Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry: Detection of Abasic Sites in Duplex DNA by EPR Spectroscopy
Site-Directed Spin-Labeling of Nucleic Acids by Click Chemistry: Detection of Abasic Sites in Duplex DNA by EPR Spectroscopy
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DOI：
10.1021/ja102797k
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发表时间：
2010-08-04
影响因子：
15
通讯作者：
Sigurdsson, Snorri Th.
中科院分区：
文献类型：
--
作者：
Jakobsen, Ulla;Shelke, Sandip A.;Sigurdsson, Snorri Th.
本文描述了一种自旋标记，它可以检测和识别双链DNA中的局部结构变形，特别是碱性位点。通过一种新的后合成方法将自旋标记引入到DNA中，该方法利用固体载体上的点击化学，简化了自旋标记寡核苷酸的合成和纯化。用较短的合成路线合成了含氮氧官能团的叠氮化合物，并与含5-乙炔基-2‘-杜的齐聚物反应。偶联反应以定量的方式进行，并在修饰的核苷酸和氮氧自旋标记之间形成了相当坚硬的连接物。自旋标记首次用于通过X-波段CW-EPR光谱检测双链DNA中的基本位置，并提供关于DNA的其他结构变形以及局部构象变化的信息。例如，在与T不匹配的对中，自旋标记的迁移率降低与自旋标记从双链中取代T是一致的。将汞(II)添加到该错对中导致自旋标记的运动显著增加，这与T和自旋标记的碱基之间的金属气的形成一致，从而导致自旋标记移动出双链并朝向溶液。因此，当自旋标记作为汞(II)控制的机械杠杆时，可以很容易地通过EPR光谱检测到重新定位。新的自旋标记的易用性和性质使其在核酸和其他大分子的EPR研究中具有吸引力。
This paper describes a spin label that can detect and identify local structural deformations in duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic approach using click-chemistry on a solid support, which simplified both the synthesis and purification of the spin-labeled oligonucleotides. A nitroxide-functionalized azide, prepared by a short synthetic route, was reacted with an oligomer containing 5-ethynyl-2'-dU. The conjugation proceeded in quantitative yield and resulted in a fairly rigid linker between the modified nucleotide and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair with T was consistent with the spin label displacing the T from the duplex. Addition of mercury(II) to this mispair resulted in a substantial increase in the motion of the spin label, consistent with formation of a metallopair between the T and the spin-labeled base that results in movement of the spin label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids and other macromolecules.