Fluorescent labeling of plasmid DNA and mRNA : gains and losses of current labeling strategies

Live-cell imaging has provided the life sciences with insights into the cell biology and dynamics. Fluorescent labeling of target molecules proves to be indispensable in this regard. In this Review, we focus on the current fluorescent labeling strategies for nucleic acids, and in particular mRNA (mRNA) and plasmid DNA (pDNA), which are of interest to a broad range of scientific fields. By giving a background of the available techniques and an evaluation of the pros and cons, we try to supply scientists with all the information needed to come to an informed choice of nucleic acid labeling strategy aimed at their particular needs

Preliminary nanopore cheminformatics analysis of aptamer-target binding strength

<p>Abstract</p> <p>Background</p> <p>Aptamers are nucleic acids selected for their ability to bind to molecules of interest and may provide the basis for a whole new class of medicines. If the aptamer is simply a dsDNA molecule with a ssDNA overhang (a "sticky" end) then the segment of ssDNA that complements that overhang provides a known binding target with binding strength adjustable according to length of overhang.</p> <p>Results</p> <p>Two bifunctional aptamers are examined using a nanopore detector. They are chosen to provide sensitive, highly modulated, blockade signals with their captured ends, while their un-captured regions are designed to have binding moieties for complementary ssDNA targets. The bifunctional aptamers are duplex DNA on their channel-captured portion, and single-stranded DNA on their portion with binding ability. For short ssDNA, the binding is merely to the complementary strand of DNA, which is what is studied here – for 5-base and 6-base overhangs.</p> <p>Conclusion</p> <p>A preliminary statistical analysis using hidden Markov models (HMMs) indicates a clear change in the blockade pattern upon binding by the single captured aptamer. This is also consistent with the hypothesis that significant conformational changes occur during the annealing binding event. In further work the objective is to simply extend this ssDNA portion to be a well-studied ~80 base ssDNA aptamer, joined to the same bifunctional aptamer molecular platform.</p

Dual Resonance Energy Transfer Nucleic Acid Probes

Dual nucleic acid probes with resonance energy transfer moieties are provided. In particular, fluorescent or luminescent resonance energy transfer moieties are provided on hairpin stem-loop molecular beacon probes that hybridize sufficiently near each other on a subject nucleic acid, e.g. mRNA, to generate an observable interaction. The invention also provides lanthanide chelate luminescent resonance energy transfer moieties on linear and stem-loop probes that hybridize sufficiently near each other on a subject nucleic acid to generate an observable interaction. The invention thereby provides detectable signals for rapid, specific and sensitive hybridization determination in vivo. The probes are used in methods of detection of nucleic acid target hybridization for the identification and quantification of tissue and cell-specific gene expression levels, including response to external stimuli, such as drug candidates, and genetic variations associated with disease, such as cancer.Georgia Tech Research Corporatio

Reflective diffractometric hydrogel sensor for biological and chemical detection

A reflective diffractometric hydrogel sensor includes an upper layer, including a microfluidic chamber formed from a substantially transparent material and configured to contain a solution, a reflective diffraction grating positioned within the microfluidic chamber, the diffraction grating including a plurality of hydrogel strips configured to change in dimension in response to a stimulus, each hydrogel strip having a top surface coated with a reflective material and a bottom surface in contact with the upper layer substrate, and a reflective surface below the reflective diffraction grating wherein when a coherent light is incident upon and reflected from the upper layer at an angle substantially normal to the upper layer an interference diffraction pattern results, including a first diffraction mode, a light intensity of which indicates the relative distance between the top surfaces of the plurality of hydrogel strips and the reflective surface.Board of Regents, University of Texas Syste

Application of Molecular Beacon Technology for the Identification of Bacteria

Veterinary Biomedical Science

A MIQE-Compliant Real-Time PCR Assay for Aspergillus Detection

PMCID: PMC3393739This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Genomic Detection Using Sparsity-inspired Tools

Genome-based detection methods provide the most conclusive means for establishing the presence of microbial species. A prime example of their use is in the detection of bacterial species, many of which are naturally vital or dangerous to human health, or can be genetically engineered to be so. However, current genomic detection methods are cost-prohibitive and inevitably use unique sensors that are specific to each species to be detected. In this thesis we advocate the use of combinatorial and non-specific identifiers for detection, made possible by exploiting the sparsity inherent in the species detection problem in a clinical or environmental sample. By modifying the sensor design process, we have developed new molecular biology tools with advantages that were not possible in their previous incarnations. Chief among these advantages are a universal species detection platform, the ability to discover unknown species, and the elimination of PCR, an expensive and laborious amplification step prerequisite in every molecular biology detection technique. Finally, we introduce a sparsity-based model for analyzing the millions of raw sequencing reads generated during whole genome sequencing for species detection, and achieve significant reductions in computational speed and high accuracy

