Click-click-click: Single to triple modification of DNA
Click-click-click: Single to triple modification of DNA
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DOI：
10.1002/anie.200705664
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
Carell, Thomas
中科院分区：
文献类型：
--
作者：
Gramlich, Philipp M. E.;Warncke, Simon;Carell, Thomas
将荧光染料或生物素分子等标记附着到DNA上对于基于DNA的分子诊断和纳米技术应用至关重要。[2,3]对这种修饰寡核苷酸的需求量很大，但标记程序背后的化学过程繁琐，而且修饰寡核苷酸的产率往往很低。目前，在固相合成寡核苷酸的过程中，这些标签被掺入作为相应的磷酰胺[4]，这往往会显著降低偶联收率。这种方法仅限于能够承受DNA合成和去保护的恶劣条件的标签。或者，通过例如相应的活化酯与氨基烷基修饰的寡核苷酸的反应，在合成后引入标签这种方法的偶联率低，使得标记寡核苷酸的纯化成为一项具有挑战性的任务。在对标记寡核苷酸的需求迅速增长的世界中，需要有效结合多种不同标记的新方法。Seela和Sirivolu[7]以及我们的团队[2,8]最近发现，由Meldal等人（[9]）和Sharpless等人（[10]）开发的铜(I)催化的叠氮化物-炔反应生成三唑，可以非常高效地功能化炔修饰的DNA核碱基。一个临界点是存在足够数量的适当的铜(I)络合配体，以防止铜催化的DNA裂解在这里，我们报告，这种化学可以扩展到标记寡核苷酸与多达三个（可能更多）不同的标签。这些功能化可以直接在树脂[13]上实现，也可以在寡核苷酸脱保护后的溶液中实现。后一种方法可用于合并极其敏感的标签与前所未有的效率。
The attachment of labels such as fluorescent dyes or biotin molecules to DNA is of paramount importance for DNA-based molecular diagnostics [1] and for nanotechnological applications.[2, 3] There is high demand for such modified oligonucleotides, but the chemistry behind the labeling procedures is cumbersome, and the modified oligonucleotides are frequently obtained in only low yields. Presently, the labels are incorporated as the corresponding phosphoramidites [4] during the solid-phase synthesis of oligonucleotides, which frequently reduces the coupling yield significantly. This method is restricted to labels that can withstand the harsh conditions of DNA synthesis and deprotection. Alternatively, the labels are introduced postsynthetically [5] by, for example, reaction of the corresponding activated esters with aminoalkyl-modified oligonucleotides.[6] This method suffers from inefficient coupling yields, making the purification of the labeled oligonucleotides a challenging task. In a world in which the demand for labeled oligonucleotides is rapidly growing, new methods for the efficient incorporation of multiple different labels are required. Seela and Sirivolu [7] and our group [2, 8] have recently discovered that the copper (I)-catalyzed version of the azide–alkyne reaction to give triazoles, developed by Meldal et al.[9] and Sharpless et al.[10] can be used to functionalize alkyne-modified DNA nucleobases with extremely high efficiency. A critical point is the presence of a sufficient amount of a proper copper (I)-complexing ligand [11] to prevent the copper-catalyzed cleavage of DNA.[12] Herein we report that this chemistry can be extended to label oligonucleotides with up to three (and possibly more) different labels. These functionalizations can be achieved either directly on the resin [13] or in solution after deprotection of the oligonucleotide. The latter method can be used to incorporate extremely sensitive labels with unprecedented efficiency.