DNA-binding properties of analogues were investigated using UV–Vis absorbance spectroscopy. All UV–Vis spectra were recorded on a JASCO V-650 spectrophotometer. In UV titration experiments, the spectra of calf-thymus DNA (CT-DNA, Sigma, St. Louis, MO, USA) in the presence of the compound have been recorded for a constant CT-DNA concentration in diverse [complex]/[CT-DNA] mixing ratios (r). The intrinsic binding constant, Kb, of the complex with CT DNA was determined through the UV spectra of the complex recorded for a constant complex concentration in the absence and presence of CT-DNA for diverse r values. Each experiment was repeated in triplicate. A solution of CT-DNA was prepared using 150 mM NaCl in 15 mM trisodium citrate at pH 7.0, followed by stirring for 48 h at 4 °C. A260/A280 ratio at 1.47 and A260/A230 ratio at 15.39 (≥2) indicated that CT-DNA was sufficiently free from organic substances and 82.5% pure of protein contamination [15 (link),16 (link)]. The concentration of duplex DNA was determined following the Beer Lambert Law, with a molar extinction coefficient of 6600 M−1cm−1 and was calculated to 59.42 μM. Analogs were diluted in a tris-citrate buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of approximately 1.0% DMSO and incubation with CT-DNA was carried out for 24h.
Ct dna
Manufactured by Merck Group
Sourced in United States
CT-DNA is a laboratory equipment product that is used for the extraction and purification of Calf Thymus Deoxyribonucleic Acid (CT-DNA). It is a commonly used DNA source for various biochemical and molecular biology applications.
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Lab products found in correlation
Human serum albumin (hsa), by Merck Group (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Ethidium bromide, by Merck Group (3 mentions)
Spectramax microplate reader, by Molecular Devices (3 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Lambda 365 uv vis spectrophotometer, by PerkinElmer (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Tensor 27, by Bruker (2 mentions)
Dimethyl sulfoxide (dmso), by Spectrochem (1 mentions)
Tris base, by Merck Group (1 mentions)
3 5 diiodosalicylaldehyde, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Glacial acetic acid, by Spectrochem (1 mentions)
Mueller hinton broth (mhb), by Merck Group (1 mentions)
Salicylaldehyde, by Merck Group (1 mentions)
4 bromoaniline, by Merck Group (1 mentions)
5 bromosalicylaldehyde, by Merck Group (1 mentions)
61 protocols using ct dna
1
Quantifying DNA-Binding Affinity of Analogues
2
DNA-binding Guanidine Derivative Synthesis
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3,5-Diiodosalicylaldehyde (Aldrich), 1,3-diaminoguanidine (Aldrich), CT-DNA, Tris base, methanol (Merck), DMSO (Spectrochem), glacial acetic acid (Spectrochem), etc. were used as received.
3
Synthesis and Characterization of Metal Complexes
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All standard chemicals, as well as CT-DNA, BSA, ethidium bromide, Hoechst 33258, Mueller-Hinton broth, were of analytical grade (or higher) and were purchased from Merck (Darmstadt, Germany) or associated companies. Solvents were used as received or dried over molecular sieves. RuCl 3 was purchased from I2CNS. Complexes 1, 2, 3 and 4 were synthesized according to published procedures from Nikolić et al. (2016) (link), Pavlović et al. (2019) (link) (Scheme 1).
4
Metal-Organic Complexation and Biomolecular Interactions
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All reagents and
solvents used in this synthesis were commercially available. Reagent-grade
chemicals were used in this experiment. Hence, no further purification
was needed. Salicylaldehyde, 5-bromo Salicylaldehyde, 4-bromo aniline,
ctDNA, HSA, EB, and DAPI were obtained from Sigma-Aldrich Chemicals.
CuCl2, ZnCl2, and triethyl amine (Et3N) were purchased from Merck. Elemental analyses were performed using
a PerkinElmer 240C elemental analyzer. Electronic absorption spectral
data were collected by using a PerkinElmer UV–vis Lambda 365
spectrophotometer at room temperature. The concentration of ctDNA
per nucleotide was determined by absorption spectroscopy using a molar
extinction coefficient (€) 6600 (M–1 cm–1) at 260 nm. ctDNA interaction studies were performed
in a citrate-phosphate (CP) buffer of 10 mM [Na+] at pH
7.40 containing 0.5 mM Na2HPO4, and HSA interaction
studies were performed in Tris buffer.
