scholarly journals Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon

2014 ◽  
Vol 25 (10) ◽  
pp. 1769-1776 ◽  
Author(s):  
Rachel E. Armstrong ◽  
Geoffrey F. Strouse
Keyword(s):  
Molecular Beacon ◽  
Binding Affinities ◽  
Stem Loop

scholarly journals DNA Detection ofToxoplasma gondiiwith a Magnetic Molecular Beacon Probe via CdTe@Ni Quantum Dots as Energy Donor

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Shichao Xu ◽  
Chen Zhang ◽  
Lei He ◽  
Tongyao Wang ◽  
Liusong Ni ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Toxoplasma Gondii ◽  
Molecular Beacon ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Xrd Analysis ◽  
Stem Loop ◽  
Current Sensing ◽  
Energy Donor ◽  
Base Pair Mismatch

A new method for detection ofToxoplasma gondiivia DNA sensing technology was developed in this study. It was based on the mechanism of fluorescence resonance energy transfer (FRET) in which multifunctional and magnetic-fluorescent CdTe@Ni quantum dots (mQDs) were utilized as energy donor and a commercial BHQ2as acceptor. The sensing probe was fabricated by labeling a stem-loopToxoplasma gondiiDNA oligonucleotide with CdTe@Ni mQDs at the 5′ end and BHQ2at 3′ end, respectively. The surface assembly of CdTe on Ni core and the formation of CdTe@Ni were confirmed by XRD analysis. The sizes of CdTe, Ni nanoparticles, and CdTe@Ni were measured via TEM and XRD methods and estimated to be~3 nm,~15 nm, and~20 nm, respectively. The sensing ability was investigated by the fluorescence spectrum (FS). An obvious fluorescence recovery was observed when the complete complimentary targetToxoplasma gondiiDNA was introduced, which did not happen in the case of the target DNA with one-base pair mismatch. Our research indicates that the current sensing probe is sensitive and specific in detection ofToxoplasma gondiiDNA and has great potential in Toxoplasmosis diagnosis.

scholarly journals Molecular Beacons: A New Approach to Plant Virus Detection

2000 ◽  
Vol 90 (3) ◽  
pp. 269-275 ◽  
Author(s):  
Alvin Jin-Cherng Eun ◽  
Sek-Man Wong
Keyword(s):  
Molecular Beacon ◽  
Virus Detection ◽  
Plant Viruses ◽  
Molecular Beacons ◽  
Cymbidium Mosaic Virus ◽  
Stem Loop ◽  
Chain Reactions ◽  
Plant Virus Detection ◽  
Virus Indexing ◽  
Fluorescent Moiety

Molecular beacons are single-stranded nucleic acid molecules with a stem-loop conformation. The stem portion consists of complementary sequences at the 5′ and 3′ terminals of the molecule, while the loop portion consists of probe sequences that are complementary to the target sequences of choice. A fluorescent moiety is attached to one end, while a quenching moiety is attached to the opposite end. Reverse transcription-polymerase chain reactions are carried out with primers that amplify specific genome sequences of interest, yielding targets complementary to their respective molecular beacons for subsequent detection. Here, we have designed four molecular beacons specific to the RNA-dependent RNA polymerase and coat protein genes of two orchid viruses, namely Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). This technology is successfully applied to detect as little as 0.5 ng of viral RNA of both orchid viruses simultaneously in 100 mg of coinfected Oncidium orchid leaves. This rapid and specific technique is applicable to the orchid industry, which routinely carries out virus indexing and screening for virus-resistant cultivars. We belief that use of this molecular beacon approach can be extended to the detection of multiple plant viruses in various crops.

