Small molecules can inhibit cellular processes such as replication and transcription by binding to the promoter regions that are prone to form G-quadruplexes. However, since G-quadruplexes exist throughout the human genome, the G-quadruplex binders suffer from specificity issues. To tackle this problem, a G-quadruplex binder (Pyridostatin, or PDS) is conjugated with a ligand (Polyamide, or PA) that can specifically recognize DNA sequences flanking the G-quadruplex forming region. The binding mechanism of this hybrid ligand to the hTERT promoter region (hTERT 5–12) is then elucidated using optical tweezers. During mechanical unfolding processes, different intermediate structures of hTERT 5–12 in presence of PDS, PA, or PA-PDS conjugate are observed. These intermediate structures are consistent with two folding patterns of G-quadruplexes in the hTERT 5–12 fragment. While the duplex DNA binder PA facilitates the folding of a hairpin-G-quadruplex structure, the PDS assists the formation of two tandem G-quadruplexes. Both replication stop assay in vitro and dual luciferase assay in vivo established the effectiveness of the PA-PDS conjugate for hTERT 5–12 targeting. We expect such a ligand dependent folding dynamics will provide guidelines to the development of drugs that not only target hTERT expressions, but also other oncogenes via interactions with specific G-quadruplex structures formed in their promotor regions.

中文翻译：

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配体对 hTERT 启动子区动态 DNA G-四链体结构的调节


小分子可以通过与容易形成 G-四链体的启动子区域结合来抑制细胞过程，例如复制和转录。然而，由于 G 四链体存在于整个人类基因组中，因此 G 四链体结合物存在特异性问题。为了解决这个问题，将 G-四链体结合物（嘧啶司他汀或 PDS）与配体（聚酰胺或 PA）偶联，该配体可以特异性识别 G-四链体形成区域两侧的 DNA 序列。然后使用光镊阐明该杂交配体与 hTERT 启动子区域 （hTERT 5-12） 的结合机制。在机械去折叠过程中，在 PDS、PA 或 PA-PDS 偶联物存在下观察到 hTERT 5-12 的不同中间结构。这些中间结构与 hTERT 5-12 片段中 G 四链体的两种折叠模式一致。双链 DNA 结合剂 PA 有助于发夹 G 四链体结构的折叠，而 PDS 则有助于形成两个串联的 G 四链体。体外复制终止测定和体内双荧光素酶测定都确定了 PA-PDS 偶联物对 hTERT 5-12 靶向的有效性。我们预计这种依赖于配体的折叠动力学将为药物开发提供指导，这些药物不仅靶向 hTERT 表达，而且通过与启动区形成的特定 G-四链体结构相互作用来靶向其他癌基因。