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A high-throughput platform to examine G-quadruplex DNA formation and recognition

Friday, September 14, 2018 — Poster Session V

12:00 p.m. – 1:30 p.m.
FAES Terrace
NCI
MOLBIO-2

Authors

  • NM Assad
  • D Tillo
  • S Ray
  • JS Schneekloth
  • C Vinson

Abstract

Nucleotide sequences of the form G3+N1–7G3+N1–7G3+N1–7G3+ (where N = A,C,G, or T) spontaneously form four-stranded helices known as G-quadruplexes (G4 DNA) in vitro in the presence of potassium that are thermodynamically more stable than double-stranded DNA. G4 DNA forming sequences have been identified in promoter regions of oncogenes and have been implicated in the regulation of transcription factors (TFs) such as c-MYC. As such, the identification of small molecules which stabilize G4 structures or inhibit their formation are of great therapeutic interest. Here, we describe the design and use of a custom microarray (“the G4 array”) to examine G4 DNA formation and recognition. Probes on the array contain DNA sequences from oncogene promoters predicted to form G4 structures, and variants of these sequences in which the location of the G4 sequence within the probe, the linker (N1–7) length, and linker nucleotide sequences are systematically varied. Our initial experiments confirm G4 formation using a fluorophore-tagged antibody (Bg4) specific to G4 DNA. We also examine nucleolin, a TF thought to bind G4 DNA. Comparison of the Bg4 and nucleolin specificity on the G4 array indicate differences in binding specificity between the two proteins. Our microarray approach provides a high-throughput platform to screen small molecule and protein binding specificity to G4 DNA.

Category: Molecular Biology and Biochemistry