scholarly journals Insights into I-motif stabilization by high resolution primer extension assays: Its strengths and limitations

2022 ◽  
Author(s):  
Jan Jamroskovic ◽  
Marco Deiana ◽  
Nasim Sabouri
Keyword(s):  
High Resolution ◽  
Small Molecules ◽  
Primer Extension ◽  
Base Pairs ◽  
Promising Candidate ◽  
Experimental Conditions ◽  
Biologically Relevant ◽  
Wide Range ◽  
G Quadruplex ◽  
The Stability

Cytosine-rich DNA can fold into four-stranded intercalated structures, i-motif (iM), in acidic pH and require hemi-protonated C:C+ base pairs to form. However, its formation and stability rely on many other factors that are not yet fully understood. In here, we combined biochemical and biophysical approaches to determine the factors influencing iM stability in a wide range of experimental conditions. By using high resolution primer extension assays, circular dichroism and absorption spectroscopies, we demonstrate that the stability of three different biologically relevant iMs are not dependent on molecular crowding agents. Instead, some crowding agents affected overall DNA synthesis. We also tested a range of small molecules to determine their effect on iM stabilization at physiological temperature, and demonstrated that the G-quadruplex-specific molecule, CX-5461, is also a promising candidate for selective iM stabilization. This work provides important insights into the requirements needed for different assays to accurately study iM stabilization, which will serve as important tools for understanding biological roles of iMs and their potential as therapeutic targets.

Laboratory Experiments on the Droplet Shattering Secondary Ice Production Mechanism

2020 ◽  
Author(s):  
Alice Keinert ◽  
Judith Kleinheins ◽  
Dominik Spannagel ◽  
Alexei Kiselev ◽  
Thomas Leisner
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Cloud Model ◽  
Pure Water ◽  
Measuring System ◽  
Striking Difference ◽  
Observation Data ◽  
Experimental Conditions ◽  
Wide Range ◽  
Ice Production

<p>Supercooled drizzle droplets may produce multiple ice particles upon freezing. This mechanism could potentially explain the high ice number concentrations outside of temperature range where the well-known Hallett-Mossop mechanism of ice multiplication would take place. Limited experimental methods in the past prevented direct observations of the shattering droplets, resulting in a wide range of experimental results, unsuitable for the development of a sophisticated cloud model parameterization. Recently, we have revived experiments on secondary ice production by levitating individual drizzle droplets in electrodynamic balance (EDB) and observing the freeze-shattering with high-speed video microscopy and high-resolution infrared thermal measuring system. In this way we have been able to identify three additional SIP mechanisms (cracking, jetting and bubble bursts) associated with the freezing of drizzle droplets (Lauber et al., 2018). <br>Additionally, we have extended the range of experimental conditions to mimick the freezing of continental (pure water) and maritime (aqueous solution of analogue sea salt) drizzle droplets suspended in the updraft of cold moist air. We report a strong enhancement of shattering probability as compared to the previous studies conducted under stagnant air conditions. The high-definition video records of shattering events reveal the coupling between various microphysical processes caused by ice propagation inside the freezing drop and reveal striking difference between freezing of pure water and SSA solution droplets. Application of high-resolution infrared microscopy allowed us to record the evolution of the droplet temperature under realistic flow conditions and thus constrain the thermodynamic parameters controlling the pressure build-up inside the droplet. Based on these new observation data and theoretical model of freezing droplet, we discuss the physical mechanism behind the shattering of drizzle droplets and its implication for mixed-phase cloud modeling.</p><p>Lauber, A., A. Kiselev, T. Pander, P. Handmann, and T Leisner (2018). “Secondary Ice Formation during Freezing of Levitated Droplets”, Journal of the Atmospheric Sciences 75, pp. 2815–2826.</p>

scholarly journals Effects of Stability of Base Pairs Containing an Oxazolone on DNA Elongation

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Masayo Suzuki ◽  
Kazuya Ohtsuki ◽  
Katsuhito Kino ◽  
Teruhiko Kobayashi ◽  
Masayuki Morikawa ◽  
...  
Keyword(s):  
Dna Replication ◽  
Oxidative Damage ◽  
Ab Initio ◽  
Base Pair ◽  
Thermal Denaturation ◽  
Primer Extension ◽  
Base Pairs ◽  
Nucleotide Incorporation ◽  
The Stability

