Netropsin binding in five duplex-dimer DNA constructs as a function of size and distance between binding sites: circular dichroism and absorption spectroscopy
Résumé
The optical activity induced on binding the drug netrospin (NET) in the minor groove of DNA is studied in five oligonucleotides (OGNs) as a function of (1) the size of the binding site in (5'-(GC)(2)AATT(GC)(2)-3')(2) (OGN 1a) versus (5'-(GC)(2)AAATTT(GC)(2)-3')(2) (OGN 1b) and (2) the distance between two AATT binding sites in (5'-(GC)(2)AATT(GC) (x) AATT(GC)(2)-3')(2), with x = 1, 2, or 3 (OGNs 2a, b, c, respectively). NET binding is monitored via the induced circular dichroism (CD) at similar to 315 nm, where the nucleic acids are optically inactive. The CD titrations, fit to a tight binding model, yield lower limits for the binding constant, K-a, a parts per thousand yen8 x 10(7) M-1 for OGN 1a and a parts per thousand yen2 x 10(8) M-1 for OGNs 2a, b, c in 1 mM buffer. In 100 mM buffer, tight binding occurs in all five OGNs with K-a a parts per thousand yen 8 x 10(7) M-1 for OGN 1a and a parts per thousand yen1 x 10(8) M-1 for OGNs 1b and 2a, b, c. In contrast, the elongated AAATTT binding site of OGN 1b results in weak binding of NET in 1 mM buffer, where competing electrostatic interactions with the solvent environment are lower. In the constructs with two binding sites, the increase in flexibility introduced by intervening GC base pairs does not induce co-operative binding, although differences in the number of binding sites, n (2.05-2.65), indicate that there may be differences in the way NET is bound in OGNs 2a, b, c. In addition, the large shifts in the absorption spectra induced in bound versus free NET, and effects on the CD spectral bands at higher energy, are discussed in terms of electrostatic and excitonic interactions.