Biophysical characterization of DNA duplexes containing modified nucleotides that influence DNA hydration and cation organization

The interaction of DNA with cationic alkylating agents and proteins is driven by the sequence dependent electrostatic atmosphere of DNA. We attempted to modulate this electrostatic environment to study the relationship between sequence dependent cation binding, hydration, DNA structure and DNA reactivity. We used a combination of spectroscopic and calorimetric techniques along with chemical probes, NMR and gel shift assays to investigate: (i) the effect of incorporation of 3-aminopropyl sidechains into DNA duplexes; (ii) the underlying mechanism behind the association of 3-aminopropyl sidechain with high cation binding sites in DNA; and (iii) the effect of substitution of a 7-deazaguanine (7-zdG) or a 7-deazaadenine (7-zdA) in the major groove of DNA duplexes.; The incorporation of a single or multiple cationic aminopropyl sidechains in a duplex DNA is destabilizing and causes reduced gel mobility that is attributed to local bending of the DNA. This reduction in mobility is sequence selective. The modified residues do not affect the global conformation or the Watson-Crick base pairing in DNA.; The association of the 3-aminopropyl sidechain with the cation binding site in DNA is driven by electrostatic attraction and the interaction is water mediated.; The inclusion of both 7-zdG and 7-zdA modifications induces thermodynamic destabilization that is enthalpy driven. The magnitude of destabilization is dependent on the type of substitution and the site of the substitution. Substitution of 7-zdG in the middle of the CGCG tract of the Dickerson dodecamer duplex leads to unstacking of the bases flanking the modified 7-zdG·C pair due to changes in hydration and cation organization. This occurs without altering the strength of the Watson-Crick base pairing at 7-zdG·dC. There is increased destabilization when the 7-zdG is present at the interface of CGCG and AATT tracks of the duplex. The incorporation of 7-zdA leads to a decrease in stacking contribution at the site of modified 7-zdA base, which is related to the organization of cations in the major groove.