Enzymatic synthesis of multiple spin-labeled DNA
Enzymatic synthesis of multiple spin-labeled DNA
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DOI：
10.1002/anie.200802314
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Marx, Andreas
中科院分区：
文献类型：
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作者：
Obeid, Samra;Yulikov, Maxim;Marx, Andreas
电子顺磁共振(EPR)谱是一种广泛应用于研究生物大分子的组织和动力学性质的技术。其中许多应用依赖于氮氧化物标记[1]对皮秒到微秒时间尺度上的动力学的敏感性，以及在纳米尺度上测量此类标记之间距离的能力。[2]这些技术适用于无序系统，比核磁共振测量更灵敏，并且比基于光激发的技术提供更详细的信息。大多数生物大分子在其天然状态下是抗磁性的，因此没有背景EPR信号。因此，自旋标记技术可以选择性地解决大分子和复杂组件中的感兴趣位置。[3]通过刚性连接体连接是必要的，以最小化自旋标记运动对光谱的影响，同时最大化生物大分子的主干动力学。[4]这种刚性连接体也有利于距离测量，因为它们导致较窄的距离分布，从而在将标记之间的距离转换为结构模型时，不确定性较小。然而，使用刚性连接物会产生干扰天然结构的风险，因为标签不能适应其环境的空间要求。为此，必须非常谨慎地设计和测试标记策略。[5]最近，EPR波谱被广泛应用于研究核酸的结构和动力学。[1，6]由于核酸不包含任何天然的顺磁中心，因此在进行EPR研究之前必须引入自旋标记。以前已经建立了几种方法来在DNA中的特定位置引入顺磁中心，例如稳定的氮氧化物。这样的自旋标记是通过在DNA自动合成过程中使用自旋标记的构建块来引入的[7]，或者首先将功能化的构建块引入生长中的DNA中，然后例如通过钯催化的偶联反应将其偶联到固体载体上的自旋标记上。然而，DNA自动合成的固有局限性限制了寡核苷酸的长度和修饰程度。就我们所知，使用这些方法将多个特定位置的自旋标记结合到DNA中还没有得到证实。其他方法是基于在核酸中加入额外的功能，这些功能是
Electron paramagnetic resonance (EPR) spectroscopy is a widespread technique for the study of the organizational and dynamic properties of biological macromolecules. Many of these applications depend on the sensitivity of nitroxide labels [1] to dynamics on the picosecond to microsecond time scales and on the ability to measure distances between such labels on the nanometer length scale.[2] These techniques are applicable in disordered systems, more sensitive than NMR measurements, and provide more detailed information than techniques based on optical excitation. Most biomacromolecules are diamagnetic in their native states and thus do not have background EPR signals. Thus, spin-labeling techniques can selectively address sites of interest in large molecules and complex assemblies.[3] Attachment through a rigid linker is necessary to minimize the effect of the motion of the spin label on the spectrum and at the same time to maximize the backbone dynamics of the biomacromolecule.[4] Such rigid linkers are also favorable for distance measurements as they lead to narrower distance distributions and thus to smaller uncertainties in translating distances between labels to structural models. However, the use of rigid linkers generates the risk of perturbating the native structure because the label cannot adapt to the steric requirements of its environment. For this reason, labeling strategies have to be designed and tested with great care.[5]Recently, EPR spectroscopy was applied extensively in studies on the structures and dynamics of nucleic acids.[1, 6] Since nucleic acids do not contain any natural paramagnetic centers, spin labels have to be introduced prior to EPR investigations. Several methods have previously been established for the introduction of a paramagnetic center, for example, a stable nitroxide, at a specific site in DNA. Such spin labels were introduced either by employment of a spinlabeled building block during automatic DNA synthesis,[7] or functionalized building blocks were introduced into the growing DNA first and subsequently coupled to a spin label on a solid support, for example, by employment of palladiumcatalyzed coupling reactions.[8] Single-labeled oligonucleotides with relatively short lengths have been synthesized by these techniques. However, the length of the oligonucleotides and their degree of modification is restricted by the inherent limitations of automatic DNA synthesis. To the best of our knowledge, multiple site-specific incorporation of spin labels into DNA by using these methods has not as yet been demonstrated. Other approaches are based on the incorporation of additional functionalities in nucleic acids that are