Grytz C M, Marko A, Cekan P, Sigurdsson S Th, Prisner T F
Institute of Physical and Theoretical Chemistry and Center of Biomolecular Magnetic Resonance, Goethe University, 60438 Frankfurt am Main, Max-von-Laue-Str. 9, Hessen, Germany.
University of Iceland, Department of Chemistry, Science Institute, Dunhaga 3, 107 Reykjavik, Iceland.
Phys Chem Chem Phys. 2016 Jan 28;18(4):2993-3002. doi: 10.1039/c5cp06158j.
The cocaine aptamer is a DNA three-way junction that binds cocaine at its helical junction. We studied the global conformation and overall flexibility of the aptamer in the absence and presence of cocaine by pulsed electron-electron double resonance (PELDOR) spectroscopy, also called double electron-electron resonance (DEER). The rigid nitroxide spin label Ç was incorporated pairwise into two helices of the aptamer. Multi-frequency 2D PELDOR experiments allow the determination of the mutual orientation and the distances between two Çs. Since Ç is rigidly attached to double-stranded DNA, it directly reports on the aptamer dynamics. The cocaine-bound and the non-bound states could be differentiated by their conformational flexibility, which decreases upon binding to cocaine. We observed a small change in the width and mean value of the distance distribution between the two spin labels upon cocaine binding. Further structural insights were obtained by investigating the relative orientation between the two spin-labeled stems of the aptamer. We determined the bend angle between this two stems. By combining the orientation information with a priori knowledge about the secondary structure of the aptamer, we obtained a molecular model describing the global folding and flexibility of the cocaine aptamer.
可卡因适体是一种在其螺旋连接处结合可卡因的DNA三向接头。我们通过脉冲电子-电子双共振（PELDOR）光谱法（也称为双电子-电子共振（DEER））研究了在不存在和存在可卡因的情况下适体的整体构象和整体柔韧性。将刚性氮氧化物自旋标记物Ç成对地掺入适体的两个螺旋中。多频二维PELDOR实验可以确定两个Ç之间的相互取向和距离。由于Ç刚性连接到双链DNA上，它直接反映了适体的动力学。可卡因结合态和非结合态可以通过它们的构象柔韧性来区分，与可卡因结合后构象柔韧性会降低。我们观察到可卡因结合后两个自旋标记物之间距离分布的宽度和平均值有微小变化。通过研究适体的两个自旋标记茎之间的相对取向，获得了进一步的结构见解。我们确定了这两个茎之间的弯曲角度。通过将取向信息与关于适体二级结构的先验知识相结合，我们获得了一个描述可卡因适体整体折叠和柔韧性的分子模型。
