Arndt Stefanie, Walter Heidi-Kristin, Wagenknecht Hans-Achim
Institute of Organic Chemistry, Karlsruhe Institute of Technology.
Institute of Organic Chemistry, Karlsruhe Institute of Technology;
J Vis Exp. 2016 Jul 6(113):54121. doi: 10.3791/54121.
In this protocol, we demonstrate a method for the synthesis of 2'-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.
在本方案中，我们展示了一种通过使用标准亚磷酰胺化学的自动化固相合成方法来合成2'-炔基修饰的脱氧核糖核酸（DNA）链的方法。寡核苷酸在合成后通过铜催化的点击化学用两种新型光稳定的花菁染料进行标记。供体染料和受体染料的合成均有描述，并分三个连续步骤进行。以DNA作为周围结构，当这两种染料通过杂交紧密靠近时会发生能量转移。因此，两条单链DNA链的退火通过荧光颜色的变化得以可视化。这种颜色变化通过荧光光谱进行表征，但也可以使用手持式紫外线（UV）灯直接观察到。双荧光颜色读数的概念使这些寡核苷酸探针成为分子成像的优秀工具，尤其是当使用所描述的光稳定染料时。由此，成像探针的光漂白得以防止，并且可以在更长的时间段内实时观察生物过程。
