title: Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation.
abstract: DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)4Ru2](4+) (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics.
authors.affiliation: Department of Physics, Northeastern University, Boston, Massachusetts.
authors.forename: Ali A
authors.initials: AA
authors.lastname: Almaqwashi
authors.affiliation: Department of Chemistry-BMC, Uppsala University, Uppsala, Sweden; Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Johanna
authors.initials: J
authors.lastname: Andersson
authors.affiliation: Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Per
authors.initials: P
authors.lastname: Lincoln
authors.affiliation: Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio.
authors.forename: Ioulia
authors.initials: I
authors.lastname: Rouzina
authors.affiliation: Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Westerlund
authors.affiliation: Department of Physics, Northeastern University, Boston, Massachusetts. Electronic address: ma.williams@neu.edu.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
mesh_terms: D004247:DNA / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D012428:Ruthenium / Q000737:chemistry; D013237:Stereoisomerism; D013997:Time Factors
chemical_list: D007364:Intercalating Agents; D008024:Ligands; D012428:Ruthenium; D004247:DNA