The nucleoside 2,2,4-triamino-5(2H)-oxazolone (Oz) can result from oxidative damage to guanine residues in DNA. Despite differences among the three polymerases (Polβ, KF exo−, and Polη) regarding nucleotide incorporation patterns opposite Oz, all three polymerases can incorporate guanine opposite Oz. Based onab initiocalculations, we proposed a structure for a stable Oz:G base pair. Here, to assess the stability of each Oz-containing base pair (Oz:G, Oz:A, Oz:C, and Oz:T) upon DNA replication, we determined the efficiency of Polβ-, KF exo−-, or Polη-catalyzed primer extension beyond each base pair. With each polymerase, extension beyond Oz:G was more efficient than that beyond Oz:A, Oz:C, or Oz:T. Moreover, thermal denaturation studies revealed that theTmvalue for the duplex containing Oz:G was significantly higher than those obtained for duplexes containing Oz:A, Oz:C, or Oz:T. Therefore, the results fromab initiocalculations along with those from DNA replication assays and thermal denaturation experiments supported the conclusion that Oz:G is the most stable of the Oz-containing base pairs.

scholarly journals Harnessing the Intrinsic Photochemistry of Isoxazoles for the Development of Chemoproteomic Crosslinking Methods

2021 ◽  
Author(s):  
Marshall G. Lougee ◽  
Vinayak Vishnu Pagar ◽  
Hee Jong Kim ◽  
Samantha X. Pancoe ◽  
Robert H. Mach ◽  
...  
Keyword(s):  
Protein Binding ◽  
Small Molecules ◽  
Functional Groups ◽  
Ex Vivo ◽  
Initial Report ◽  
Biologically Relevant ◽  
Fine Grained ◽  
Wide Range ◽  
Unique Method

Photo-crosslinking is a powerful technique for identifying both coarse- and fine-grained information on protein binding by small molecules. However, the scope of useful functional groups remains limited, with most studies focusing on diazirine, aryl azide, or benzophenone-containing molecules. Here, we report a unique method for photo-crosslinking, employing the intrinsic photochemistry of the isoxazole, a common heterocycle in medicinal chemistry, to offer an alternative to existing strategies using more perturbing, extrinsic crosslinkers. In this initial report, this technique is applied both in vitro and ex vivo, used in a variety of common chemoproteomic workflows, and validated across multiple proteins, demonstrating the utility of isoxazole photo-crosslinking in a wide range of biologically relevant experiments.

scholarly journals New (Iso)quinolinyl-pyridine-2,6-dicarboxamide G-Quadruplex Stabilizers. A Structure-Activity Relationship Study

2021 ◽  
Vol 14 (7) ◽  
pp. 669
Author(s):  
Enrico Cadoni ◽  
Pedro R. Magalhães ◽  
Rita M. Emídio ◽  
Eduarda Mendes ◽  
Jorge Vítor ◽  
...  
Keyword(s):  
Small Molecules ◽  
Gene Promoter ◽  
Ras Gene ◽  
Dynamic Simulations ◽  
Dna Polymerization ◽  
Structure Activity ◽  
Parallel Structures ◽  
Wide Range ◽  
G Quadruplex ◽  
Potential Applications

G-quadruplex (G4)-interactive small molecules have a wide range of potential applications, not only as drugs, but also as sensors of quadruplex structures. The purpose of this work is the synthesis of analogues of the bis-methylquinolinium-pyridine-2,6-dicarboxamide G4 ligand 360A, to identify relevant structure–activity relationships to apply to the design of other G4-interactive small molecules bearing bis-quinoline or bis-isoquinoline moieties. Thermal denaturation experiments revealed that non-methylated derivatives with a relative 1,4 position between the amide linker and the nitrogen of the quinoline ring are moderate G4 stabilizers, with a preference for the hybrid h-Telo G4, a 21-nt sequence present in human telomeres. Insertion of a positive charge upon methylation of quinoline/isoquinoline nitrogen increases compounds’ ability to selectively stabilize G4s compared to duplex DNA, with a preference for parallel structures. Among these, compounds having a relative 1,3-position between the charged methylquinolinium/isoquinolinium nitrogen and the amide linker are the best G4 stabilizers. More interestingly, these ligands showed different capacities to selectively block DNA polymerization in a PCR-stop assay and to induce G4 conformation switches of hybrid h-Telo G4. Molecular dynamic simulations with the parallel G4 formed by a 21-nt sequence present in k-RAS gene promoter, showed that the relative spatial orientation of the two methylated quinoline/isoquinoline rings determines the ligands mode and strength of binding to G4s.

