Application Notes

3 Figure 2: A) Van´t Hoff plots of the DNA-DNA hybridization reactions. The template-PM, template-MM1 and template-MM2 interactions are shown in black, red and blue, respectively (n=2). B) Comparison between experimentally determined and calculated ΔH and ΔS values (PM= perfect match, MM=mismatch). Conclusion Using MST, we determined ΔH and ΔS for the hybridization reaction of a DNA template with three different complementary oligonucleotides. All values were well within the margin of error compared to the values calculated by the IDT Biophysics tool. Thus MST is a feasible method to determine thermodynamic parameters of biomolecular interactions with minimal sample consumption. This app note thus provides a guideline of how to perform thermodynamic measurements - be it for DNA and/or protein interactions. Material and Methods Experimental setup Hybridization experiments were carried out in DNA buffer (20 mM HEPES, 100 mM NaCl, 0.05 % Tween-20). Standard enhanced gradient capillaries were used in all experiments because they reduce the risk of convection effects. Due to the strong thermophoresis of DNA, DNA oligomers are more prone to convection when compared to proteins. Also, the use of high MST power should be avoided to prevent convection. In order to minimize buffer evaporation during the experiment, the capillaries were sealed with NT Wax Tight Sealing. Additionally, the NT Anti Photobleaching Kit (A001) was used to prevent photobleaching. 5 mM EDTA was added to prevent enzymatic DNA decay during the measurement. The fluorescence intensities for the first measurements at low temperatures were adjusted to be well above the minimal fluorescence required for MST measurements, (the upper third of the detection range), since fluorescence decreased at higher temperatures. DNA hybridization: MST measurements All measurements were performed at LED power 90% and MST power 15% on a NT.115 Pico device. The concentration of the 5´Cy5-labelled DNA 20- mer was kept constant at 1 nM and its perfect match or one of two mismatches were diluted in a range from 1600 nM down to 0.05 nM, respectively. Measurements were performed over a temperature range of 25 °C to 45 °C increasing in increments of 1 °C. For each temperature, MST measurements were started 120 seconds after reaching the desired temperature. The temperature-jump signals of the MST traces were fitted to obtain the K d values. DNA hybridization: thermodynamics The thermodynamic parameters of the DNA-DNA hybridization were calculated by using the relationship between K d and K a (3)

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