Binuclear ruthenium complex linker length tunes DNA threading intercalation kinetics

Ali A Almaqwashi; Micah J McCauley; Johanna Andersson; Ioulia Rouzina; Fredrik Westerlund; Per Lincoln; Mark C Williams

doi:10.1016/j.bpj.2025.01.002

Binuclear ruthenium complex linker length tunes DNA threading intercalation kinetics

Biophys J. 2025 Jan 9:S0006-3495(25)00002-5. doi: 10.1016/j.bpj.2025.01.002. Online ahead of print.

Authors

Ali A Almaqwashi¹, Micah J McCauley², Johanna Andersson³, Ioulia Rouzina⁴, Fredrik Westerlund⁵, Per Lincoln⁶, Mark C Williams⁷

Affiliations

¹ Physics Department, College of Science and Arts, King Abdulaziz University, Rabigh, Saudi Arabia.
² Department of Physics, Northeastern University, Boston, MA, 02115, USA.
³ Department of Chemistry-BMC, Uppsala University, Uppsala, Sweden; Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, SE-41296, Sweden.
⁴ Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, 43210, USA.
⁵ Department of Life Sciences, Chalmers University of Technology, Gothenburg, SE-41296, Sweden.
⁶ Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, SE-41296, Sweden.
⁷ Department of Physics, Northeastern University, Boston, MA, 02115, USA. Electronic address: ma.williams@neu.edu.

PMID: 39797403
DOI: 10.1016/j.bpj.2025.01.002

Abstract

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA base pairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semi-rigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically-relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semi-rigid linker provides enough molecular spacing to allow a single monomer to fully stack with base pairs, comparable to the monomeric parent ruthenium complex. The DNA base pairs in the equilibrium threading intercalated state are likely intact and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.

Keywords: DNA binding; DNA threading intercalation; force spectroscopy; single molecule; structural dynamics.