Development of a Novel Stem-loop-type Molecular Beacon Probe Possessing Polyamine-connected Deoxyuridine and Silylated Pyrene

2014 ◽  
Vol 43 (12) ◽  
pp. 1915-1917 ◽  
Author(s):  
Jakir Ahmed Chowdhury ◽  
Tomohisa Moriguchi ◽  
Kazuo Shinozuka
Keyword(s):  
Molecular Beacon ◽  
Stem Loop

scholarly journals Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Paul J. Bujalowski ◽  
Wlodzimierz Bujalowski ◽  
Kyung H. Choi
Keyword(s):  
Dengue Virus ◽  
Untranslated Region ◽  
Hemorrhagic Fever ◽  
Magnesium Concentration ◽  
Binding Affinities ◽  
Stem Loop ◽  
Kinetic And Thermodynamic Parameters ◽  
Virus Life Cycle ◽  
Association Reaction ◽  
Medical Importance

ABSTRACT The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5′ untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA70 and SLA80, which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides. IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5′ untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the energetics of intrinsic affinities, characterize the stoichiometry of the complex of NS5 and SLA, and determine how solution conditions such as magnesium and sodium concentrations and temperature influence NS5-SLA interactions in solution. Quantitatively characterizing dengue virus NS5-SLA interactions will facilitate the design and assessment of antiviral therapeutics that target this essential step of the dengue virus life cycle.

scholarly journals An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-17 ◽  
Author(s):  
Dmitry M. Kolpashchikov
Keyword(s):  
Molecular Beacon ◽  
Signal To Noise Ratio ◽  
Probe Design ◽  
Rna Sequences ◽  
Molecular Sensing ◽  
Stem Loop ◽  
Nucleic Acid Analysis ◽  
Loop Shape ◽  
Folded Rna ◽  
Rna And Dna

Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA.

Synthesis and Properties of Molecular Beacon DNA Probe Bearing Novel Silylated Pyrene Derivative

2011 ◽  
Vol 497 ◽  
pp. 47-50 ◽  
Author(s):  
M. Gias Uddin ◽  
Tomohisa Moriguchi ◽  
Mayumi Ichimura ◽  
Kazuo Shinozuka
Keyword(s):  
Molecular Beacon ◽  
Dna Probe ◽  
Stem Segment ◽  
Excimer Emission ◽  
Stem Loop ◽  
Complementary Dna ◽  
Single Base ◽  
Fluorescent Signal ◽  
Modified Dna ◽  
Dna Strand

A novel stem-loop structured fluorescent oligoDNA probe (molecular beacon probe) bearing a silylated pyrene derivative at C-5 position of deoxyuridine has been synthesized. The fluorescently labeled modified nucleoside has been incorporated into two consecutive positions in the stem segment of the DNA using an automated DNA synthesizer. The resulting modified DNA exhibited an excimer fluorescent signal upon binding to the fully matched complementary DNA strand. The excimer emission was, however, effectively quenched while it stays alone or it hybridizes to a single base mismatched complementary target.

Specificity determinants of phosphoprotein phosphatases controlling kinetochore functions

2020 ◽  
Vol 64 (2) ◽  
pp. 325-336 ◽  
Author(s):  
Dimitriya H. Garvanska ◽  
Jakob Nilsson
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Activity Level ◽  
Phosphorylation Sites ◽  
Binding Affinities ◽  
Mitotic Kinases ◽  
Anaphase Onset ◽  
Phosphoprotein Phosphatases ◽  
Linear Motifs ◽  
Temporal And Spatial ◽  
Kinetochore Function

Abstract Kinetochores are instrumental for accurate chromosome segregation by binding to microtubules in order to move chromosomes and by delaying anaphase onset through the spindle assembly checkpoint (SAC). Dynamic phosphorylation of kinetochore components is key to control these activities and is tightly regulated by temporal and spatial recruitment of kinases and phosphoprotein phosphatases (PPPs). Here we focus on PP1, PP2A-B56 and PP2A-B55, three PPPs that are important regulators of mitosis. Despite the fact that these PPPs share a very similar active site, they target unique ser/thr phosphorylation sites to control kinetochore function. Specificity is in part achieved by PPPs binding to short linear motifs (SLiMs) that guide their substrate specificity. SLiMs bind to conserved pockets on PPPs and are degenerate in nature, giving rise to a range of binding affinities. These SLiMs control the assembly of numerous substrate specifying complexes and their position and binding strength allow PPPs to target specific phosphorylation sites. In addition, the activity of PPPs is regulated by mitotic kinases and inhibitors, either directly at the activity level or through affecting PPP–SLiM interactions. Here, we discuss recent progress in understanding the regulation of PPP specificity and activity and how this controls kinetochore biology.

