A G-quadruplex (G4) is the most frequently observed non-canonical structures of nucleic acids formed from G-rich sequences. Bioinformatic studies have identified 3.7 × 10⁵ G4-forming sequences in the human genome, corresponding to around 7.4 × 10⁵ nucleotides. However, the human genome contains 6 × 10⁹ nucleotides, the most of which fold into the canonical duplex. Thus, G4 ligands are required to have not only high affinity but also high selectivity for G4 for a practical use in cells.
Fast and reliable screening methods are required to develop G4-selective ligands. Until now, various screening systems have been developed to identify G4 ligands, such as G4-fluorescent intercalator displacement (G4-FID) and fluorescence-based thermal melting assay. These screening methods are powerful to develop G4 ligands which have high affinity with a G4. However, a screening method to obtain G4 ligands with both of the high affinity and the high selectivity simultaneously is not reported yet.
Here, we developed a novel screening system using Thioflavin T (ThT). We reported that ThT specifically bound to G4 and enhanced fluorescence (V. Gaberica, et al., Biochemistry 52, 5620 (2013)). ThT screening system is similar with G4-FID assay which utilizes Thiazole orange (TO) as a fluorescence indicator (D. Monchaud, et al., Bioorg. Med. Chem. Lett. 16, 4842 (2006)). However, unlike TO, ThT neither bind to nor emit fluorescence with DNA duplex. Therefore, ThT enables screen even in the presence of excess DNA duplex (Figure). This method does not require pre-modification and is expected to be easy and fast. In addition, this ThT screening system can determine the dissociation constants (K_d) of the selected ligands and the target G4 by measuring the decrement of ThT fluorescence upon the addition of the ligand. Series of molecules that can bind to G4 even in the presence of excess DNA duplex and their K_d will be shown in the presentation.

Schematic representation of ThT displacement assay in the presence of DNA duplex

Nagisa Takamiya

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