DNA G-quadruplexes play important roles in biological processes and have been identified as promising drug targets, e.g. in anticancer research. In an all-RESOLV interdisciplinary collaboration, the groups of Guido Clever and Müge Kasanmascheff, both at TU Dortmund University, have developed a method for the precise distance measurement in pairs of G‑quadruplexes - with or without bound drug-like molecules. The method allows the researchers to elucidate the structure of such DNA adducts with high accuracy and provides evidence for intercalated compounds. Clever and Kasanmascheff just published their results in an open access paper in the journal Angewandte Chemie. The publication was selected as Very Important Paper - VIP, a label awarded to only 5% of the journal's publications.
Human DNA usually adopts a helical, double stranded structure. However, also four-stranded variants (so-called G‑quadruplexes) are possible. They form from guanine-rich sequences, resembling columnar stacks.
In the following interview, Clever and Kasanmascheff explain their research on G-quadruplexes and why it is important.
What is the new discovery that you made - and why is it exciting?
DNA G-quadruplexes are four-stranded DNA molecules. They can assemble pairwise to form so-called dimers and bind small molecules in between them - forming sandwich-like complexes held together by non-covalent interactions. Up to now, it has been very difficult to obtain precise spatial information about such G-quadruplex assemblies in aqueous solution. Therefore, we decided to tackle this question using EPR spectroscopy, a technique that can deliver accurate distance measurements between unpaired electrons - a sort of ruler for nanometer distances in molecules. We designed an extremely rigid spin label based on copper ions, incorporated it into a DNA G-quadruplex and measured the distances within the formed pairs with EPR. In this way, we could confirm that sandwich-like structures can form, using either a drug-like compound or a natural product as "filling". We even discovered new binding motifs. Moreover, we could show for the first time that the natural nucleobase guanine, usually having only negligible solubility in water, can be brought into solution by sneaking between these G-quadruplexes.
Why is it important?
DNA G-quadruplexes have biological relevance and play roles in cancer and diseases such as HIV and malaria. Studying their aggregation and non-covalent interaction with small molecules will help to understand their biological behaviour and medicinal implications. The tool we developed bears potential to find new DNA-binding molecules, identify contacts between different DNA species and even reveal DNA-protein complex formation. The method is so accurate that it can deliver distance information with precision high enough to study minor structural changes and dynamic processes.
Is this related to Solvation Science? If yes, how?
The formation of higher-order DNA structures from individual G-quadruplexes and flat aromatic molecules is driven by attractive π-stacking interactions. While single crystal X-ray analysis has revealed such structures in the solid state before, aggregation in solution is more dynamic and competes with other processes. This is especially the case for sandwich-type complexes where uncharged binders (like the natural macrocycle telomestatin or free guanine tetrads) intercalate under the influence of hydrophobic effects. Our experiments could show that good water solubility is not a prerequisite for such intercalators: For example, cyclic quartets of guanines can interact with the G‑quadruplex dimers that in turn bring them in solution. This opens the question whether this observation can be linked to biological processes involving DNA and free nucleosides or bases. Ongoing work within RESOLV may further reveal effects of molecular crowding and pressure changes on non-covalent adduct formation involving G-quadruplexes, aimed at understanding processes in densely packed chromosomal DNA environments.
How did your collaboration start and how will it proceed?
While our labs are literally door-to-door, it was our common interest in using advanced spectroscopic techniques for the elucidation of biomolecular structures in solution that brought us together here to tackle questions on DNA aggregation phenomena that could not be answered before. We merged the expertise in the synthesis of chemically modified DNA, G-quadruplex assembly and transition metal binding of the Clever Lab with the high competence in pulsed EPR techniques by the Kasanmascheff Lab, resulting in this highly interdisciplinary study. Within the RESOLV research consortium, we were able to join our experimental and theoretical backgrounds in close collaboration, supported by state-of-the-art instrumentation. This work is just the beginning of several joint projects focusing on the solution study of higher-order DNA structures and adducts!
Original Publication: L. M. Stratmann, Y. Kutin, M. Kasanmascheff, G. H. Clever, Precise Distance Measurements in DNA G‐Quadruplex Dimers and Sandwich Complexes by Pulsed Dipolar EPR Spectroscopy, Angew. Chem. Int. Ed. 2020, accepted, DOI: 10.1002/anie.202008618 (VIP Paper)