A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures.

Marco Deiana,Nasim Sabouri,Ikenna Obi,Shanmugam Tamilselvi,Erik Chorell,Karam Chand,Måns Andreasson

doi:10.1021/acschembio.1c00134

Abstract

G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.

Highlights

The compounds were synthesized in a single step through Knoevenagel condensation of commercially available substituted ortho-hydroxyl benzaldehydes (1a-c) with ethyl cyanoacetate in the presence of ammonium acetate via in situ formation of 3-cyanocoumarin, which on subsequent reduction led to the formation of the desired 3-amidinocoumarin derivatives (2a-c) (Scheme 1)
We first determined the spectroscopic properties of the coumarin derivatives 2a−2c and performed solvent-dependent UV/vis absorption and emission measurements in order to determine how different solvent polarities affect the optical properties of the compounds
We focused our attention on a small water-soluble fluorescent light-up probe capable of targeting parallel G4 structures over antiparallel and non-G4 structures

Summary

■ INTRODUCTION

G-quadruplexes (G4s) are four-stranded non-B DNA helical structures formed by the stacking of four in-plane guanine bases (G-quartets) stabilized through Hoogsteen-type hydrogen bonding and coordinated by a central metal ion (usually K+ or Na+).[1−4] Extensive and detailed biophysical and structural studies have highlighted an impressive diversity of. We found that the fluorescence signal of the 2a-c-MYC Pu22 system was efficiently quenched and blue-shifted by the addition of increasing concentrations of Phen-DC3 (Figure S20B in the Supporting Information) These results highlight the strong competitive behavior of the two molecules for the same binding sites and suggest that 2a binds to c-MYC G4 by stacking on the terminal G-tetrad. The light-up ability of 2a titrated in the presence of the competitive complementary c-MYC sG4 C-rich sequence (sC4 = 5′CCCACCCTACCCACCC-3′) and duplex DNA was nearly unchanged (∼15-fold emission enhancement) (Figure S21C) These results highlight the high selectivity of 2a for parallel G4 structures in a complex and highly competitive environment. The decreased replication rate might result from slower replication fork progression, due to 2a’s ability to stabilize G4s

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES