scholarly journals The Phenanthridine-modified Tyrosine Dipeptide

2019 ◽  
Vol 92 (2) ◽  
pp. 249-258
Author(s):  
Antonija Erben ◽  
Josipa Matić ◽  
Nikola Basarić ◽  
Ivo Piantanida
Keyword(s):  
Unnatural Amino Acids ◽  
Thermal Denaturation ◽  
Specific Binding ◽  
Quinone Methide ◽  
Melting Temperatures ◽  
Double Helices ◽  
Fluorescence Titrations ◽  
Alkylate Dna ◽  
Ct Dna ◽  
Phenanthridine Derivative

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.

Studies on the Melting of DNA

1974 ◽  
Vol 29 (3-4) ◽  
pp. 130-132
Author(s):  
Gokul Chandra Das
Keyword(s):  
Ionic Strength ◽  
Melting Temperature ◽  
Thermal Denaturation ◽  
Spectrophotometric Methods ◽  
Melting Temperatures

Abstract The thermal denaturation of the native DNA in solvents of varying salt concentrations was studied by viscometric and spectrophotometric methods. It was observed that within the molarity range of 0.02 ᴍ to 0.3 ᴍ, the melting temperatures obtained by the two independent methods agreed well, but that at lower ionic strength the agreement was not satisfactory. Both the visco­metric and the spectrophotometric measurements showed an increase of the melting temperature with increasing counterion concentration and a levelling off effect in the neighbourhood of 0.3 ᴍ.

scholarly journals Optimization of the experimental parameters of the ligase cycling reaction

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Niels Schlichting ◽  
Felix Reinhardt ◽  
Sven Jager ◽  
Michael Schmidt ◽  
Johannes Kabisch
Keyword(s):  
Secondary Structure ◽  
Thermal Denaturation ◽  
Screening System ◽  
Experimental Procedure ◽  
Melting Temperatures ◽  
Assembly Method ◽  
High Efficient ◽  
Experimental Parameters ◽  
Molecular Crosstalk ◽  
Validation Experiments

Abstract The ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as dimethyl sulfoxide and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.

scholarly journals Interaction between Chromosomal Protein HMGB1 and DNA Studied by DNA-Melting Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Elena V. Chikhirzhina ◽  
Starkova J. Tatiana ◽  
Alexander M. Polyanichko
Keyword(s):  
Circular Dichroism ◽  
Thermal Denaturation ◽  
Circular Dichroism Spectroscopy ◽  
Dna Melting ◽  
Chromosomal Protein ◽  
Melting Temperatures ◽  
Melting Analysis ◽  
Circular Dichroïsm ◽  
Nonhistone Chromosomal Protein ◽  
Biphasic Process

Interaction of HMGB1 nonhistone chromosomal protein with DNA was studied using circular dichroism spectroscopy and thermal denaturation of DNA. Melting DNA in the complex was shown to be a biphasic process. The characteristic melting temperatures of unbound DNA and the DNA bound to HMGB1 in 0.25 mM EDTA solutions were found to beTmI=44.0±0.5°C andTmII=62.0±1°C, respectively. It was shown that the binding of the HMGB1 molecule affects the melting of the DNA region approximately 30 b.p. long.

scholarly journals The accumulation and compartmentalization of isometamidium chloride in Trypanosoma congolense, monitored by its intrinsic fluorescence

1995 ◽  
Vol 312 (1) ◽  
pp. 319-327 ◽  
Author(s):  
J M Wilkes ◽  
A S Peregrine ◽  
D Zilberstein
Keyword(s):  
Binding Sites ◽  
Thermal Denaturation ◽  
Specific Binding ◽  
Trypanosoma Congolense ◽  
Emission Peak ◽  
Intrinsic Fluorescence ◽  
Drug Uptake ◽  
Membrane Transporter ◽  
High Affinity ◽  
Dnase 1

