Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA.
Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA.
复制标题
硫代磷酸酯 DNA 的快速、廉价、序列独立的荧光标记。
DOI：
10.1016/j.bpj.2023.02.011
复制
发表时间：
2023
影响因子：
3.4
通讯作者：
Erie,DorothyA
中科院分区：
文献类型：
--
作者：
Satusky,MatthewJ;Johnson,CaitlinV;Erie,DorothyA
荧光标记的寡核苷酸是表征DNA过程的有力工具；然而，它们的使用受到当前标记技术的成本和序列要求的限制。在这里，我们开发了一种简单，廉价，序列无关的方法，用于位点特异性标记DNA寡核苷酸。我们利用商业合成的含有硫代二酯的寡核苷酸，其中非桥接氧被硫（PS-DNA）取代。硫代磷酰硫相对于磷酰氧的亲核性增强，使得硫代磷酰硫与碘乙酰胺化合物具有选择性反应活性。因此，我们利用一种存在已久的双功能连接剂N，N ' -双（α-碘乙酰基）-2-2 ' -双硫代（乙胺）（BIDBE），它与ps - dna反应，留下一个自由的硫醇，允许缀合各种商业马来酰亚胺功能化化合物。我们优化了BIDBE的合成及其与PS-DNA的附着，然后使用标记半胱氨酸的标准方案对BIDBE-PS-DNA进行荧光标记。我们纯化了单个外显体，并使用单分子Förster共振能量转移（FRET），我们发现FRET效率与外显体附着无关。随后，我们证明了双标记Holliday结（HJs）的外聚体混合物可以用来表征它们在没有和存在结构特异性内切酶edrosophila melanogasterGen的情况下的构象性质。最后，我们使用生化活性测定来证明这种双标记的HJ对Gen的切割有功能，并且双标记的HJ允许在一次实验中识别多个DNA物种。总之，我们的研究结果表明，染料标记的bidbe - ps - dna与商业标记的dna相当，且成本显著降低。值得注意的是，该技术可以应用于其他马来酰亚胺功能化化合物，如自旋标记，生物素和蛋白质。标记的序列独立性，加上其易用性和低成本，可以无限制地探索染料的放置和选择，为创建差异标记的DNA文库提供了潜力，并开辟了以前无法进入的实验途径。
Fluorescently labeled oligonucleotides are powerful tools for characterizing DNA processes; however, their use is limited by the cost and sequence requirements of current labeling technologies. Here, we develop an easy, inexpensive, and sequence-independent method for site-specifically labeling DNA oligonucleotides. We utilize commercially synthesized oligonucleotides containing phosphorothioate diester(s) in which a nonbridging oxygen is replaced with a sulfur (PS-DNA). The increased nucleophilicity of the thiophosphoryl sulfur relative to the phosphoryl oxygen permits selective reactivity with iodoacetamide compounds. As such, we leverage a long-existing bifunctional linker,N,N′-bis(α-iodoacetyl)-2-2′-dithiobis(ethylamine) (BIDBE), that reacts with PS-DNAs to leave a free thiol, allowing conjugation of the wide variety of commercial maleimide-functionalized compounds. We optimized BIDBE synthesis and its attachment to PS-DNA and then fluorescently labeled the BIDBE-PS-DNA using standard protocols for labeling cysteines. We purified the individual epimers, and using single-molecule Förster resonance energy transfer (FRET), we show that the FRET efficiency is independent of the epimeric attachment. Subsequently, we demonstrate that an epimeric mixture of double-labeled Holliday junctions (HJs) can be used to characterize their conformational properties in the absence and presence of the structure-specific endonucleaseDrosophila melanogasterGen. Finally, we use a biochemical activity assay to show that this double-labeled HJ is functional for cleavage by Gen and that the double-labeled HJ allows multiple DNA species to be identified in a single experiment. In conclusion, our results indicate that dye-labeled BIDBE-PS-DNAs are comparable to commercially labeled DNAs at a significantly reduced cost. Notably, this technology could be applied to other maleimide-functionalized compounds, such as spin labels, biotin, and proteins. The sequence independence of labeling, coupled with its ease and low cost, enables unrestricted exploration of dye placement and choice, providing the potential for creation of differentially labeled DNA libraries and opening previously inaccessible experimental avenues.