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Structural Insight into the Unbound State of the DNA Analogue of the PreQ1 Riboswitch: A Thermodynamic Approach
Biochemistry ( IF 2.9 ) Pub Date : 2017-11-09 00:00:00 , DOI: 10.1021/acs.biochem.7b00596
Calliste Reiling-Steffensmeier 1 , Luis A. Marky 1
Affiliation  

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.

中文翻译:

PreQ 1核糖开关的DNA类似物的未结合状态的结构洞察:一种热力学方法。

preQ 1核糖开关适体结构域在未结合状态下非常动态,能够形成多种结构:发夹,亲吻发夹和伪结状结构。这项研究的目的是确定DNA类似物(PreQ 1)是否能够形成与报道的RNA适体相似的结构。使用热力学方法,我们报告了在不同盐条件下使用差示扫描量热法进行结构测定的报告。对主要序列的进一步分析使我们能够设计修饰的分子,以确定在此单链DNA类似物中形成哪些潜在结构。我们发现,在16 mM Na +解决方案中,PreQ 1T发生三个跃迁M值为14.8、19.4和26.2°C,总ΔH为-44.7 kcal / mol。随着盐浓度增加到116 mM,T M值为22.3、28.7和38.9°C,ΔH为-69.1 kcal / mol,而在216 mM时,三个转变的T M值为24.4, 31.6和42.9°C,总ΔH为-71.5kcal / mol。因此,焓的增加归因于额外碱基对堆叠的形成。修饰后的分子具有抑制假结形成,亲吻发夹和内部环相互作用的特性,已得到充分表征,并与天然DNA类似物进行了比较。对平衡离子的焓和差异结合的分析使我们得出结论,在生理条件下这种单链DNA类似物没有形成假结状结构。取而代之的是,两种可能的结构,即Compact-HairpinKissing-Complex,更可能处于平衡状态。
更新日期:2017-11-09
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