Live Cell Detection of Specific Messenger RNA for Molecular Analysis of Plaque Formation

2007 ◽  
Author(s):  
Wonjong Rhee ◽  
Hanjoong Jo ◽  
Gang Bao
Keyword(s):  
Shear Flow ◽  
Molecular Analysis ◽  
Messenger Rna ◽  
Molecular Beacon ◽  
Mrna Level ◽  
Live Cell ◽  
Molecular Beacons ◽  
Cell Detection ◽  
Unstable Flow ◽  
Fluorescent Intensity

The ability to visualize mRNA in single living cells and monitor in real-time the changes of mRNA level and localization in response to shear flow can provide unprecedented opportunities for the molecular analysis of atherosclerosis. We carried out an extensive study of the design of molecular beacons to target BMP-4 mRNA, which plays important roles in proatherogenic development in response to unstable flow conditions. Specifically, we selected an optimal molecular beacon design, and found that the fluorescent intensity from targeting BMP-4 mRNA correlated well with the GFP signal after up-regulating BMP-4 and co-expressing GFP using adenovirus. The knock-down of BMP-4 mRNA using siRNA significantly reduced the beacon signal, further demonstrating detection specificity. We found that, due to target accessibility, molecular beacons designed with different target sequences gave very different signal levels, and establishing molecular beacon design rules has significant implications to live cell mRNA detections, especially to the studies of BMP-4 mRNA in endothelial cells under shear flow.

scholarly journals Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons

2008 ◽  
Vol 36 (5) ◽  
pp. e30-e30 ◽  
Author(s):  
W. J. Rhee ◽  
P. J. Santangelo ◽  
H. Jo ◽  
G. Bao
Keyword(s):  
Live Cell ◽  
Molecular Beacons ◽  
Cell Detection ◽  
Signal Specificity

scholarly journals PinMol: Python application for designing molecular beacons for live cell imaging of endogenous mRNAs

2018 ◽  
Author(s):  
Livia V. Bayer ◽  
Omar S. Omar ◽  
Diana P. Bratu ◽  
Irina E. Catrina
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Molecular Beacon ◽  
Structural Information ◽  
Live Cell ◽  
Molecular Beacons ◽  
Intramolecular Interactions ◽  
Live Cells ◽  
Target Rna

ABSTRACTMolecular beacons are nucleic acid oligomers labeled with a fluorophore and a quencher that fold in a hairpin-shaped structure, which fluoresce only when bound to their target RNA. They are used for the visualization of endogenous mRNAs in live cells. Here, we report a Python program (PinMol) that designs molecular beacons best suited for live cell imaging by using structural information from secondary structures of the target RNA, predicted via energy minimization approaches. PinMol takes into account the accessibility of the targeted regions, as well as the inter- and intramolecular interactions of each selected probe. To demonstrate its applicability, we synthesized an oskar mRNA-specific molecular beacon (osk1236), which is selected by PinMol to target a more accessible region than a manually designed oskar-specific molecular beacon (osk2216). We previously demonstrated osk2216 to be efficient in detecting oskar mRNA in in vivo experiments. Here, we show that osk1236 outperformed osk2216 in live cell imaging experiments.

scholarly journals Complexation design of cationized gelatin and molecular beacon to visualize intracellular mRNA

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245899
Author(s):  
Sho Takehana ◽  
Yuki Murata ◽  
Jun-ichiro Jo ◽  
Yasuhiko Tabata
Keyword(s):  
Messenger Rna ◽  
Molecular Beacon ◽  
Fluorescent Protein ◽  
Apparent Size ◽  
Sequence Specificity ◽  
Fluorescent Intensity ◽  
Mixing Ratios ◽  
Preparation Conditions ◽  
Gapdh Mrna ◽  
Green Fluorescent

