Cary 300 bio uv vis spectrophotometer

DNA thermal melting experiments were performed on a Cary 300 Bio UV-vis spectrophotometer (Varian). The concentration of each hairpin DNA sequence was 3 μM in TNE 100 using 1 cm quartz cuvettes. The solutions of DNA and ligands were tested with a ratio of 2:1 [ligand] / [DNA]. All samples were increased to 95 °C and cooled down to 25 °C slowly before each experiment. The spectrophotometer was set at 260 nm with a 0.5 °C/min increase beginning at 25 °C, which is below the DNA melting temperature and ending above it at 95 °C. The absorbance of the buffer was subtracted, and a graph of normalized absorbance versus temperature was created using KaleidaGraph 4.0 software. The ΔT_m values were calculated using a combination of the derivative function and estimation from the normalized graphs.

UV-vis measurements were conducted on a Varian Cary 300 Bio UV-vis spectrophotometer equipped with thermoelectrically controlled multicell holders. Samples were prepared in a buffer containing 10mM Tris-HCl (pH 7) and 0.1 mM Na₂EDTA. For LiCl experiments, a stock solution of 10 mM Li₂EDTA was used. Thermal experiments were conducted in the presence of either 100 mM KCl or 100 mM LiCl. The solutions were heated to 95 °C followed by cooling to 15 °C at a rate of 1 °C min⁻¹. The samples were allowed to equilibrate for 5 minutes at 15 °C and then heated back to 95 °C at the same rate. The absorbance at 260 nm for complementary binding or 295nm for G-quadruplex formation was recorded at an interval of 0.5 °C. All samples contained concentrations of 4 μM PNA and DNA repeat sequences, for example 2 μM γP₁₂ (2 repeat sequences) + 2 μM Telo-2 (2 repeat sequences) or 1 μM Telo-4 (4 repeat sequence); 4 μM γP₆ or Quantitation of FISH results (1 repeat sequence) + 4 μM Telo-1, 2 μM Telo-2, 1.3 μM Telo-3 or 1 μM Telo-4. All experiments were run in triplicate and the averaged data were plotted as the fraction of melted PNA-DNA duplex as a function of temperature. Melting transition temperature (T_m), the temperature at which PNA-DNA hybrid was half-melted, was determined as described by Marky and Breslauer.^{40 (link)}

Octanol/water partition coefficients were calculated using
XLOGP3.^{23 (link)} Molecular
volume was calculated using the Molinspiration property calculation toolkit
(Molinspiration Cheminformatics). The density of fropofol was determined from
replicate measurements of the volume/mass relationship. The measurement of the
UV−vis absorbance (Varian Cary 300 Bio UV−vis spectrophotometer)
of AziFo showed a maximum diazirine absorbance at 317 nm with
additional aromatic absorption maxima at 267 and 273 nm. The extinction
coefficient (Σ₂₇₃ = 1600 M⁻¹cm⁻¹) was calculated from UV absorption measurements from
the aromatic absorption at 273 nm in methanolic solutions of known
concentrations. The extinction coefficient was used to calculate the maximal
water solubility of AziFo after 24 h of sonication in double
distilled water (ddH₂O) and filtration with a 0.22
μm polyvinylidene difluoride (PVDF) syringe (MidSci,
St. Louis, MO).