Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States of America.
Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States of America; Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30303, United States of America.
Biophys Chem. 2019 Aug;251:106177. doi: 10.1016/j.bpc.2019.106177. Epub 2019 Apr 30.
The ends of nucleic acids oligomers alter the statistics of interior nonspecific ligand binding and act as binding sites with altered properties. While the former aspect of "end effects" has received much theoretical attention in the literature, the physical nature of end-binding, and hence its potential impact on a wide range of studies with oligomers, remains poorly known. Here, we report for the first time end-binding to DNA using a model helix-turn-helix motif, the DNA-binding domain of ETV6, as a function of DNA sequence length. Spectral analysis of ETV6 intrinsic tryptophan fluorescence by singular value decomposition showed that end-binding to nonspecific fragments was negligible at >0.2 kbp and accumulated to 8% of total binding to 23-bp oligomers. The affinity for end-binding was insensitive to salt but tracked the affinity of interior binding, suggesting translocation from interior sites rather than free solution as its mechanism. As the presence of a cognate site in the 23-bp oligomer suppressed end-binding, neglect of end-binding to the short cognate DNA does not introduce significant error. However, the same applies to nonspecific DNA only if longer fragments (>0.2 kbp) are used.
寡核苷酸的末端改变了内部非特异性配体结合的统计特性，并充当具有改变性质的结合位点。虽然“末端效应”的前一方面在文献中受到了很多理论关注，但末端结合的物理性质，以及它对广泛的寡核苷酸研究的潜在影响，仍然知之甚少。在这里，我们首次报道了使用模型螺旋-转角-螺旋基序（即 ETV6 的 DNA 结合结构域）作为 DNA 序列长度的函数，研究 DNA 上的末端结合。通过奇异值分解对 ETV6 固有色氨酸荧光的光谱分析表明，>0.2 kbp 时非特异性片段的末端结合可以忽略不计，而 23 个碱基对寡聚物的总结合中有 8%是末端结合。末端结合的亲和力对盐不敏感，但与内部结合的亲和力相关，表明其机制是从内部位点而不是游离溶液中进行易位。由于 23 个碱基对寡聚物中存在同源结合位点会抑制末端结合，如果忽略对短同源 DNA 的末端结合，则不会引入显著误差。然而，只有当使用更长的片段（>0.2 kbp）时，这种情况才适用于非特异性 DNA。