The objective of this study is to prepare cationized gelatin-molecular beacon (MB) complexes for the visualization of intracellular messenger RNA (mRNA). The complexes were prepared from cationized gelatins with different extents of cationization and different mixing ratios of MB to cationized gelatin. The apparent size of complexes was almost similar, while the zeta potential was different among the complexes. Irrespective of the preparation conditions, the complexes had a sequence specificity against the target oligonucleotides in hybridization. The cytotoxicity and the amount of complexes internalized into cells increased with an increase in the cationization extent and the concentration of cationized gelatin. After the incubation with complexes prepared from cationized gelatin with the highest extent of cationization and at mixing ratios of 10 and 20 pmole MB/μg cationized gelatin, a high fluorescent intensity was detected. On the other hand, the complex prepared with the mixing ratio at 20 pmole/μg did not show any cytotoxicity. The complex was the most effective to visualize the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA endogenously present. In addition, even for enhanced green fluorescent protein (EGFP) mRNA exogenously transfected, the complex permitted to effectively detect it as well. It is concluded that both the endogenous and exogenous mRNA can be visualized in living cells by use of cationized gelatin-MB complexes designed.

scholarly journals Novel nucleic acid origami structures and conventional molecular beacon–based platforms: a comparison in biosensing applications

2021 ◽  
Author(s):  
Noemi Bellassai ◽  
Roberta D’Agata ◽  
Giuseppe Spoto
Keyword(s):  
Nucleic Acid ◽  
Structural Properties ◽  
Molecular Beacon ◽  
Dna Origami ◽  
Molecular Beacons ◽  
Conformational Polymorphism ◽  
Functional Systems ◽  
Similarities And Differences ◽  
Nucleic Acid Structures ◽  
Nucleic Acid Sequences

AbstractNucleic acid nanotechnology designs and develops synthetic nucleic acid strands to fabricate nanosized functional systems. Structural properties and the conformational polymorphism of nucleic acid sequences are inherent characteristics that make nucleic acid nanostructures attractive systems in biosensing. This review critically discusses recent advances in biosensing derived from molecular beacon and DNA origami structures. Molecular beacons belong to a conventional class of nucleic acid structures used in biosensing, whereas DNA origami nanostructures are fabricated by fully exploiting possibilities offered by nucleic acid nanotechnology. We present nucleic acid scaffolds divided into conventional hairpin molecular beacons and DNA origami, and discuss some relevant examples by focusing on peculiar aspects exploited in biosensing applications. We also critically evaluate analytical uses of the synthetic nucleic acid structures in biosensing to point out similarities and differences between traditional hairpin nucleic acid sequences and DNA origami. Graphical abstract

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics

The Analyst ◽  
2017 ◽  
Vol 142 (1) ◽  
pp. 147-155 ◽  
Author(s):  
Y. Ma ◽  
X. Dai ◽  
T. Hong ◽  
G. B. Munk ◽  
M. Libera
Keyword(s):  
Real Time ◽  
Bloodstream Infection ◽  
Molecular Diagnostics ◽  
Molecular Beacon ◽  
Molecular Beacons ◽  
Infection Model ◽  
Rna Amplification ◽  
Microbial Detection

Gel-tethered molecular beacons coupled with NASBA RNA amplification enable real-time microbial detection and differentiation in a bloodstream infection model.

Tissue Factor Expression in Vital Organs during Murine Traumatic Shock 

1999 ◽  
Vol 91 (6) ◽  
pp. 1844-1844 ◽  
Author(s):  
Valerie E. Armstead ◽  
Irina L. Opentanova ◽  
Alexander G. Minchenko ◽  
Allan M. Lefer
Keyword(s):  
Small Intestine ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Messenger Rna ◽  
Mrna Level ◽  
Regulatory Elements ◽  
Traumatic Shock ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Nf Kappab