968: Gelsolin Gene Silencing Involving Unusual Chromatin Structures and a Novel Stem Loop Structure of the Promoter Region in Human Bladder Cancer

2004 ◽  
Vol 171 (4S) ◽  
pp. 256-257
Author(s):  
Kazunori Haga ◽  
Ataru Sazawa ◽  
Toru Harabayashi ◽  
Nobuo Shinohara ◽  
Minoru Nomoto ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Gene Silencing ◽  
Promoter Region ◽  
Loop Structure ◽  
Human Bladder Cancer ◽  
Human Bladder ◽  
Stem Loop ◽  
Stem Loop Structure

Automated Assessment of Binding Affinity via Alchemical Free Energy Calculations

2020 ◽  
Author(s):  
Maximilian Kuhn ◽  
Stuart Firth-Clark ◽  
Paolo Tosco ◽  
Antonia S. J. S. Mey ◽  
Mark Mackey ◽  
...  
Keyword(s):  
Free Energy ◽  
Protein Structures ◽  
Free Energy Calculations ◽  
Distinct Advantage ◽  
Binding Affinities ◽  
Structure Based Drug Design ◽  
Energy Calculations ◽  
Kendall’S Τ ◽  
Experimental Values ◽  
Better Than

Free energy calculations have seen increased usage in structure-based drug design. Despite the rising interest, automation of the complex calculations and subsequent analysis of their results are still hampered by the restricted choice of available tools. In this work, an application for automated setup and processing of free energy calculations is presented. Several sanity checks for assessing the reliability of the calculations were implemented, constituting a distinct advantage over existing open-source tools. The underlying workflow is built on top of the software Sire, SOMD, BioSimSpace and OpenMM and uses the AMBER14SB and GAFF2.1 force fields. It was validated on two datasets originally composed by Schrödinger, consisting of 14 protein structures and 220 ligands. Predicted binding affinities were in good agreement with experimental values. For the larger dataset the average correlation coefficient Rp was 0.70 ± 0.05 and average Kendall’s τ was 0.53 ± 0.05 which is broadly comparable to or better than previously reported results using other methods. <br>

SAMPL7 TrimerTrip Host-Guest Binding Poses and Binding Affinities from Spherical-Coordinates-Biased Simulations

2020 ◽  
Author(s):  
Zhaoxi Sun
Keyword(s):  
Free Energy ◽  
Computational Modelling ◽  
Free Energy Calculations ◽  
Energy Estimates ◽  
Binding Affinities ◽  
Spherical Coordinates ◽  
Collective Variables ◽  
Guest Binding ◽  
Starting Point ◽  
Sampl Challenge

Host-guest binding remains a major challenge in modern computational modelling. The newest 7<sup>th</sup> statistical assessment of the modeling of proteins and ligands (SAMPL) challenge contains a new series of host-guest systems. The TrimerTrip host binds to 16 structurally diverse guests. Previously, we have successfully employed the spherical coordinates as the collective variables coupled with the enhanced sampling technique metadynamics to enhance the sampling of the binding/unbinding event, search for possible binding poses and predict the binding affinities in all three host-guest binding cases of the 6<sup>th</sup> SAMPL challenge. In this work, we employed the same protocol to investigate the TrimerTrip host in the SAMPL7 challenge. As no binding pose is provided by the SAMPL7 host, our simulations initiate from randomly selected configurations and are proceeded long enough to obtain converged free energy estimates and search for possible binding poses. The predicted binding affinities are in good agreement with the experimental reference, and the obtained binding poses serve as a nice starting point for end-point or alchemical free energy calculations.

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