Caution! Mercury salts are extremely toxic, so proper caution should be taken
before use.
solvents used in this synthesis were commercially available. Reagent-grade
chemicals were used in this experiment. Hence, no further purification
was needed. Salicylaldehyde, 5-bromo Salicylaldehyde, 4-bromo aniline,
ctDNA, HSA, EB, and DAPI were obtained from Sigma-Aldrich Chemicals.
CuCl2, ZnCl2, and triethyl amine (Et3N) were purchased from Merck. Elemental analyses were performed using
a PerkinElmer 240C elemental analyzer. Electronic absorption spectral
data were collected by using a PerkinElmer UV–vis Lambda 365
spectrophotometer at room temperature. The concentration of ctDNA
per nucleotide was determined by absorption spectroscopy using a molar
extinction coefficient (€) 6600 (M–1 cm–1) at 260 nm. ctDNA interaction studies were performed
in a citrate-phosphate (CP) buffer of 10 mM [Na+] at pH
7.40 containing 0.5 mM Na2HPO4, and HSA interaction
studies were performed in Tris buffer.
before use.
5
Spectroscopic Characterization of Oligonucleotides
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Absorption measurements were made on a Perkin-Elmer ultraviolet-visible spectrometer. Emission spectra were obtained on a Varian Cary-Eclipse or Horiba Jobin Yvon Fluorolog fluorescence spectrometer. The oligonucleotides used were purchased RP-cartridge purified from Eurogentec. CT-DNA was obtained from Sigma-Aldrich. pH was measured using a Mettler Toledo pH meter.
6
Characterization of Calf Thymus DNA-Surfactant Interactions
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ct-DNA, NOS (98%), SDS,
and phosphate salts (sodium monophosphate and biphosphate) were obtained
from Sigma-Aldrich. The stock solution of ctDNA was prepared by dissolving
in phosphate buffer (10 mM, pH 7.0) and stored in −20 °C,
as described in the literature.18 (link) The ctDNA
concentration was determined spectrophotometrically using extinction
coefficients (ε) 6600 M–1 cm–1 at 260 nm and the purity of ctDNA was confirmed by observing absorbance
ratio (A260 nm/A280 nm) which is observed between the range of 1.8–1.9.19 (link) NOS solution was prepared in the phosphate buffer.
The freshly prepared micellar solution of SDS was utilized after confirming
its cmc by using a Langmuir tensiometer (Kibron, Helsinki, Finland).
The cmc of SDS in water at room temperature was observed at 8.2 mM,
which is matches well with reported values.5 (link) Ultrapure water was used throughout the experiments obtained from
a Millipore water purification system. The buffer solution was filtered
through Millipore filters of 0.22 μm pore size. All other reagents
were of analytical reagent grade and used without further purification.
and phosphate salts (sodium monophosphate and biphosphate) were obtained
from Sigma-Aldrich. The stock solution of ctDNA was prepared by dissolving
in phosphate buffer (10 mM, pH 7.0) and stored in −20 °C,
as described in the literature.18 (link) The ctDNA
concentration was determined spectrophotometrically using extinction
coefficients (ε) 6600 M–1 cm–1 at 260 nm and the purity of ctDNA was confirmed by observing absorbance
ratio (A260 nm/A280 nm) which is observed between the range of 1.8–1.9.19 (link) NOS solution was prepared in the phosphate buffer.
The freshly prepared micellar solution of SDS was utilized after confirming
its cmc by using a Langmuir tensiometer (Kibron, Helsinki, Finland).
The cmc of SDS in water at room temperature was observed at 8.2 mM,
which is matches well with reported values.5 (link) Ultrapure water was used throughout the experiments obtained from
a Millipore water purification system. The buffer solution was filtered
through Millipore filters of 0.22 μm pore size. All other reagents
were of analytical reagent grade and used without further purification.
