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Molecular beacon

Aleksei Agapov, Daria Esyunina, Danil Pupov, Andrey Kulbachinskiy
The Gre-family transcription factors bind within the secondary channel of bacterial RNA polymerase (RNAP) directly modulating its catalytic activities. Universally conserved Gre factors activate RNA cleavage by RNAP, by chelating catalytic metal ions in the RNAP active site, and facilitate both promoter escape and transcription elongation. Gfh factors are Deinococcus/Thermus -specific homologues of Gre factors whose transcription functions remain poorly understood. Recently, we found that Gfh1 and Gfh2 proteins from Deinococcus radiodurans dramatically stimulate RNAP pausing during transcription elongation in the presence of Mn(2+) but not Mg(2+) ions...
October 17, 2016: Biochemical Journal
Dmitry A Tsybulsky, Maksim V Kvach, Dmitry Y Ryazantsev, Ilya O Aparin, Alexander A Stakheev, Igor A Prokhorenko, Yury V Martynenko, Sergey V Gontarev, Andrey A Formanovsky, Timofei S Zatsepin, Vadim V Shmanai, Vladimir A Korshun, Sergey K Zavriev
Molecular beacons carrying JOE dye (4',5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein) on a rigid or flexible linker and one or two BHQ1 quenchers have been prepared and tested in real-time PCR using Fusarium avenaceum elongation factor 1α DNA template. The probes were different in their structures (loop size and stem length), linkers for dye attachment (6-aminohexanol or trans-4-aminocyclohexanol), quencher composition (single and double BHQ1) to elucidate the influence of all these features. Fluorogenic properties of the probes were studied and compared to those of FAM (fluorescein)-based probes...
October 6, 2016: Molecular and Cellular Probes
Meltem Göksel, Mahmut Durmuş, Devrim Atilla
A series of phthalocyanine-peptide-quencher conjugates (6-9) were synthesized as photosensitizers for photodynamic therapy in cancer treatment. The photophysical, photochemical and photobiological properties of these activatable molecular beacons were also investigated in this study. For this purpose, the fluorescence, singlet oxygen and photodegradation quantum yields and fluorescence lifetime values of the compounds were determined in DMSO solutions. The phototoxicity and cytotoxicity of the systems were studied against the cervical cancer cell line named HeLa for an evaluation of their suitability for photodynamic therapy...
October 5, 2016: Photochemical & Photobiological Sciences
C Y Song, Y J Yang, B Y Yang, Y Z Sun, Y P Zhao, L H Wang
Simultaneous detection of multiple trace cancer associated serum miRNA biomarkers is considered as a feasible method for early cancer screening and diagnosis. In the present work, an ultrasensitive SERS sensor was prepared based on an Ag nanorod array SERS substrate by assembling special hairpin-shaped molecular beacons (MBs) for the detection of multiple lung cancer-related miRNA biomarkers. The portable SERS sensor exhibits excellent performance for the qualitative and quantitative detection of miRNAs, with advantages of ultra-sensitivity, good specificity, uniformity, reproducibility and stability, as well as remarkable reusability...
October 6, 2016: Nanoscale
Huning Jia, Ying Bu, Bingjie Zou, Jianping Wang, Shalen Kumar, Janet L Pitman, Guohua Zhou, Qinxin Song
Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA...
September 30, 2016: Analyst
Tairong Kuang, Lingqian Chang, Xiangfang Peng, Xianglong Hu, Daniel Gallego-Perez
Heterogeneities and oncogenesis essentially result from proteomic disorders orchestrated by changes in DNA and/or cytoplasmic mRNA. These genetic fluctuations, however, cannot be decoded through conventional label-free methods (e.g., patch clamps, electrochemical cellular biosensors, etc.) or morphological characterization. Molecular beacons (MBs) have recently emerged as efficient probes for interrogating biomarkers in live cancer cells. MBs hybridize with their intracellular targets (e.g., mRNAs, DNAs, or proteins), emitting a fluorescent signal that can be quantified and correlated with the expression levels of their targets...
