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 http://biophysics.idtdna.com/). 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.
