Single-stranded DNA (ssDNA) containing 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 sequence specificity. Here, we use custom DNA microarrays to examine the binding specificities of proteins, small molecules, and antibodies across ~15,000 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 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.
Overall design: Binding microarray experiments were performed for G-quadruplex (G4) DNA interacting proteins (nucleolin, IGF2, CNBP), helicases (BLM, FANCJ, DHX36, PIF1, WRN), the anti-G4 antibody BG4, and the small molecules pyridostatin (PDS), TmPyP4, and DC-34. Briefly, the binding experiments involved incubating GST-tagged proteins or Cy5-conjugated molecules (BG4, PDS) to 180K feature Agilent microarrays pre-incubated with 100mM potassium chloride (KCl) in order to determine their binding preferences to G4-forming sequences.
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