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Custom DNA Microarrays Reveal Diverse Binding Preferences of Proteins and Small Molecules to Thousands of G-Quadruplexes.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2020-04-07 , DOI: 10.1021/acschembio.9b00934
Sreejana Ray 1 , Desiree Tillo 1 , Robert E Boer 2 , Nima Assad 1 , Mira Barshai 3 , Guanhui Wu 4 , Yaron Orenstein 3 , Danzhou Yang 4 , John S Schneekloth 2 , Charles Vinson 1
Affiliation  

Single-stranded DNA (ssDNA) containing four guanine repeats can form G-quadruplex (G4) structures. While cellular proteins and small molecules can bind G4s, it has been difficult to broadly assess their DNA-binding specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ∼15,000 potential G4 structures. Molecules used include fluorescently labeled pyridostatin (Cy5-PDS, a small molecule), BG4 (Cy5-BG4, a G4-specific antibody), and eight proteins (GST-tagged nucleolin, IGF2, CNBP, FANCJ, PIF1, BLM, DHX36, and WRN). Cy5-PDS and Cy5-BG4 selectively bind sequences known to form G4s, confirming their formation on the microarrays. Cy5-PDS binding decreased when G4 formation was inhibited using lithium or when ssDNA features on the microarray were made double-stranded. Similar conditions inhibited the binding of all other molecules except for CNBP and PIF1. We report that proteins have different G4-binding preferences suggesting unique cellular functions. Finally, competition experiments are used to assess the binding specificity of an unlabeled small molecule, revealing the structural features in the G4 required to achieve selectivity. These data demonstrate that the microarray platform can be used to assess the binding preferences of molecules to G4s on a broad scale, helping to understand the properties that govern molecular recognition.

中文翻译:

定制 DNA 微阵列揭示了蛋白质和小分子与数千个 G-四链体的不同结合偏好。

包含四个鸟嘌呤重复的单链 DNA (ssDNA) 可以形成 G-四链体 (G4) 结构。虽然细胞蛋白和小分子可以结合 G4,但很难广泛评估它们的 DNA 结合特异性。在这里,我们使用定制的 DNA 微阵列来检查蛋白质、小分子和抗体在约 15,000 个潜在 G4 结构中的结合特异性。使用的分子包括荧光标记的吡咯他汀(Cy5-PDS,一种小分子)、BG4(Cy5-BG4,一种 G4 特异性抗体)和八种蛋白质(GST 标记的核仁素、IGF2、CNBP、FANCJ、PIF1、BLM、DHX36、和警告)。Cy5-PDS 和 Cy5-BG4 选择性地结合已知形成 G4 的序列,确认它们在微阵列上的形成。当使用锂抑制 G4 形成或当微阵列上的 ssDNA 特征被制成双链时,Cy5-PDS 结合减少。类似的条件抑制了除 CNBP 和 PIF1 之外的所有其他分子的结合。我们报告说蛋白质具有不同的 G4 结合偏好,表明具有独特的细胞功能。最后,竞争实验用于评估未标记小分子的结合特异性,揭示实现选择性所需的 G4 结构特征。这些数据表明,微阵列平台可用于广泛评估分子与 G4 的结合偏好,有助于了解控制分子识别的特性。竞争实验用于评估未标记小分子的结合特异性,揭示实现选择性所需的 G4 结构特征。这些数据表明,微阵列平台可用于广泛评估分子与 G4 的结合偏好,有助于了解控制分子识别的特性。竞争实验用于评估未标记小分子的结合特异性,揭示实现选择性所需的 G4 结构特征。这些数据表明,微阵列平台可用于广泛评估分子与 G4 的结合偏好,有助于了解控制分子识别的特性。
更新日期:2020-04-23
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