RNA binding small molecules: Studies on t-RNA binding by cytotoxic plant alkaloids berberine, palmatine and the comparison to ethidium

Biophysical studies on the base specificity and energetics of the DNA interaction of photoactive dye thionine: Spectroscopic and calorimetric approach

“…The excess heat capacities as a function of temperature were measured on a Microcal VP-differential scanning calorimeter (DSC) (MicroCal, Inc., Northampton, MA, USA) as described previously [25,30]. In a series of DSC scans, both the sample and reference cells were loaded with buffer solution, equilibrated at 20°C for 15 min and scanned from 40°to 120°C at a scan rate of 60°C/h.…”

“…All measurements were performed keeping an excitation and emission band pass of 3 nm at 20 ± 1.0°C. The amount of free and bound thionine was determined as described in details previously [25]. Binding data obtained from spectrophotometric and spectrofluorimetric titrations were cast into Scatchard plots of r/C f versus r. All the Scatchard plots revealed negative slope at low r values characteristic of non-cooperative binding isotherms and hence were analyzed using the following McGhee-von Hippel equation [26],…”

“…Steady state fluorescence measurements were performed on a Shimadzu RF5301PC spectrofluorimeter (Shimadzu Corporation, Kyoto, Japan) in fluorescence free quartz cuvettes of 1 cm path length as described previously [23,25]. The excitation wavelength was 596 nm.…”

“…Job plot [28,29] was constructed to determine the binding stoichiometry in each case from fluorescence spectroscopy results at 20 ± 1°C as described previously [25,30]. At constant temperature, the fluorescence signal was recorded for mixture of solutions where the concentrations of both DNA and thionine were varied while the sum of their concentrations was kept constant.…”

“…Melting curves of DNA and DNA-thionine complexes were measured on a Shimadzu Pharmaspec 1700 unit equipped with a peltier controlled TMSPC-8 model accessory (Shimadzu Corporation, Kyoto, Japan) as described earlier [25,30]. DNA sample (20 μM) was mixed with varying concentrations of the thionine in the degassed buffer in to the eight chamber micro optical cuvette of 1 cm path length and the temperature of the microcell accessory was raised at a heating rate of 0.5°C/min.…”