and HP3. The target-analogue embedded in HP3 can be activated accompanied by the dissolving of HP3 and go on to initiate another RCA reaction, named Cycle II. Subsequently, the RCA product of Cycle II can initiate a new round of Cycle I in a feedback manner. Overall, the triple-hairpin structures serve as the pivot to power the THF-RCA reaction continues autonomously. THF-RCA based cycle system can be used to detect miR-21 down to 1 fM, and target miRNA can be identified obviously from mutation versions and no-target miRNAs. Notably, the THF-RCA based strategy allows not only for in vitro detection of miRNAs without reverse transcription, but also for intracellular imaging of miRNA with higher sensitivity compared with miRNA-FISH technology. THF-RCA based strategy is expected to provide new doors for developing novel signal amplification technologies and biosensors applied in early tumor diagnosis and early treatment.

A One-Two-Three Multifunctional System for Enhanced Imaging and Detection of Intracellular MicroRNA and Chemogene Therapy.

Simultaneous imaging, diagnosis, and therapy can offer an effective strategy for cancer treatment. However, the complex probe design, poor drug release efficiency, and multidrug resistance remain tremendous challenges to cancer treatment. Here, a novel one-two-three system is built for enhanced imaging and detection of miRNA-21 (miR-21) overexpressed in cancer cell and chemogene therapy. The system consists of dual-mode DNA robot nanoprobes assembled by two types of hairpin DNAs and three-way branch DNAs modified on gold nanoparticles, with intercalating anticancer drugs (doxorubicin), into DNA duplex GC base pairs. In the system, via intracellular ATP-accelerated cyclic reaction triggered by miR-21, fluorescence and SERS signals were alternated with DNA structure switch, and the precise SERS detection of miRNA and fluorescence imaging oriented "on-demand" release of two types of anticancer drugs (anti-miR-21 and Dox) are achieved. Thus, "one-two-three" means one kind of miR-21-triggered endogenous substance accelerated cyclic reaction, two modes of signal switch, and three functions including enhanced imaging, detection, and comprehensive treatment. The one-two-three system has some notable merits. First, ATP as an endogenous substance promotes DNA structure switching and accelerates the cyclic reaction. Second, the treatment with a dual-mode signal switch is more reliable and accurate and can provide more abundant information than a single-mode treatment platform. Thus, the imaging and detection of intracellular miRNA and effective comprehensive therapy are realized. In vivo results indicate that the system can provide new insights and strategies for diagnosis and therapy.

MicroRNA-Initiated and Intracellular Na+-Fueled DNAzyme Motor for Differentiating Molecular Subtypes of Nonsmall Cell Lung Cancer.

Synthetic DNAzyme motors or machines hold great potential in the detection of intracellular microRNA (miRNA) and mRNA. However, to make intracellular DNAzyme motors or machines operate efficiently, adding exogenous metal ion cofactors as fuel is imperative, which limits their applications. Here, we reported a Na+-specific DNAzyme-based DNAzyme motor differentiating cell subtypes of nonsmall cell lung cancer by simultaneously sensing intracellular miRNA-21 and miRNA-205. The DNAzyme motor could be fueled by intracellular Na+, which avoids the necessity of adding exogenous cofactors. It could be also designed to detect other miRNAs or mRNAs by changing 12-nt DNA domain. Meanwhile, our DNAzyme motor had high sensitivity, excellent specificity, high biostability, and little cytotoxicity. Therefore, the miRNA-initiated and intracellular Na+-fueled DNAzyme motor can expand the application of DNAzyme motors or machines in sensing miRNA and has potential value in cancer clinical diagnosis and prognosis.

MicroRNA Detection with Turnover Amplification via Hybridization-Mediated Staudinger Reduction for Pancreatic Cancer Diagnosis.

The occurrence of and development in the early pathological stage of pancreatic cancer has proved to be associated with microRNAs. However, it remains a great challenge to directly monitor low-expression, and downregulation of, microRNA among living cells, tissues, and serum samples. In this work, Staudinger reduction is first applied in intracellular microRNA detection, establishing a set of smart hybridization-mediated Staudinger reduction probes (HMSR-probe) which contain designed oligonucleotide sequences. Meanwhile, 40 serum samples (healthy people (6), patients with pancreatitis (22), and pancreatic cancer patients (12)) are tested for exploring the potential clinical application. Of note, the molecules bound to nucleic acid confine the reactive site to close proximity in a compact space, and nonconnected product from Staudinger reaction facilitates turnover amplification to an ameliorative detection limit (1.3 × 10-15 M). Moreover, compared with qRT-PCR, a low false positive signal and an excellent specificity makes the probe more suitable and convenient for pancreatic cancer diagnosis in blood samples. For practical applications, HMSR-probe enable accurate differentiation in cell and tissue samples under both 488 and 785 nm and have good coherence to known research. As a proof of concept, the reliable results in distinguishing pancreatic cancer patients from different morbid stages might supply a feasible method for endogenous microRNA detection in fundamental research and clinical diagnostics.

