Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration

Abstract

We report on the physicochemical effects resulting from incorporating a 5-(3-aminopropyl) side chain onto a 2'-deoxyuridine (dU) residue in a short DNA hairpin. A combination of spectroscopy, calorimetry, density and ultrasound techniques were used to investigate both the helix-coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC) [N = dU, deoxythymidine (dT) or 5-(3-aminopropyl)-2'-deoxyuridine (dU*)], and the interaction of each hairpin with Mg(2+). All three molecules undergo two-state transitions with melting temperatures (T(M)) independent of strand concentration that indicates their intramolecular hairpin formation. The unfolding of each hairpin takes place with similar T(M) values of 64-66 degrees C and similar thermodynamic profiles. The unfavorable unfolding free energies of 6.4-6.9 kcal/mol result from the typical compensation of unfavorable enthalpies, 36-39 kcal/mol, and favorable entropies of approximately 110 cal/mol. Furthermore, the stability of each hairpin increases as the salt concentration increases, the T(M)-dependence on salt yielded slopes of 2.3-2.9 degrees C, which correspond to counterion releases of 0.53 (dU and dT) and 0.44 (dU*) moles of Na(+) per mole of hairpin. Absolute volumetric and compressibility measurements reveal that all three hairpins have similar hydration levels. The electrostatic interaction of Mg(2+) with each hairpin yielded binding affinities in the order: dU > dT > dU*, and a similar release of 2-4 electrostricted water molecules. The main result is that the incorporation of the cationic 3-aminopropyl side chain in the major groove of the hairpin stem neutralizes some local negative charges yielding a hairpin molecule with lower charge density.

Figure 1

Sequence of deoxyribonucleotide and structure of base modifications.

Figure 2

Thermodynamic characterization of the helix–coil transition of dU (circles), dT (squares) and dU* (triangles) in 10 mM sodium phosphate buffer at pH 7. (A) UV melting curves at oligonucleotide concentrations of 5 µM. (B) DSC curves at oligonucleotide concentrations of ∼225 µM (in strands). (C) Dependence of T_M on strand concentration.

Figure 3

UV melting curves of oligonucleotides, at constant strand concentration of 5 µM, as a function of salt concentration in 10 mM sodium phosphate buffer at pH 7. (A) Typical melting curves over a NaCl concentration range of 16–200 mM. (B) Dependence of T_M on salt concentration, dU (circles), dT (squares) and dU* (triangles).

Figure 4

Ultrasonic titration curves of DNA hairpins with MgCl₂ in 10 mM HEPES buffer pH 7.5 and at oligonucleotide concentrations of ∼150 µM (in strands).