An Oligo-Library-Based Approach for Mapping DNA–DNA Triplex Interactions In Vitro,ACS Synthetic Biology

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An Oligo-Library-Based Approach for Mapping DNA–DNA Triplex Interactions In Vitro
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-08-10 , DOI: 10.1021/acssynbio.1c00122
Beate Kaufmann ₁ , Or Willinger ₁ , Nanami Kikuchi ₁ , Noa Navon ₁ , Lisa Kermas ₁ , Sarah Goldberg ₁ , Roee Amit _{1,

2}

Affiliation

We present Triplex-seq, a deep-sequencing method that systematically maps the interaction space between an oligo library of ssDNA triplex-forming oligos (TFOs) and a particular dsDNA triplex target site (TTS). We demonstrate the method using a randomized oligo library comprising 67 million variants, with five TTSs that differ in guanine (G) content, at two different buffer conditions, denoted pH 5 and pH 7. Our results show that G-rich triplexes form at both pH 5 and pH 7, with the pH 5 set being more stable, indicating that there is a subset of TFOs that form triplexes only at pH 5. In addition, using information analysis, we identify triplex-forming motifs (TFMs), which correspond to minimal functional TFO sequences. We demonstrate, in single-variant verification experiments, that TFOs with these TFMs indeed form a triplex with G-rich TTSs, and that a single mutation in the TFM motif can alleviate binding. Our results show that deep-sequencing platforms can substantially expand our understanding of triplex binding rules and aid in refining the DNA triplex code.

中文翻译：

一种基于寡核苷酸库的方法，用于在体外绘制 DNA-DNA 三重相互作用图谱

我们提出了 Triplex-seq，这是一种深度测序方法，可系统地映射 ssDNA 三链体形成寡核苷酸 (TFO) 的寡核苷酸库与特定 dsDNA 三链体靶位点 (TTS) 之间的相互作用空间。我们使用包含 6700 万个变体的随机寡核苷酸库演示了该方法，其中 5 个 TTS 的鸟嘌呤 (G) 含量不同，在两种不同的缓冲条件下，表示为 pH 5 和 pH 7。我们的结果表明，富含 G 的三链体在这两种情况下都形成pH 5 和 pH 7，其中 pH 5 设置更稳定，表明有一部分 TFO 仅在 pH 5 时形成三链体。此外，通过信息分析，我们确定了三链体形成基序 (TFM)，它们对应到最小的功能性 TFO 序列。我们在单变体验证实验中证明，具有这些 TFM 的 TFO 确实与富含 G 的 TTS 形成了三联体，并且 TFM 基序中的单个突变可以减轻结合。我们的结果表明，深度测序平台可以大大扩展我们对三链体结合规则的理解，并有助于完善 DNA 三链体代码。

更新日期：2021-08-20

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