Linkage between proton binding and folding in RNA:: A thermodynamic framework and its experimental application for investigating pKa shifting
Linkage between proton binding and folding in RNA:: A thermodynamic framework and its experimental application for investigating pKa shifting
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DOI：
10.1261/rna.7177505
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发表时间：
2005-02-01
期刊：
影响因子：
4.5
通讯作者：
Bevilacqua, PC
中科院分区：
文献类型：
--
作者：
Moody, EM;Lecomte, JTJ;Bevilacqua, PC
pK（a）值的扰动可以改变生物pH下核碱基的有利质子化状态，从而调节RNA和DNA分子的功能。为了了解核酸特有的pK（a）位移的驱动力，我们开发了一个热力学框架，该框架将质子与核碱基的结合和螺旋-卷曲转变联系起来。从治疗中出现的关键特征是对质子结合对RNA折叠的所有作用的全面描述：螺旋的酸和碱变性以及折叠状态下的pK（a）转移。从热变性实验中测量pK（a）s的实用实验方法的开发。未折叠状态的微观pk（a）值（其中k（a）是酸解离常数）直接通过对非结构化寡核苷酸的实验来确定，这导致未折叠状态的总体的宏观pK（a）向中性移动。然后将形式主义应用于模型DNA寡核苷酸的pH依赖性UV熔解数据。折叠状态的pKa值与pH滴定的结果吻合良好，并且很好地描述了酸性和碱性变性区域。这里发展的形式类似于德雷珀和他的同事关于Mg 2+与RNA结合的形式，除了由于碱基上存在特异性质子结合位点而明确描述了未折叠状态。一个主要的结论是，通过设计RNA分子协同折叠，应该可以获得大的pK（a）位移。
Perturbation of pK(a) values can change the favored protonation stales of the nucleobases at biological pH and thereby modulate the function of RNA and DNA molecules. In an effort to understand the driving forces for pK(a) shifting specific to nucleic acids, we developed a thermodynamic framework that relates proton binding to the nucleobases and the helix-coil transition. Key features that emerge from the treatment are a comprehensive description of all the actions of proton binding on RNA folding: acid and alkaline denaturation of the helix and pK(a) shifting in the folded state. Practical experimental approaches for measuring pK(a)s from thermal denaturation experiments are developed. Microscopic pk(a) values (where k(a) is the acid dissociation constant) for the unfolded state were determined directly by experiments on unstructured oligonucleotides, which led to a macroscopic pK(a) for the ensemble of unfolded states shifted toward neutrality. The formalism was then applied to pH-dependent UV melting data for model DNA oligonucleotides. Folded-state pka values were in good agreement with the outcome of pH titrations, and the acid and alkaline denaturation regions were well described. The formalism developed here is similar to that of Draper and coworkers for Mg2+ binding to RNA, except that the unfolded state is described explicitly owing to the presence of specific proton-binding sites on the bases. A principal conclusion is that it should be possible to attain large pK(a) shifts by designing RNA molecules that fold cooperatively.