Effect of Cu Salt Molarity on the Nanostructure of CuO Prolate Spheroid

2016 ◽  
Vol 16 (03) ◽  
pp. 1650034
Author(s):  
Sabah H. Sabeeh ◽  
Hashim Abed Hussein ◽  
Hadia Kadhim Judran
Keyword(s):  
Size Range ◽  
Prolate Spheroid ◽  
Promising Candidate ◽  
Sulfate Pentahydrate ◽  
Copper Sulfate Pentahydrate ◽  
Wide Range ◽  
Pure Phase ◽  
Potential Applications ◽  
The Stability ◽  
Zeta Potential Analysis

Copper sulfate pentahydrate was used as a source of Cu ion with five different molarities (0.02, 0.05, 0.1, 0.15, 2 and 0.25[Formula: see text]M). XRD, FE-SEM and TEM techniques all showed that CuO samples have polycrystalline monoclinic structure. CuO prolate spheroid is assembled from nanoparticles as building units. It was demonstrated that the purity, morphology, size range of prolate spheroid and density of nano building units are significantly influenced by Cu precursor’s molarity. The pure phase of CuO prolate spheroid was produced via molarity of 0.2[Formula: see text]M with crystallite size of 15.1565[Formula: see text]nm while the particle size of building units ranges from 16[Formula: see text]nm to 21[Formula: see text]nm. The stability of CuO nanosuspension or nanofluid was evaluated by zeta potential analysis. The obtained properties of specific structure with large surface area of CuO prolate spheroid make it a promising candidate for wide range of potential applications as in nanofluids for cooling purposes.

scholarly journals Insight into the mechanism of nonenzymatic RNA primer extension from the structure of an RNA-GpppG complex

2017 ◽  
Vol 114 (29) ◽  
pp. 7659-7664 ◽  
Author(s):  
Wen Zhang ◽  
Chun Pong Tam ◽  
Travis Walton ◽  
Albert C. Fahrenbach ◽  
Gabriel Birrane ◽  
...  
Keyword(s):  
High Resolution ◽  
Slow Rate ◽  
Primer Extension ◽  
Base Pairs ◽  
X Ray ◽  
The Origin Of Life ◽  
The Relationship ◽  
Self Replication ◽  
Template Complex ◽  
Insight Into

The nonenzymatic copying of RNA templates with imidazole-activated nucleotides is a well-studied model for the emergence of RNA self-replication during the origin of life. We have recently discovered that this reaction can proceed through the formation of an imidazolium-bridged dinucleotide intermediate that reacts rapidly with the primer. To gain insight into the relationship between the structure of this intermediate and its reactivity, we cocrystallized an RNA primer–template complex with a close analog of the intermediate, the triphosphate-bridged guanosine dinucleotide GpppG, and solved a high-resolution X-ray structure of the complex. The structure shows that GpppG binds the RNA template through two Watson–Crick base pairs, with the primer 3ʹ-hydroxyl oriented to attack the 5ʹ-phosphate of the adjacent G residue. Thus, the GpppG structure suggests that the bound imidazolium-bridged dinucleotide intermediate would be preorganized to react with the primer by in-line SN2 substitution. The structures of bound GppG and GppppG suggest that the length and flexibility of the 5ʹ-5ʹ linkage are important for optimal preorganization of the complex, whereas the position of the 5ʹ-phosphate of bound pGpG explains the slow rate of oligonucleotide ligation reactions. Our studies provide a structural interpretation for the observed reactivity of the imidazolium-bridged dinucleotide intermediate in nonenzymatic RNA primer extension.

scholarly journals Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Paulo H. da Silva Santos ◽  
João R. Vieira Manechini ◽  
Michael S. Brito ◽  
Elisson Romanel ◽  
Renato Vicentini ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Floral Induction ◽  
Photoperiodic Induction ◽  
Experimental Conditions ◽  
Wide Range ◽  
The Stability ◽  
Stability And Consistency

AbstractAlthough reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.

