Yuzhe Ding, Ziyu Zhang, Yunus A Kaiyum, Yicheng Heng, Philip E Johnson, Juewen Liu
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引用次数: 0
摘要
在典型的适配体选择实验中,缓冲液分子的使用浓度在 10 至 50 mM 之间,而目标分子的使用浓度要低得多,甚至在低μM 水平。因此,人们对缓冲液结合适配体的潜在富集能力存在疑虑,尤其是对于无法验证富集序列结合能力的失败选择。在本研究中,我们使用了两种常见的缓冲液分子(Tris 和 HEPES)作为目标分子。我们成功地分离出了与 Tris 缓冲液结合的适配体,但生成与 HEPES 缓冲液结合的适配体的尝试却失败了。硫黄素 T(ThT)荧光光谱显示特里斯缓冲液适配体的解离常数(Kd)为 2.9 mM,而等温滴定量热法显示 Kd 为 43 μM。核磁共振光谱也证实了合体的结合。最后,我们讨论了这项缓冲液选择工作的意义,并建议使用某些缓冲液。
DNA aptamers for common buffer molecules: possibility of buffer interference in SELEX.
During a typical aptamer selection experiment, buffer molecules are used at the 10 to 50 mM range, whereas target molecules could be used at much lower concentrations even in low μM levels. Therefore, doubts existed regarding the potential enrichment of buffer binding aptamers, particularly for failed selections that cannot validate binding of enriched sequences. In this study, we used two common buffer molecules, Tris and HEPES, as target molecules. While we successfully isolated aptamers for Tris buffer, our attempts to generate aptamers for HEPES buffer failed. Thioflavin T (ThT) fluorescence spectroscopy showed the dissociation constant (Kd) of the Tris buffer aptamer to be 2.9 mM, while isothermal titration calorimetry showed a Kd of 43 μM. NMR spectroscopy also confirmed aptamer binding. Finally, we discussed the implications of this buffer selection work and recommended the use of certain buffers.