September 28, 2016: Trends in Biotechnology
Qing Xu, Xinhui Lou, Lei Wang, Xiaofan Ding, Haixiang Yu, Yi Xiao
The controlled attachment of thiolated DNA to gold nanoparticles (AuNPs) dictates many applications. This is typically achieved by either "aging-salting" processes or low-pH method, where either Na(+) or H(+) is used to minimize charge repulsion and facilitate attachment of thiolated DNA onto AuNPs. However, the "aging-salting" process takes a long time, and is prone to aggregation when used with larger AuNPs. Surfactants are needed to precoat and thereby enhance the stability of AuNPs. The low-pH method can disrupt the structural integrity of DNAs...
October 12, 2016: ACS Applied Materials & Interfaces
Qianwen Ding, Qiuqiang Zhan, Xiaoming Zhou, Tao Zhang, Da Xing
Remote optical detection and imaging of specific tumor-related biomarkers and simultaneous activation of therapy according to the expression level of the biomarkers in tumor site with theranostic probes should be an effective modality for treatment of cancers. Herein, an upconversion nanobeacon (UCNPs-MB/Dox) is proposed as a new theranostic nanoprobe to ratiometrically detect and visualize the thymidine kinase 1 (TK1) mRNA that can simultaneously trigger the Dox release to activate the chemotherapy accordingly...
September 20, 2016: Small
Clara Valero, Laura de la Cruz-Villar, Óscar Zaragoza, María José Buitrago
The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFIs diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time...
September 14, 2016: Journal of Clinical Microbiology
Haisheng Liu, Changbei Ma, Meijuan Zhou, Hanchun Chen, Hailun He, Kemin Wang
This work demonstrates a novel method for DNA methyltransferase (MTase) activity detection with a quencher-free molecular beacon (MB) probe based on exonuclease (Exo) III-assisted signal amplification. In the presence of Dam MTase and DpnI endonuclease, the elaborately designed hairpin substrate (MB1) was cleaved into two parts (part A and part B). Exo III can then digest part A and release a single-stranded target of the 2-aminopurine-labeled MB (MB2). Subsequently, the MB2 can hybridize with its target to form a double-stranded structure with a protruding 3'-terminus and then trigger the digestion of MB2 by Exo III...
November 2016: Analytical and Bioanalytical Chemistry
Yan Huang, Wenqian Wang, Tingting Wu, Li-Ping Xu, Yongqiang Wen, Xueji Zhang
A rapid, sensitive, and accurate detection strategy for microRNA 16 (miR-16) was developed, which combined the convenience of lateral flow biosensors (LFBs), the design flexibility of Y-shaped junction DNA probe, and the enhancement ability of endonuclease-assisted target recycling amplification. The system is composed of a molecular beacon (MB) probe, an assistant probe, and endonuclease Nt.BbvCI, which plays the role of signal translation and amplification. In the presence of the target microRNAs (miRNAs), three chains of nucleic acid could hybridize with each other to form a Y-shaped junction structure, which could be recognized by the endonuclease Nt...
November 2016: Analytical and Bioanalytical Chemistry
Hui Zhou, Chao Yang, Huifang Chen, Xun Li, Yongdong Li, Xiaolin Fan
MicroRNAs (miRNAs) family members are usually different from each other in one-base variation. The high sequence homology poses a challenge for miRNA analysis with single-base selectivity. On the basis of G-quadruplex molecular beacons (G4MB) and duplex-specific nuclease (DSN), we developed a simple and highly selective amplification biosensor for miRNA detection. G4MB with a G4 motif stem is used as recognition probe. In the present of target miRNAs, G4MB hybridizes with target miRNA perfectly and forms a G4MB-miRNA duplex...
July 19, 2016: Biosensors & Bioelectronics
Kalyan D Chavda, Michael J Satlin, Liang Chen, Claudia Manca, Stephen G Jenkins, Thomas J Walsh, Barry N Kreiswirth
We developed and evaluated multiplexed molecular beacon probes in a real-time PCR assay to identify prominent extended-spectrum β-lactamase, plasmid-mediated AmpC β-lactamase (pAmpC), and carbapenemase genes directly from perianal swab specimens within six hours. We evaluated this assay on 158 perianal swabs collected from hematopoietic stem cell transplant recipients and found that this assay was highly sensitive and specific for detection of CTX-M-, pAmpC-, and KPC-producing Enterobacteriaceae, compared to culture onto chromogenic agar...
