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Melting curve by CD spectroscopy

RS Ranajay Saha
SV Samuel Verbanic
IC Irene A. Chen
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Temperature-dependent CD spectra were acquired using a JASCO J-1500 spectrophotometer (JASCO International Co. Ltd, Tokyo, Japan) equipped with a Peltier-controlled cell holder (model PTC-517, JASCO). A sample containing 3.8 μM RNA, 12 μM MG, 1.7 mM Tris, 10 mM HEPES, 1 mM Mg-Citrate, 100 mM KCl, 0.2 M bicine, pH 8.5 was prepared. The CD signal of the aptamer was monitored at 264 nm in 5 °C increments from 1 to 91 °C, with a 10 min incubation at each interval. The spectra were recorded using a 1 mm path length cuvette. Melting curves were fitted in Origin Pro 9 software using the Boltzmann sigmoidal equation θ = θmin + (θmax − θmin)/(1 + exp((Tm − T)/s)), where θ refers to the ellipticity at 264 nm and θmin and θmax are the minimum and maximum θ, respectively, T is temperature, Tm is the melting temperature, and s is a fitting parameter.

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