Counting the Number of Magnesium Ions Bound to the Surface-Immobilized Thymine Oligonucleotides That Comprise Spherical Nucleic Acids

Label-free studies carried out under aqueous phase conditions quantify the number of Mg²⁺ ions binding to surface-immobilized T₄₀ 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 T₄₀ 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 T₄₀ 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)°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)°C form if 18.5–19.0 Mg²⁺ ions are bound, corresponding to saturation of the T₄₀ strand.