September 6, 2016: Antimicrobial Agents and Chemotherapy
Marc C Rideout, Benjamin Liet, Didier Gasparutto, Nathalie Berthet
We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA...
November 15, 2016: Analytical Biochemistry
Kai Zhang, Ke Wang, Xue Zhu, Fei Xu, Minhao Xie
MicroRNA (miRNA) has become an important biomarker candidate for cancer diagnosis, prognosis, and therapy. In this study, we have developed a novel fluorescence method for sensitive and specific miRNA detection via duplex specific nuclease (DSN) signal amplification and demonstrated its practical application in biological samples. Malachite green (MG) was employed as a "label-free" signal transducer since fluorescence of MG could be enhanced by 100-fold when MG were binding to a G-quadruplex structure formed within the d(G2T)13G sequence...
August 26, 2016: Biosensors & Bioelectronics
Eoin Brennan, Roisin D Moriarty, Tia E Keyes, Robert J Forster
The ability of a molecular beacon to detect miR-132, a microRNA associated with the childhood cancer neuroblastoma is reported in solution and within live cells. The stem-loop structure comprises a sequence complementary to miR-132, modified with a 6-FAM dye and dabcyl quencher on either end. In the absence of the target, self-binding occurs bringing the luminophore and quencher into close proximity significantly decreasing the emission intensity. In the presence of miR-132, the signal is greatly enhanced, with a linear increase in intensity for mole ratios of beacon:target between 0...
September 1, 2016: Bioconjugate Chemistry
Hongling Li, Jianguo Xu, Zhenmeng Wang, Zai-Sheng Wu, Lee Jia
An increasingly branched rolling circle amplification (IB-RCA) which contains a padlock probe (PP) and a structurally tailored molecular beacon (MB) was innovatively developed for highly sensitive detection of cancer gene, Kras gene codon 12. In this system, the PP can be circularized after hybridization with the precisely-matched target DNA, while the stem of MB can be also opened by target DNA, resulting in hybridization with the circularized PP to generate a long tandem single-stranded DNA (ssDNA) product...
December 15, 2016: Biosensors & Bioelectronics
Francesco Ricci, Alexis Vallée-Bélisle, Anna J Simon, Alessandro Porchetta, Kevin W Plaxco
The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclusion of cumbersome washing and dilution steps or via careful control experimental conditions. Nature, in contrast, cannot afford the luxury of washing and dilution steps, nor can she arbitrarily change the conditions (temperature, pH, ionic strength) under which binding occurs in the homeostatically maintained environment within the cell...
September 20, 2016: Accounts of Chemical Research
Malgorzata Kloc, Szczepan Bilinski, Jacek Z Kubiak
The majority of oocyte functions involves and depends on the cytoskeletal elements, which include microtubules and actin and cytokeratin filaments. Various structures and molecules are temporarily or permanently bound to the cytoskeletal elements and their functions rely on cytoskeleton integrity and its timely assembly. Thus the accurate visualization of cytoskeleton is often crucial for studies and analyses of oocyte structure and functions. Here we describe several reliable methods for microtubule and/or microfilaments preservation and visualization in Xenopus oocyte extracts, and in situ in live and fixed insect and frog (Xenopus) oocytes...
2016: Methods in Molecular Biology
Yanan Zhao, Yoji Nagasaki, Milena Kordalewska, Ellen G Press, Ryan K Shields, M Hong Nguyen, Cornelius J Clancy, David S Perlin
A novel and highly accurate diagnostic assay platform was established for rapid identification of FKS mutations associated with echinocandin resistance in Candida glabrata. The assay platform uses allele-specific molecular beacon and DNA melt analysis following asymmetric PCR. A dual assay for FKS1 and FKS2 was developed to identify within 3 h the most common and clinically relevant resistance associated mutations including 8 FKS1 HS1 (WT, S629P, F625S, D632Y, D632E <T1896G>, D632E <T1896A>, I634V, and F625F) and 7 FKS2 HS1 (WT, F659del, F659S, F659V, F659L, S663P, S663F) genotypes...
August 22, 2016: Antimicrobial Agents and Chemotherapy
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