Application Notes

Thermodynamic characterization of DNA hybridization

Issue link:

Contents of this Issue


Page 1 of 3

2 phenomenon however does not compromise the MST experiments. After calculating the K d of DNA hybridization for each temperature, a van´t Hoff plot (Figure 2A) was used to deduce the thermodynamic parameters ΔH and ΔS as described in detail in the Material and Methods section (see below). In addition, we performed experiments with two oligonucleotides carrying one or two mismatches, respectively (Figure 1A). As expected, the hybridization affinity was greatly reduced for the mismatch-nucleotides, and the free enthalpy and entropy where higher compared to the perfect match hybridization reaction (Figure 2B). One of the big benefits of using DNA hybridization as a model system is that all interactions can be simulated in detail. We compared the experimentally determined thermodynamic parameters to calculated ones (using the IDT Biophysics tool The comparison showed an excellent agreement of the parameters for the perfect match- as well as the mismatch hybridizations. We note that the parameters obtained for the mismatch construct with two mismatches shows the biggest divergence from the calculated parameters, which could however be due to limitations of the simulation software rather than due to a larger experimental error. Figure 1: A) Sequences of the DNA oligomers. Single base substitutions of the mismatches are highlighted in red, 5'-terminal Cy5 in blue. B) Exemplary MST timetraces for a single-stranded (ssDNA) and a double-stranded DNA (dsDNA). ssDNA shows a stronger MST response then dsDNA. C) Fitted sigmoidal binding curve of a temperature-jump MST signal for a template-PM interaction at 24 °C. D) Temperature-jump binding curves over a temperature range from 38 °C to 45 °C; increments 1 °C.

Articles in this issue

Links on this page

view archives of Application Notes - Thermodynamic characterization of DNA hybridization