7
Quantification of Anti-Cytokine Antibodies
8
Spectrophotometric Characterization of Biomolecules
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CT-DNA, DAPI, and NB were purchased from
Sigma-Aldrich Corporation (St. Louis, MO, USA). A spectrophotometer
was used to determine their concentrations. Molar extinction coefficients
of CT-DNA (ε = 6600 M–1 cm–1 at 260 nm)30 (link) (expressed in terms of nucleotide
phosphates) and DAPI (ε = 27,000 M–1 cm–1 at 353 nm) were used to determine concentrations
in solution. The concentration of NB was measured in 100% ethanol
using molar extinction coefficients 76,800 M–1 cm–1 at 627.5 nm.43 (link) The same
concentration was also measured in CP buffer pH 7.0, [Na+] 10 mM and found that the molar extinction coefficient in that medium
is 54,185 M–1 cm–1 at 635 nm.
No deviation from Beer’s law was observed in the concentration
range at 0–12 μM for NB used in our study.
All
the experiments were conducted in CP buffer medium (10 mM [Na+]) at pH 7.0, containing 5.0 mM Na2HPO4. The pH of the medium was adjusted using citric acid.31 (link) The pH values of the solutions were measured
with a calibrated Orion-Ross combined electrode system (model 81-02).
All buffer solutions were filtered through Millipore filters (Millipore,
India Pvt. Ltd., Bangalore, India) of 0.45 μM, before use.
Sigma-Aldrich Corporation (St. Louis, MO, USA). A spectrophotometer
was used to determine their concentrations. Molar extinction coefficients
of CT-DNA (ε = 6600 M–1 cm–1 at 260 nm)30 (link) (expressed in terms of nucleotide
phosphates) and DAPI (ε = 27,000 M–1 cm–1 at 353 nm) were used to determine concentrations
in solution. The concentration of NB was measured in 100% ethanol
using molar extinction coefficients 76,800 M–1 cm–1 at 627.5 nm.43 (link) The same
concentration was also measured in CP buffer pH 7.0, [Na+] 10 mM and found that the molar extinction coefficient in that medium
is 54,185 M–1 cm–1 at 635 nm.
No deviation from Beer’s law was observed in the concentration
range at 0–12 μM for NB used in our study.
All
the experiments were conducted in CP buffer medium (10 mM [Na+]) at pH 7.0, containing 5.0 mM Na2HPO4. The pH of the medium was adjusted using citric acid.31 (link) The pH values of the solutions were measured
with a calibrated Orion-Ross combined electrode system (model 81-02).
All buffer solutions were filtered through Millipore filters (Millipore,
India Pvt. Ltd., Bangalore, India) of 0.45 μM, before use.
9
Characterization of Biomolecular Interactions
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Diflunisal, bipy, bipyam, phen, Hpko, ZnCl2, KOH, trisodium citrate, NaCl, CT DNA, BSA, HSA, EB, DPPH, ABTS, sodium linoleate, butylated hydroxytoluene (BHT), 6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (trolox), nordihydroguaiaretic (NDGA) and caffeic acid were purchased from Sigma-Aldrich and all solvents were purchased from Merck. All the chemicals and solvents were reagent grade and were used as purchased.
DNA stock solution was prepared by dilution of CT DNA to buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) followed by exhaustive stirring at 4°C for three days, and kept at 4°C for no longer than two weeks. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) in the range 1.85-1.90, indicating that the DNA was sufficiently free of protein contamination [47] . The concentration of CT DNA was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M -1 cm -1 [48] .
Infrared (IR) spectra (400-4000 cm -1 ) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr pellets. UV-visible (UV-vis) spectra were recorded as nujol mulls and in solution at concentrations in the range 10 -5 -10 mol -1 , 1 mM in DMSO).
DNA stock solution was prepared by dilution of CT DNA to buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) followed by exhaustive stirring at 4°C for three days, and kept at 4°C for no longer than two weeks. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) in the range 1.85-1.90, indicating that the DNA was sufficiently free of protein contamination [47] . The concentration of CT DNA was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M -1 cm -1 [48] .
Infrared (IR) spectra (400-4000 cm -1 ) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr pellets. UV-visible (UV-vis) spectra were recorded as nujol mulls and in solution at concentrations in the range 10 -5 -10 mol -1 , 1 mM in DMSO).
10
Preparation and Characterization of ct DNA
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