Speeding up G-Quadruplex drug discovery with synchrotron light

G-quadruplexes (G4s) are unusual DNA and RNA structures formed when guanine-rich sequences fold into stacked G-tetrads. These structures are implicated in cancer, viral or bacterial infections, and gene regulation, making them compelling targets for drug discovery. However, testing small molecules (ligands) for their ability to bind and modulate G4s has traditionally required large sample volumes or slow techniques like NMR, or fluorescence assays. In a new study published in Molecules, an international team of researchers demonstrates a high-throughput method to screen G4 ligands using High-Throughput Synchrotron Radiation Circular Dichroism (HT-SRCD) at Diamond's B23 beamline.  

(A) Image of the optical elements (exit slit, 45° plane mirror, focusing lens, polarizer prism, PEM, iris, X–Y motorised stage, horizontal sample and PMT detector). The sample is placed on the motorised X–Y stage. (B) Picture of the 96-well plate held in the X–Y motorised stage inside the vertical sample chamber unit, redrawn from Hussain et al., 2016

G4 structures can regulate key biological processes: they form in telomeres (ends of chromosomes), in promoter regions of oncogenes, and in viral genomes. Ligands that stabilise - or destabilise - G4s have potential therapeutic applications (anticancer, antiviral). But finding effective ligands is not trivial. Conventional screening methods may consume a lot of material or fail to capture subtle binding-driven conformational changes. To accelerate discovery, the authors developed a library of small tetrazole-based molecules and also used a known G4-binding peptide (Rhau25). They wanted a fast, low-material assay that can detect not just binding but also changes in G4 conformation (topology) — a critical piece for drug development. 

HT-SRCD spectra of G4 sequences in the presence of Rhau25 peptide in 10 mM Tris-HCl buffer, pH 7.4, in the presence of 70 mM K+ or Na+ ions, acquired using 2 string plates using B23 module A. Each box contains the spectrum of the G4 alone (black), the measured (blue) and the calculated (red) spectra of the ligand–G4 complex and the spectrum of the ligand alone (green line).

The team used HT-SRCD on the B23 Beamline at Diamond Light Source. This technique enables circular dichroism (CD) measurements on very small volumes (in 96-well microplates), thanks to the highly collimated synchrotron beam. Circular dichroism (CD) spectroscopy is a biophysical method widely used to evaluate the secondary structure, folding and binding interaction properties of chiral biopolymers, including nucleic acids. In this work, HT-SRCD has been used a powerful tool for studying and characterising the interaction of G4 with ligands. The authors screened three biologically relevant G4 sequences: HTelo1 (human telomeric), G3T3, and T95-2T, in the presence of either potassium or sodium ions, which influence G4 folding.  

They synthesised a panel of α-amino tetrazole derivatives (via a four-component Ugi reaction) to test as potential G4 ligands. Using HT-SRCD, they measured spectral changes when ligands were added - changes in CD signal and spectral shape indicated unambiguously binding with conformational effects. Importantly, the method is label-free and doesn’t require immobilisation, unlike many other screening strategies such as SPR and fluorescence.  

The results showed that different ligands produced distinct spectral signatures depending on the G4 sequence and the ionic environment (Na⁺ vs K⁺). Some ligands induced relatively small changes, while others altered intensity more significantly - suggesting different binding modes or affinities. They also tested the Rhau25 peptide and found that it behaves differently depending on the G4 sequence and the ionic context.  

This work demonstrates that HT-SRCD at Diamond is a powerful, rapid, and low-consumption tool for screening G4–ligand interactions. Its ability to monitor conformational (topological) changes in G4s, not just binding, is particularly valuable: many G4-targeted ligands’ biological effects depend not only on how tightly they bind, but also on how they reshape G4 structure. 

Looking ahead, this method could accelerate drug discovery pipelines for anticancer or antiviral molecules targeting G4s, by enabling fast triage of large ligand libraries unattainable with bespoke benchtop CD instruments. It could also be used to study G4-binding peptides, aptamers, or even proteins.