Background Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation that has been shown to have a role in the pathophysiology of sepsis and reperfusion injury. The purpose of this study was to investigate TF expression in vital organs and to determine possible regulatory mechanisms of TF expression in the lung during traumatic shock in rats. Methods Noble-Collip drum trauma was induced in anesthetized Sprague-Dawley rats. Anesthetized rats without trauma served as controls. TF activity was measured in plasma and lung tissue. TF messenger RNA (mRNA) was measured in the lung, liver, and small intestine using ribonuclease protection assays. Electromobility shift assays were used to quantify binding of nuclear extracts from lung to TF-specific consensus domains for transcription factors NF-kappaB and AP-1. Results TF activity in plasma increased up to 14-fold and +232% in the lung (P < 0.001 for plasma and lung) 2 h after trauma. TF mRNA level was significantly increased in the lungs (P < 0.01), small intestine (P < 0.01), and liver (P < 0.05) 1 h after trauma compared to sham-operated control rats. TF mRNA expression continued to increase in the lungs and the liver (both, P < 0.001) 2 h after trauma TF sequence-specific complex binding to AP-1 and NF-kappaB domains was enhanced in the lungs of trauma rats (+395%, P < 0.001 and +168%, P < 0.001, respectively). Conclusions These results suggest that TF may play an important role in the pathophysiology of severe trauma and that regulatory elements AP-1 and NF-kappaB may be involved in the regulation of TF mRNA expression in traumatic shock.

scholarly journals A Rapid and Easy-to-Perform Method of Nucleic-Acid Based Dengue Virus Diagnosis Using Fluorescence-Based Molecular Beacons

Biosensors ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 479
Author(s):  
Soumi Sukla ◽  
Prasenjit Mondal ◽  
Subhajit Biswas ◽  
Surajit Ghosh
Keyword(s):  
Nucleic Acid ◽  
Dengue Virus ◽  
Magnetic Beads ◽  
Molecular Beacon ◽  
Detection System ◽  
Denv Infection ◽  
Molecular Beacons ◽  
Nucleic Acid Amplification ◽  
Complementary Sequence ◽  
Reverse Transcription Pcr

Detecting dengue virus (DENV) infection in patients as early as possible makes the disease management convenient. Conventionally, DENV infection is diagnosed by ELISA-based methods, but sensitivity and specificity are major concerns. Reverse-transcription-PCR (RT-PCR)-based detection confirms the presence of DENV RNA; however, it is expensive, time-consuming, and skilled personnel are required. A fluorescence-based detection system that detects DENV RNA in patient’s serum directly, without any nucleic acid amplification step, has been developed. The method uses target-specific complementary sequence in the molecular beacon, which would specifically bind to the DENV RNA. The molecular beacons are approximately 40 bases long hairpin structures, with a fluorophore-quencher system attached at the terminal ends of the stem. These probes are biotinylated in the stem region, so that they can be immobilized on the streptavidin-tagged magnetic beads. These magnetic beads, coupled with biotinylated molecular beacons, are used for the detection of the target RNA in the serum by incubating the mixture. After incubation, beads are separated and re-suspended in a buffer. The measurement of fluorescence is taken in fluorometer after 15 min incubation at 50 °C. The whole work is carried out in a single tube. This rapid method can precisely detect dengue RNA within two hours, confirming ongoing DENV replication in the patient.

Detection of DNA methyltransferase activity using allosteric molecular beacons

The Analyst ◽  
2016 ◽  
Vol 141 (2) ◽  
pp. 579-584 ◽  
Author(s):  
Weiting Zhang ◽  
Xiaolong Zu ◽  
Yanling Song ◽  
Zhi Zhu ◽  
Chaoyong James Yang
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Molecular Beacon ◽  
Molecular Beacons ◽  
Sensitive Detection ◽  
Methyltransferase Activity ◽  
Mtase Activity ◽  
Adenine Methylation ◽  
Methylation Patterns

Abnormal DNA methylation patterns caused by altered DNA methyltransferase (MTase) activity are closely associated with cancer. Herein, using DNA adenine methylation methyltransferase (Dam MTase) as a model analyte, we designed an allosteric molecular beacon (aMB) for sensitive detection of Dam MTase activity.

scholarly journals Live-cell Molecular Analysis of Akt Activation Reveals Roles for Activation Loop Phosphorylation

2007 ◽  
Vol 282 (50) ◽  
pp. 36634-36641 ◽  
Author(s):  
Bharath Ananthanarayanan ◽  
Matthew Fosbrink ◽  
Meghdad Rahdar ◽  
Jin Zhang
Keyword(s):  
Molecular Analysis ◽  
Live Cell ◽  
Activation Loop ◽  
Akt Activation
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