A PELDOR-based nanometer distance ruler for oligonucleotides
A PELDOR-based nanometer distance ruler for oligonucleotides
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DOI：
10.1021/ja0393877
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发表时间：
2004-05-12
影响因子：
15
通讯作者：
Prisner, TF
中科院分区：
文献类型：
--
作者：
Schiemann, O;Piton, N;Prisner, TF
用一系列双链DNA研制了一种寡核苷酸脉冲电子顺磁共振（EPR）光谱尺。自旋标记在寡核苷酸的固相合成期间利用5-碘-2 '-脱氧尿苷和刚性自旋标记2，2，5，5-四甲基-吡咯啉-1-氧基-3-乙炔（TPA）之间的钯催化的交叉偶联反应完成。4-然后使用脉冲电子双共振（PELDOR）通过偶极耦合来测量分子内自旋-自旋距离，得到19.2、23.3、34.7、44.8和52.5埃的自旋-自旋距离。采用一个全原子力场与明确的水，分子动力学（MD）模拟相同的自旋标记的寡核苷酸在其双链体B-形式的自旋-自旋距离分别为19.6，21.4，33.0，43.3，和52.5埃，在非常好的协议与测量的距离。这表明寡核苷酸在冷冻缓冲水溶液中也采用B型双链体结构。它还表明，结合使用定点自旋标记，PELDOR实验，和MD模拟可以产生一个关于寡核苷酸的整体结构的微观图片。该技术也适用于更复杂的系统，如核酶或DNA/RNA-蛋白质复合物，这些系统很难通过NMR或X射线晶体学获得。
A pulsed electron paramagnetic resonance (EPR) spectroscopic ruler for oligonucleotides was developed using a series of duplex DNAs. The spin-labeling is accomplished during solid-phase synthesis of the oligonucleotides utilizing a palladium-catalyzed cross-coupling reaction between 5-iodo-2'-deoxyuridine and the rigid spin-label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA). 4-Pulse electron double resonance (PELDOR) was then used to measure the intramolecular spin-spin distances via the dipolar coupling, yielding spin-spin distances of 19.2, 23.3, 34.7, 44.8, and 52.5 Angstrom. Employing a full-atom force field with explicit water, molecular dynamic (MD) simulations on the same spin-labeled oligonucleotides in their duplex B-form gave spin-spin distances of 19.6, 21.4, 33.0, 43.3, and 52.5 Angstrom, respectively, in very good agreement with the measured distances. This shows that the oligonucleotides adopt a B-form duplex structure also in frozen aqueous buffer solution. It also demonstrates that the combined use of site-directed spin-labeling, PELDOR experiments, and MD simulations can yield a microscopic picture about the overall structure of oligonucleotides. The technique is also applicable to more complex systems, like ribozymes or DNA/RNA-protein complexes, which are difficult to access by NMR or X-ray crystallography.