Abstract：
The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5\' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5\' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.
摘要：
小分子表小檗碱（EPI）是一种天然生物碱，具有多种生物活性，可抵抗多种疾病，包括癌症和细菌感染。EPI可以诱导在具有四个端粒重复序列（Q4）的人端粒G-四链体（HTG）序列的5'侧形成独特的结合口袋，产生纳摩尔结合亲和力(KD约26nM),在结合时具有显著的荧光增强。重要的是要理解(1)EPI结合如何影响HTG结构稳定性和(2)如何通过DNA结合袋的工程化来实现增强的EPI结合。在这项工作中,EPI结合诱导的HTG结构稳定效应通过肽核酸(PNA)侵袭试验结合一系列生物物理技术进行检测.我们表明，基于PNA侵入的方法可用于表征在生理条件下与DNA（和RNA）结构结合的化合物，而无需改变溶液温度或缓冲液成分。这通常是结构稳定性表征所需的。重要的是,理论建模和实验量化的结合使我们能够通过在5'末端简单地将双链结构扩展到Q4来成功地设计Q4衍生物Q4-ds-A。Q4-ds-A是一种出色的EPI粘合剂，KD为8nM，通过结合袋的形成和降低的解离速率实现结合增强。Q4和Q4-ds-A与EPI的紧密结合使我们能够开发一种新型的基于磁珠的亲和纯化系统，以有效地从黄连（黄连）提取物中提取EPI。
