Enhanced G-quadruplex ligand screening exploiting high-throughput synchrotron radiation circular dichroism

G-quadruplexes (G4s) represent rigid arrangements of DNA/RNA, composed of stacked guanine tetrads held together by Hoogsteen-type hydrogen bonds, and stabilized by metal cations, predominantly K+ . These structures can adopt parallel, anti-parallel, or hybrid topologies depending on the orientation of connecting strands, exhibiting distinct CD spectral features useful for diagnostic purposes [1]. Their presence in crucial genomic regions like proto-oncogenes and telomeres, along with their conservation across diverse organisms including bacteria, viruses such as SARS-CoV-2 [2], underscores their significance in gene expression regulation. Consequently, there is considerable interest in exploring specific ligands, including peptides and peptidomimetics, that can be readily synthesized and tailored to target G4s effectively. CD spectroscopy serves as a valuable tool for characterizing G4 topologies in various environments, evaluating ligand interactions and ligand-induced thermal stability [3]. Despite peptides being chiral molecules, their dichroic signal in the near-UV region is relatively small compared to nucleic acids, particularly in sequences lacking aromatic residues. To assess the binding affinity of peptides and peptide mimetics to G4 strands, advanced techniques such as High-Throughput Synchrotron Radiation Circular Dichroism (HT-SRCD) have been employed, notably utilizing the cutting-edge setup developed at beamline B23 of the Diamond Light Source synchrotron. This setup features a highly collimated SR micro-beam capable of measuring custom quartz 96-cell plates of suitable pathlength for limited sample volumes, while the high photon flux enhances signal-to-noise ratios. The conformational behavior of three G4-forming strands was analyzed in TRIS-HCl buffer, both in the presence of 75 mM NaCl and 75 mM KCl, ensuring DNA absorption around 1 a.u. at 260 nm. Subsequently, we will discuss conformational stability and ligand-binding capabilities with selected ligands under varied experimental conditions, including diverse metal ion compositions and concentration.