Cadoni Enrico, Moerman Hanne, Madder Annemieke
Organic and Biomimetic Chemistry Research Group, Department of Organic and Macromolecular Chemistry, Ghent University Krijgslaan 281-S4 9000 Gent Belgium
RSC Chem Biol. 2024 Nov 28;6(1):56-64. doi: 10.1039/d4cb00185k. eCollection 2025 Jan 2.
In this study, we developed a simple pull-down assay using peptide nucleic acids (PNAs) equipped with a His-Tag and a G-quadruplex (G4) ligand for the selective recognition and quantification of G4-forming DNA sequences. Efficient and specific target recovery was achieved using optimized buffer conditions and magnetic Ni-NTA beads, while quantification was realized by employing the enzyme-like properties of the G4/hemin complex. The assay was validated through HPLC analysis and adapted for a 96-well plate format. The results show that higher recovery can be achieved using His-Tag with Ni-NTA magnetic beads as compared to the more common biotin-streptavidin purification. The inclusion of the G4-ligand as an additional selectivity handle was shown to be beneficial for both recovery and selectivity.
在本研究中，我们开发了一种简单的下拉分析法，该方法使用配备有His标签和G-四链体（G4）配体的肽核酸（PNA），用于选择性识别和定量形成G4的DNA序列。使用优化的缓冲条件和磁性镍-次氮基三乙酸（Ni-NTA）磁珠实现了高效且特异的靶标回收，同时通过利用G4/血红素复合物的类酶特性实现了定量分析。该分析方法通过高效液相色谱（HPLC）分析进行了验证，并适用于96孔板形式。结果表明，与更常用的生物素-链霉亲和素纯化方法相比，使用带有Ni-NTA磁珠的His标签可实现更高的回收率。作为额外的选择性手段加入G4配体被证明对回收率和选择性均有益处。