An Analysis of Dissociation of Molecules in a High Resolution Electron Microscope

1973 ◽  
Vol 31 ◽  
pp. 486-487
Author(s):  
Mihir Parikh
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Cross Sections ◽  
Resolving Power ◽  
Peak Intensity ◽  
Molecular Film ◽  
Resolution Electron ◽  
Dissociation Cross Section ◽  
High Resolution Electron Microscope ◽  
The Stability

It is well known that the resolution of bio-molecules in a high resolution electron microscope depends not just on the physical resolving power of the instrument, but also on the stability of these molecules under the electron beam. Experimentally, the damage to the bio-molecules is commo ly monitored by the decrease in the intensity of the diffraction pattern, or more quantitatively by the decrease in the peaks of an energy loss spectrum. In the latter case the exposure, EC, to decrease the peak intensity from IO to I’O can be related to the molecular dissociation cross-section, σD, by EC = ℓn(IO /I’O) /ℓD. Qu ntitative data on damage cross-sections are just being reported, However, the microscopist needs to know the explicit dependence of damage on: (1) the molecular properties, (2) the density and characteristics of the molecular film and that of the support film, if any, (3) the temperature of the molecular film and (4) certain characteristics of the electron microscope used

Development of a conical anode Fe-gun for low voltage SEM

1988 ◽  
Vol 46 ◽  
pp. 978-979
Author(s):  
T. Miyokawa ◽  
S. Norioka ◽  
S. Goto
Keyword(s):  
High Resolution ◽  
Field Emission ◽  
Low Voltage ◽  
Irradiation Damage ◽  
Cold Cathode ◽  
Virtual Source ◽  
Source Position ◽  
Electrostatic Lens ◽  
Electrostatic Lenses ◽  
Wide Range

Field emission SEMs (FE-SEMs) are becoming popular due to their high resolution needs. In the field of semiconductor product, it is demanded to use the low accelerating voltage FE-SEM to avoid the electron irradiation damage and the electron charging up on samples. However the accelerating voltage of usual SEM with FE-gun is limited until 1 kV, which is not enough small for the present demands, because the virtual source goes far from the tip in lower accelerating voltages. This virtual source position depends on the shape of the electrostatic lens. So, we investigated several types of electrostatic lenses to be applicable to the lower accelerating voltage. In the result, it is found a field emission gun with a conical anode is effectively applied for a wide range of low accelerating voltages.A field emission gun usually consists of a field emission tip (cold cathode) and the Butler type electrostatic lens.

Three-fold astigmatism: An important TEM aberration

1993 ◽  
Vol 51 ◽  
pp. 972-973 ◽  
Author(s):  
O.L. Krivanek ◽  
M.L. Leber
Keyword(s):  
High Resolution ◽  
Spatial Frequency ◽  
Field Emission ◽  
Azimuthal Angle ◽  
Hollow Cone ◽  
Small Probe ◽  
Wide Range ◽  
High Resolution Images ◽  
Image Position

Three-fold astigmatism resembles regular astigmatism, but it has 3-fold rather than 2-fold symmetry. Its contribution to the aberration function χ(q) can be written as:where A3 is the coefficient of 3-fold astigmatism, λ is the electron wavelength, q is the spatial frequency, ϕ the azimuthal angle (ϕ = tan-1 (qy/qx)), and ϕ3 the direction of the astigmatism.Three-fold astigmatism is responsible for the “star of Mercedes” aberration figure that one obtains from intermediate lenses once their two-fold astigmatism has been corrected. Its effects have been observed when the beam is tilted in a hollow cone over a wide range of angles, and there is evidence for it in high resolution images of a small probe obtained in a field emission gun TEM/STEM instrument. It was also expected to be a major aberration in sextupole-based Cs correctors, and ways were being developed for dealing with it on Cs-corrected STEMs.

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