Interaction of the trypanocide isometamidium chloride with components of Trypanosoma congolense results in characteristic shifts in the intrinsic fluorescence of the drug. The specificity of this interaction was investigated by analysing the effects of various physicochemical manipulations on its fluorescence properties. The characteristic shifts involved a preferential increase in the intensity of one emission peak over the other, resulting in a systematic increase in the ratio of fluorescence intensities. These effects were apparently due to constraints on fluorophore free rotation in the solution (that is, viscosity). Purified DNA produced similar effects in a saturable manner displaying high affinity for the drug, indicating that the constraint involves binding of the drug to high-affinity binding sites within the DNA. Such binding sites were demonstrated in lysates derived from trypanosomal cells. The binding sites were associated with macromolecular species (M(r) > 12000), and were partly disrupted by thermal denaturation and proteolysis. Treatment with DNase 1 produced high levels of disruption of the binding sites (> 85%), indicating an involvement of DNA in the binding. BSA demonstrated weak non-specific binding of the drug. Entry of drug into live trypanosomal cells (monitored by 14C-labelled drug uptake) was paralleled by fluorescence shifts observed under comparable conditions of drug concentration and buffer conditions. Both systems (fluorescence shifts and accumulation of labelled drug) indicated the presence of a saturable membrane transporter with high affinity for the drug. We conclude that monitoring the fluorescence shifts of isometamidium constitutes a sensitive and highly specific probe for entry of the drug into trypanosomal cells, thereby enabling resolution of the transport events involved.

The Role of DNA Concentration

1969 ◽  
Vol 24 (5) ◽  
pp. 511-514 ◽  
Author(s):  
Samarendra Basu
Keyword(s):  
Thermal Denaturation ◽  
Melting Profile ◽  
Dna Concentration ◽  
Melting Temperatures

Melting temperatures (Tm) of several DNA’s do not vary with the change of DNA concentration. However, at any temperature above Tm, the fractional hyperchromism was always greater at the lower concentration. This result was also evident in the presence of formaldehyde.Reversibility on renaturation decreased with the progress of thermal denaturation along the melting profile. The effect was much enhanced at low DNA concentration and for a DNA of low G—C content.The occurrence of some permanent changes on the dissociated molecules has been suspected to be the only cause for the observed effects.

scholarly journals Interaction of Ruthenium(II)-dipyridophenazine Complexes with CT-DNA: Effects of the Polythioether Ancillary Ligands

2001 ◽  
Vol 8 (3) ◽  
pp. 125-136 ◽  
Author(s):  
Teresa M. Santos ◽  
João Madureira ◽  
Brian J. Goodfellow ◽  
Michael G. B. Drew ◽  
Júlio Pedrosa de Jesus ◽  
...  
Keyword(s):  
Steady State ◽  
Thermal Denaturation ◽  
Noncovalent Interactions ◽  
Structural Data ◽  
Dna Intercalation ◽  
Ancillary Ligands ◽  
High Affinity ◽  
Order Of Magnitude ◽  
Ct Dna ◽  
Van Der Waals Contacts

The complexes [Ru([9]aneS3)(dppz)Cl]Cl 1 and [Ru([12]aneS4)(dppz)]Cl2,2 ([9]aneS3 = 1,4,7- trithiaciclononane and [12]aneS4 = 1,4,7,10-tetrathiaciclododecane) were synthesised and fully characterised . These complexes belong to a small family of dipyridophenazine complexes with non-polypyridyl ancillary ligands . Interaction studies of these complexes with CT-DNA (UV/Vis titrations, steady-state emission and thermal denaturation) revealed their high affinity for DNA . Intercalation constants determined by UV/Vis titrations are of the same order of magnitude (106) as other dppz metallointercalators, namely [Ru(II)(bpy)2dppz]S2+. Differences between l and2 were identified by steady-state emission and thermal denaturation studies . Emission results are in accordance with structural data, which indicate how geometric distortions and different donor and/or acceptor ligand abilities affect luminescence . The possibility of noncovalent interactions between ancillary ligands and nucleobases by van der Waals contacts and H-bridges is discussed . Furthermore, complex l undergoes aquation under intra-cellular conditions and an equilibrium with the aquated form l' is attained . This behaviour may increase the diversity of available interaction modes.

scholarly journals Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds

2015 ◽  
Vol 11 ◽  
pp. 784-791 ◽  
Author(s):  
Michaela Mühlberg ◽  
Michael G Hoesl ◽  
Christian Kuehne ◽  
Jens Dernedde ◽  
Nediljko Budisa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Specific Binding ◽  
Protein Scaffolds ◽  
Binding Studies ◽  
Scaffold Design ◽  
Thermophilic Lipase

To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at theN-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding toErythrina cristagallilectin by SPR binding studies.

