4 years ago

Structural Insight into the Unbound State of the DNA Analogue of the PreQ1 Riboswitch: A Thermodynamic Approach

Structural Insight into the Unbound State of the DNA Analogue of the PreQ1 Riboswitch: A Thermodynamic Approach
Luis A. Marky, Calliste Reiling-Steffensmeier
The preQ1 riboswitch aptamer domain is very dynamic in its unbound state with the ability to form multiple structures: a hairpin, kissing hairpins, and pseudoknot-like structure. The aim of this study is to determine whether the DNA analogue (PreQ1) is able to form structures similar to that of the reported RNA aptamer. Using a thermodynamic approach, we report on structural determination using differential scanning calorimetry under different salt conditions. Further analysis of the primary sequence allowed us to design modified molecules to determine what potential structures are forming in this single-stranded DNA analogue. We found, in a 16 mM Na+ solution, PreQ1 has three transitions with TM values of 14.8, 19.4, and 26.2 °C and a total ΔH of −44.7 kcal/mol. With the increase in salt concentration to 116 mM, there are TM values of 22.3, 28.7, and 38.9 °C and a ΔH of −69.1 kcal/mol, while at 216 mM, the three transitions have TM values of 24.4, 31.6, and 42.9 °C with a total ΔH of −71.5 kcal/mol. Therefore, the increase in enthalpy is due to the formation of additional base-pair stacks. The modified molecules, which would inhibit pseudoknot formation, kissing hairpins, and internal loop interactions, were fully characterized and compared to the native DNA analogue. The analysis of the enthalpy and differential binding of counterions allows us to conclude this single-stranded DNA analogue under physiological conditions is not forming a pseudoknot-like structure. Instead, two potential structures, Compact-Hairpin and Kissing-Complex, are more likely and could be in equilibrium.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00596

DOI: 10.1021/acs.biochem.7b00596

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