A DNA Polymerase Stop Assay for Characterization of G-Quadruplex Formation and Identification of G-Quadruplex-Interactiv
Guanine-rich DNA sequences are able to spontaneously fold into G-quadruplex structures in the presence of certain metal cations. In the human genome, the majority of DNA G-quadruplexes form at the telomeres and regulatory regions of cancer-related genes.
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Introduction The formation of DNA G-quadruplexes (G4s) can influence critical nuclear processes, such as transcription [1–3], DNA replication [4], and DNA repair [5–7]. The G4 structures arise from DNA sequences containing consecutive runs of guanine, which are predominantly restricted in telemetric regions and regulatory regions of cancer-related genes in genomes [8]. Stabilization of G4 structures using G4-interactive ligands can disrupt the interaction between single-stranded or double-stranded DNA binding proteins and their target sequences, thereby inhibiting gene transcription and impairing cellular homeostasis [9, 10]. G-quadruplexes
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_12, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Guanhui Wu and Haiyong Han
can also induce replication fork stalls during DNA replication and eventually result in DNA double-strand breaks [6, 11]. Since they are more prevalent in cancer cells than in normal cells [3], G4 structures have gained significant attraction as new targets for cancer drug discovery. Biophysical techniques, such as Fo¨rster resonance energy transfer (FRET) and circular dichroism (CD) spectroscopy have been widely used to characterize G4 formation in particular short G-rich oligonucleotides, but their use is limited in the determination of G4-forming region in a long DNA sequence especially when a sequence consisting of more than four runs of contiguous guanines. In this chapter, we describe a biochemical method using DNA polymerases for investigating the G4 formation in a relatively long (70–80 nt) DNA strand [12]. DNA polymerases are enzymes that synthesize DNA strands during DNA replication. They move along the DNA template strand in the 30 –50 direction and create a new strand from a 50 to 30 direction. In the polymerase stop assay, the template DNA containing G4 forming region is annealed with radiolabeled primers. The primer-annealed DNA templates are then utilized by Taq DNA polymerase for primer extension. The presence of G4 structures in the template strand can cause DNA polymerase to stall immediately before these structures (Fig. 1a). Resolving these paused products on a denaturing PAGE gel can provide a simple and rapid way to identify DNA secondary structures in the template strand. This assay has also been widely used to test/identify potential G-quadruplex-interactive small molecules [12–14].
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Materials All solutions are prepared with ultrapure deionized water and stored at room temperature unless indicated otherwise.
2.1 Radioactive 50 -End Labeling with T4 Polynucleotide Kinase
1. Oligonucleotide primer: stock solution 20 μM (see Note 1). 2. T4 polynucleotide kinase (PNK) (10 units/μL, NEB, Ipswich, MA, USA). 3. T4 PNK kinase reaction buffer (10): 70 mM Tris–HCl (pH 7.6), 100 mM MgCl2, 50 mM DTT (NEB). 4. Adenosine 50 -gamma 32P triphosphate (10 BLU502A, PerkinElmer, Waltham
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