Application Notes

Thermodynamic characterization of DNA hybridization

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1 DNA-DNA Interaction Analysis Application Note NT-MO-018 Thermodynamic characterization of DNA hybridization Timon André 1 and Dennis Breitsprecher 1 1 NanoTemper Technologies GmbH, Munich, German Abstract Here we report a thermodynamic analysis of biomolecular interactions using MicroScale Thermophoresis (MST). Using DNA hybridization with fluorescently labeled oligonucleotides as a well-characterized model system, we monitor the shift in the dissociation (K d ) constant of the interaction over a range of temperatures and calculate the free enthalpy (ΔH) and entropy (ΔS) for the hybridization reaction. The obtained values were in excellent agreement with theoretically calculated values, demonstrating that MST is feasible to perform thermodynamic analysis of biomolecules. Introduction Biomolecular interactions display a remarkable degree of specificity, which is determined by distinct molecular recognition events. Binding between two interacting partners has enthalpic (ΔH) and entropic (TΔS) components, corresponding to changes in both, structure and dynamics of each counterpart. In general, binding occurs only when the associated Gibbs free energy (ΔG) is negative (see below), which can be achieved by an entropy- and/or an enthalpy-driven process. The information content from thermodynamic parameters is vast. The parameters are instrumental to elucidate the molecular mechanism of an interaction. Moreover, this knowledge can be used for the rational design of interactions, paving the way for novel pharmaceuticals or biomaterials. Importantly, thermodynamic parameters are not only accessible through the direct measurement of reaction temperatures, but can also be extracted from the temperature dependence of the dissociation constant as shown by the following relations: Given a reversible, bimolecular reaction L+M ↔ LM the dissociation constant K d that is defined as which in turn directly depends on the Gibbs free energy change: Thus, by measuring K d s over a temperature range, ΔG, ΔH and ΔS can be calculated. Here we demonstrate that MicroScale Thermophoresis (MST) can precisely recapitulate thermodynamic parameters of DNA hybridization reactions. Results In the initial experiments, a perfect-match oligo nucleotide was titrated against Cy5-labeled template (Figure 1A), and the dissociation constant was calculated from the differing MST signals for single-stranded (ssDNA) and double- stranded DNA (dsDNA) (Figure 1B). The binding curves showed a typical sigmoidal shape, with the ssDNA displaying a stronger MST response than the dsDNA (Figure 1C). Increasing the temperature resulted in a clear shift of the K d towards higher values. We moreover note that the Cy5-fluorescence intensity dropped significantly with increasing temperature (Figure 1D), which is due to shorter fluorescence lifetimes and reduced quantum yields at higher temperature. This K d = [ L] [ M ] [ LM ] (1) ΔG= RT ln ( K d )( 2.1)

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