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Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

A. A. Almaqwashi ; Johanna Andersson (Institutionen för kemi och kemiteknik, Farmaceutisk teknologi) ; Per Lincoln (Institutionen för kemi och kemiteknik, Fysikalisk kemi) ; I. Rouzina ; Fredrik Westerlund (Institutionen för biologi och bioteknik, Kemisk biologi) ; M. C. Williams
Biophysical Journal (0006-3495). Vol. 110 (2016), 6, p. 1255-1263.
[Artikel, refereegranskad vetenskaplig]

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 [mu-dppzip(phen)(4)Ru-2](4+) (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)(2)dppz) and the distal subunit (Ru(phen)(2)ip), respectively, each of which can be either right-handed (Delta) or left-handed (Lambda). 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 Delta x(eq), and the dynamic DNA structural deformations during ligand association x(on) and dissociation x(off). We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a K-d of 27 +/- 3 nM for (Delta,Lambda)-Piz to a K-d of 622 +/- 55 nM for (Lambda,Delta)-Piz. The striking affinity decrease is correlated with increasing Delta x(eq) from 0.30 +/- 0.02 to 0.48 +/- 0.02 nm and x(on) from 0.25 +/- 0.01 to 0.46 +/- 0.02 nm, but limited x(off) 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.

Nyckelord: binuclear ruthenium complex; binding-kinetics; noncooperative binding; force spectroscopy; optical tweezers; actinomycin-d; single dna; molecule; equilibrium; affinity; Biophysics

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Denna post skapades 2016-05-04. Senast ändrad 2017-02-09.
CPL Pubid: 235964


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