Label-free studies carried out under aqueous phase conditions quantify the number of Mg2+ ions binding to surface-immobilized T-40 sequences, the subsequent reordering of DNA on the surface, and the consequences of Mg2+ binding for DNA DNA interactions. Second harmonic generation measurements indicate that, within error, 18-20 Mg2+ ions are bound to the T-40 strand at saturation and that the metal DNA interaction is associated with a near 30% length contraction of the strand. Structural reordering, evaluated using vibrational sum frequency generation, atomic force microscopy, and dynamic light scattering, is attributed to increased charge screening as the Mg2+ ions bind to the negatively charged DNA, reducing repulsive Coulomb forces between nucleotides and allowing the DNA single strands to collapse or coil upon themselves. The impact of Mg2+ binding on DNA hybridization and duplex stability is assessed with spherical nucleic acid (SNA) gold nanoparticle conjugates in order to determine an optimal working range of Mg2+ concentrations for DNA DNA interactions in the absence of NaCl. The findings are consistent with a charge titration effect in which, in the absence of NaCl, (1) hybridization does not occur at room temperature if an average of 17.5 or less Mg2+ ions are bound per T-40 strand, which is not reached until the bulk Mg2+ concentration approaches 0.5 mM; (2) hybridization proceeds, albeit with low duplex stability having an average T-m of 31(3)degrees C, if an average of 17.5-18.0 Mg2+ ions are bound; and (3) highly stable duplexes having a T-m of 64(2)degrees C form if 18.5-19.0 Mg2+ ions are bound, corresponding to saturation of the T-40 strand.