Spectroscopic studies on the thermodynamic and thermal denaturation of the ct-DNA binding of methylene blue

2010 ◽  
Vol 77 (2) ◽  
pp. 528-534 ◽  
Author(s):  
Mudasir ◽  
Endang Tri Wahyuni ◽  
Daryono H. Tjahjono ◽  
Naoki Yoshioka ◽  
Hidenari Inoue
Keyword(s):  
Methylene Blue ◽  
Dna Binding ◽  
Thermal Denaturation ◽  
Spectroscopic Studies ◽  
Ct Dna

scholarly journals Optimization of the experimental parameters of the ligase cycling reaction

2019 ◽  
Author(s):  
Niels Schlichting ◽  
Felix Reinhardt ◽  
Sven Jager ◽  
Michael Schmidt ◽  
Johannes Kabisch
Keyword(s):  
Secondary Structure ◽  
Thermal Denaturation ◽  
Screening System ◽  
Experimental Procedure ◽  
Melting Temperatures ◽  
Assembly Method ◽  
High Efficient ◽  
Experimental Parameters ◽  
Molecular Crosstalk ◽  
Validation Experiments

ABSTRACTThe ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as DMSO and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.

scholarly journals INTERACTION OF IRON(II) MIXED-LIGAND COMPLEXES WITH DNA: BASE-PAIR SPECIFICITY AND THERMAL DENATURATION STUDIES

2010 ◽  
Vol 4 (3) ◽  
pp. 174-179 ◽  
Author(s):  
Mudasir Mudasir ◽  
Karna Wijaya ◽  
Endang Tri Wahyuni ◽  
Hidenari Inoue
Keyword(s):  
Dna Binding ◽  
Electrostatic Interaction ◽  
Base Pair ◽  
Thermal Denaturation ◽  
Spectrophotometric Titration ◽  
Mixed Ligand ◽  
Mixed Ligand Complexes ◽  
Dna Base ◽  
Dna Base Pair ◽  
Ct Dna

A research about base-pair specificity of the DNA binding of [Fe(phen)3]2+, [Fe(phen)2(dip)]2+ and [Fe(phen)(dip)2]2+ complexes and the effect of calf-thymus DNA (ct-DNA) binding of these metal complexes on thermal denaturation of ct-DNA has been carried out. This research is intended to evaluate the preferential binding of the complexes to the sequence of DNA (A-T or G-C sequence) and to investigate the binding strength and mode upon their interaction with DNA. Base-pair specificity of the DNA binding of the complexes was determined by comparing the equilibrium binding constant (Kb) of each complex to polysynthetic DNA that contain only A-T or G-C sequence. The Kb value of the interaction was determined by spectrophotometric titration and thermal denaturation temperature (Tm) was determined by monitoring the absorbance of the mixture solution of each complex and ct-DNA at λ =260 nm as temperature was elevated in the range of 25 - 100 oC. Results of the study show that in general all iron(II) complexes studied exhibit a base-pair specificity in their DNA binding to prefer the relatively facile A-T sequence as compared to the G-C one. The thermal denaturation experiments have demonstrated that Fe(phen)3]2+ and [Fe(phen)2(dip)]2+ interact weakly with double helical DNA via electrostatic interaction as indicated by insignificant changes in melting temperature, whereas [Fe(phen)2(dip)]2+  most probably binds to DNA in mixed modes of interaction, i.e.: intercalation and electrostatic interaction. This conclusion is based on the fact that the binding of [Fe(phen)2(dip)]2+ to ct-DNA moderately increase the Tm value of ct- DNA   Keywords: DNA Binding, mixed-ligand complexes

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