Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University, Bonn, Germany.
Methods Mol Biol. 2022;2439:91-101. doi: 10.1007/978-1-0716-2047-2_7.
During the last decades, various functional oligonucleotides have been discovered including DNAzymes, ribozymes, and riboswitches. Their function is based on their ability to form and change their three-dimensional structure. Binding of divalent ions to specific binding pockets was found to be important for the global structure and function. Here, we present a protocol that allows counting the number of Mn-binding sites and to determine their dissociation constants by means of continuous wave X-band Electron Paramagnetic Resonance (EPR) spectroscopy. In this method, Mn is titrated into the oligonucleotide-containing sample and the intensity of the EPR spectrum is recorded. By comparison with a Mn-only reference sample, the binding isotherm can be constructed and fitted to binding models yielding the number and affinities of the binding sites. This method has been successfully applied to several functional oligonucleotides.
在过去的几十年中，已经发现了各种功能寡核苷酸，包括 DNA 酶、核酶和核糖开关。它们的功能基于它们形成和改变三维结构的能力。研究发现，二价离子与特定结合口袋的结合对于全局结构和功能很重要。在这里，我们提出了一种通过连续波 X 波段电子顺磁共振（EPR）光谱来计数 Mn 结合位点数量并确定其离解常数的方案。在该方法中，将 Mn 滴定到含寡核苷酸的样品中，并记录 EPR 光谱的强度。通过与仅含 Mn 的参比样品进行比较，可以构建结合等温线，并拟合到结合模型中，得到结合位点的数量和亲和力。该方法已成功应用于几种功能寡核苷酸。
