UV Melting Analysis

Oligonucleotides were dissolved in a buffer containing 100 mM potassium chloride, 20 mM sodium cacodylate, and 0.5 mM Na₂EDTA (pH 7.0). Single-stranded oligonucleotide concentrations were calculated based on the absorbance measured above 80°C,57 and the extinction coefficients were calculated using Oligo Calculator (http://www.ribotask.com). The samples were denatured at 95°C for 5 min and then cooled to room temperature overnight. The measurements were performed for nine different concentrations of the G-quadruplexes in the concentration range of 10⁻⁴ to 10⁻⁶ M. Absorbance versus temperature curves were obtained using the UV melting method at 295 nm in the temperature range of 4°C to 90°C with a heating rate of 0.2°C/min on a JASCO V-650 spectrophotometer equipped with a thermoprogrammer. Melting curves (Supplemental Information; Figure S3) were analyzed and the thermodynamic parameters determined by nonlinear curve fitting using the MeltWin 3.5 software. Melting temperatures calculated for a 10⁻⁴ M concentration of oligonucleotide are denoted by T_M, and melting points for any other concentration of oligonucleotide are denoted by T_m.