Custom DNA Microarrays Reveal Diverse Binding Preferences of Proteins and Small Molecules to Thousands of G-Quadruplexes

Sreejana Ray, Desiree Tillo, Robert E. Boer, Nima Assad, Mira Barshai, Guanhui Wu, Yaron Orenstein, Danzhou Yang, John S. Schneekloth, Charles Vinson

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)925-935
Number of pages11
JournalACS Chemical Biology
Volume15
Issue number4
DOIs
StatePublished - Apr 17 2020
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

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