Detection of intracellular microRNA using a self-assembling magnetic resonance beacon

MicroRNAs (miRNAs or miRs) are implicated in several biological processes such as proliferation, differentiation, apoptosis, development and disease. A number of miRNA imaging tools have been developed due to the scientific and clinical significance of miRNA. Current miRNA imaging techniques are based on reporter gene and molecular beacon systems, and most of these techniques utilize fluorescent probes. These techniques have been successfully used to study the function and interaction of miRNAs in the cellular environment. However, they have limited clinical applicability as imaging systems, mainly due to the inability of fluorescent probes to penetrate tissue. Magnetic resonance imaging (MRI) provides high resolution in three dimensions to analyze the anatomy and structure of tissues. We developed a self-assembling magnetic nanoparticle based molecular beacon (miR124a MR beacon) to detect miR124a in mammalian cells and tissues during neuronal differentiation by T2-weighted magnetic resonance (MR). The self-assembled structure of the miR124a MR beacons was induced by incubating 3’-adaptor, 5’-adaptor and miR linker containing the miR124a binding sequence and adaptor binding sequence. When the miR124a expression level was low in cells and tissues, the MR signal of the miR124a MR beacons was quenched. As the concentration of miR124a increased during neuronal differentiation, selective hybridization occurred between miR124a and the miR124a binding sequence of the miR124a MR beacon. This ultimately induced nanoparticle disassembly and a gradual increase in the MR signal. Our findings suggest that this method can be used to monitor the expression of miRNAs, as well as other intracellular genetic and cellular processes, by MR imaging in vivo .

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

MicroRNAs (miRNAs) are a class of short, endogenous, noncoding regulatory RNAs (approximately 18–25 nucleotides), encoded in the genomes of plants, animals, and viruses. They partially complement the 3’ untranslated region of target mRNAs, causing mRNA cleavage or inhibiting protein synthesis within the Dicer/Argonaute complex. MiRNAs play key regulatory roles in a diverse range of biological processes, including cell development, differentiation, metabolism, and apoptosis. In particular, distinct miRNA expression patterns are associated with various cancer phenotypes. Therefore, miRNAs are also an emerging class of useful diagnostic and prognostic markers. However, miRNA analysis is challenging owing to the unique characteristics of miRNAs, such as their small size, sequence similarity among family members, low abundance, susceptibility to degradation, and the technical impediments in delivering miRNAs across the plasma membrane of cells. An improved delivery strategy is thus needed to successfully monitor intracellular miRNA levels in situ. Generally, transmission of oligonucleotides for gene therapy falls into two broad categories, viral vectors or nonviral carriers. Viral vectors exhibit high transfection efficiency but also some fundamental problems, such as immunogenicity and toxicity. These problems limit their broad application. Currently, the nonviral vectors, such as liposomes, cationic polymers, dendrimers, polypeptides, and nanomaterials have attracted significant interest, owing to their good biocompatibility and potential for large-scale production, in contrast to viral vectors. However, many of these carrier systems do not inhibit genes in a cell-specific manner and cannot be used to monitor their intracellular delivery or therapeutic response. An excellent nonviral vector should efficiently facilitate cell-specific gene-probe (a single-stranded oligonucleotide designed to recognize a single target-nucleotide sequence) uptake and gene-probe endosomal escape for intracellular delivery. Fluorescent nanoparticles are highly attractive materials for gene-probe delivery because of their unique properties, including uniform size, superior imaging characteristics, and facile surface modification. The rational functionalization of these nanomaterials with target-specific moieties, such as antibodies, aptamers, and other molecules, to recognize receptors on the cell surface has led to versatile theragnostic nanosystems. These multifunctional nanosystems not only allow efficient delivery of gene probes to target cells with fewer side effects, but also allow the simultaneous monitoring of delivery and the therapeutic response of the targeted genes, using the advanced optical properties of the nanosystem. However, these nanosystems have not been used for in situ detection of intracellular miRNAs yet, because the detection strategy has not been optimized. In this study we design a novel multifunctional SnO2 nanoprobe (mf-SnO2), which contains a cell-targeting moity, as well as a conjugated gene probe to specifically recognize the target sequence, thus providing a detection strategy or inhibitor. Also, visualization of the delivery and intracellular response is possible through fluorescence of the SnO2. As shown in Scheme 1, cell-specific delivery is achieved by functionalizing SnO2 nanoparticles (SnO2NPs) with folic acid (FA), which targets cancer cells; a gene probe, in this case a molecular beacon (MB) to detect target miRNAs, is conjugated by a disulfide linkage, which is sensitive to pH values. Cleavage of the disulfide linkage between the gene probe and the nanoparticle enhances the efficiency of intracellular delivery. Using miRNA-21 in HeLa cells as a model, a method for in situ detection of intracellular miRNA by the multifunctional nanoprobe is reported. To verify the practicality of this approach, another nanoprobe was also designed, by substituting the MB with an anti-miR of miRNA-21, to down-regulate the expression of a target miRNA. The proposed method, with a MB as the recognition probe, can be used subsequently to monitor the change in miRNA levels from negligible cytotoxicity, and to monitor the ability of the multifunctional nanoprobe to [*] Dr. H. Dong, Prof. J. Lei, Prof. H. Ju, Z. Zhu State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University Nanjing 210093 (P.R. China) E-mail: hxju@nju.edu.cn

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO2 Nanoprobe

Target‐Cell‐Specific Delivery, Imaging, and Detection of Intracellular MicroRNA with a Multifunctional SnO₂ Nanoprobe