G-Quadruplex Aptamer Beacon for Detection of Prostate Cancer Biomarker

The prostate is the major male reproductive gland involved in male fertility and plays an important role in triggering of molecular pathways relevant to fertility success. Unfortunately, in Portugal prostate cancer is the most common cancer type among men, being asymptomatic in earlier stages. Thus, is important early detection of disease. NCL is a multifunctional protein involved in multiple biological processes under both physiological and pathological processes and can have several cellular localizations. Cell surface protein overexpression was found restricted to cancer cells, namely in prostate cancer cells. Thus, we can consider NCL as a potential biomarker for cancer diagnosis and a target for cancer treatment. The AS1411 is an aptamer capable to recognise and binds specifically NCL and have a therapeutic effect on cancer cells through of induction of antiproliferative activity. Beyond its therapeutic use, AS1411 can be used in imaging and diagnostic, particularly on aptasensors development. One of the most relevant characteristics of this aptamer is the ability to fold in a G4 conformation, a secondary structure of nucleic acids. G4 structure confers stabilization to sequence and availability to bind NCL. Thus, in this work is presented the first approach of use AS1411 aptamer to prostate cancer diagnosis, namely through the design of molecular beacon (MB) designated by AS1411N5. Initially, biophysical characterization of AS1411-N5 was done by circular dichroism, nuclear magnetic resonance or fluorometric spectroscopies. Additionally, it was performed microfluidic experiments, to detect NCL using AS1411-N5 in biological samples. The results demonstrated that the proposed AS1411-N5 adopt a G4 structure and it is capable to bind with specificity and selectivity NCL, even in plasma of human patients with prostate cancer.A próstata é a maior glândula reprodutiva masculina e tem um papel importante nas vias moleculares relevantes para o sucesso da fertilização. Infelizmente, em Portugal o cancro da próstata é o cancro mais comum entre os homens, sendo assintomático em estadios iniciais. Assim é imperativo a deteção precoce da doença. A nucleolina (NCL) é uma proteína multifuncional envolvida em múltiplos processos biológicos sob condições fisiológicas e patológicas, podendo ter várias localizações celulares. A sobre-expressão da proteína na superfície das células é apenas encontrada em células cancerosas, nomeadamente as do cancro da próstata. Assim a NCL pode ser considerada como um potencial biomarcador para o diagnóstico e tratamento do cancro da próstata. O AS411 é um aptamero capaz de reconhecer e ligar especificamente a esta proteína, e de ter um efeito terapêutico nas células cancerosas ao induzir atividade antiproliferativa. Além do uso terapêutico, a sequência pode ser utilizada na imagiologia e diagnóstico, particularmente através do desenvolvimento de aptasensores. Uma das características mais relevantes do aptamero AS1411 é a capacidade de adotar a configuração de G-quadruplex (G4), uma estrutura secundária dos ácidos nucleicos. As estruturas G4 conferem estabilização à sequência e capacidade de ligar à NCL quando adota esta estrutura. Assim, neste trabalho é apresentada uma primeira abordagem do uso do AS1411 no diagnóstico do cancro da próstata, nomeadamente através da construção de uma sonda a partir da sequência deste aptamero designado por AS1411N5. Inicialmente foi efetuada a caracterização biofísica do AS1411-N5 a nível da estrutura e interação com o alvo, recorrendo às espectroscopias dicroísmo circular e ressonância magnética nuclear, e ensaios fluorométricos. Adicionalmente foram efetuadas experiências de microfluídica, para o uso do AS1411N5 como sonda de deteção da NCL. Estes resultados demonstraram, que o AS1411-N5adota a estrutura G4 e é capaz de ligar especificamente e com seletividade com a NCL, mesmo em amostras biológicas

DNA glycosylases as modulators of chemotherapeutic response

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Median survival is less than two years due to several factors, including challenges in surgical removal and chemotherapy resistance, underlining the need for more effective therapeutic options. To identify genes that contribute to chemotherapy resistance, we conducted a synthetic lethal screen in a chemotherapy-resistant GBM derived cell line (T98G) with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored towards “druggable” targets. This screen for TMZ-sensitizing genes indicated that a subset of genes that were over-expressed in GBM cells increased the cell’s sensitivity to TMZ when knocked down. An ubiquitin ligase, UBE3B, and a DNA glycosylase, UNG, were among the TMZ-sensitizing genes identified using the siRNA library. We demonstrate that UBE3B and UNG are sensitizing genes in the screen validation studies using unique siRNA and shRNA sequences. Although UNG is one of four human DNA glycosylases that remove uracil lesions, UNG was the only uracil removing glycosylase to sensitize GBM cells in the validation studies. Notably, analysis of archived transcription datasets revealed that over-expression of UNG was correlated with poor outcomes in glioma patients. In order to uncover functional groupings of TMZ-sensitizing proteins, we conducted in situ pathway analysis of gene candidates for synthetic lethal functions from our screen. This analysis discovered statistically significant enrichment of ontogeny clusters related to base excision repair (BER), response to DNA damage, cellular proliferation and protein modification. Interestingly, this pathway topography overlapped with TMZ-sensitizing genes identified from similar experiments in yeast and bacteria. In order to facilitate rapid in vitro identification of lesion-specific repair activity in cancer cells, we developed a novel fluorescent assay that extends the state of the art. The molecular beacon assay measures real-time DNA repair rates of specific DNA lesions by defined DNA repair proteins. These studies reveal that GBM up-regulates several TMZ-sensitizing genes that correlate with poor patient survival and inhibiting these genes may increase TMZ cytotoxicity in a tumor specific manner. These TMZ-sensitizing genes are not only potential targets for adjuvant therapy, but also represent potential biomarkers to predict TMZ response