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bioRxiv · 10.1101/730432

Using a novel web application for thermodynamic characterization of Tel22 G-quadruplex unfolding

Prislan, I.·Sajko, S.·Poklar Ulrih, N.·Fuerst, L.
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Abstract

Measuring and quantifying thermodynamic parameters that determine stability of and interactions between biological macromolecules is an essential and necessary complement to structural studies. Although several laboratories are able to obtain basic thermodynamic parameters for the observed process, the data interpretation and analysis quality of reported data can be extremely tedious. We have started to develop a web application that will help users to perform thermodynamic characterization of G-quadruplex unfolding. The application can perform global fitting of calorimetric and spectroscopic data, and it uses a three-state equilibrium model to obtain thermodynamic parameters for each transition step: the Gibbs energy, the enthalpy, and the heat capacity. As well as these, the application can define the number of K+ ions and the number of water molecules being released or taken up during the unfolding. To test our application, we used UV spectroscopy, circular dichroism, and differential scanning calorimetry, to monitor folding and unfolding of a model 22-nucleotide-long sequence of human 3-telomeric overhang, known as Tel22. The obtained data was fed to the web application and global fit revealed that unfolding of Tel22 involves at least one intermediate state, and that K+ ions are released during the unfolding, whereas water molecules are taken up.\n\nSTATEMENT OF SIGNIFICANCEThe laws of thermodynamics provide tools for the use of elegant mathematical expressions to describe stabilities and interactions of biological macromolecule. Even though thermodynamic profiles of simple transitions (e.g., two state) can be obtained in a relatively straightforward manner, performing thermodynamic analysis of complex/ multistep transitions or global analysis of several experimental data requires some experiences and skills. In the present study we are demonstrating how newly developed web application can be used to provide better understanding of driving forces responsible for the structural interconversion of G-quadruplex structures. We have tested this web application with experimental data obtained from monitoring thermal folding/ unfolding of the 5-AG3T2AG3T2AG3T2AG3-3 (Tel22) DNA sequence. We believe that this application can be used as a research and/or teaching tool, and it will allow comparisons of the thermodynamic parameters obtained between different laboratories.

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Prislan, I., Sajko, S., Poklar Ulrih, N., Fuerst, L.. 2019-08-08. Using a novel web application for thermodynamic characterization of Tel22 G-quadruplex unfolding. https://doi.org/10.1101/730432

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