Fig. 1: DNA double helix embedded in water (angled small molecules, not to scale). The dark red spheres on the helix surface represent oxygen atoms of the negatively charged PO₂^- units, the blue spheres positively charged ions in the environment.

Positive ions are arranged in different geometries around DNA and RNA: in so-called site-bound or contact-pair geometries, a positive ion is located in direct contact with an oxygen atom of a phosphate group. In contrast, the so-called outer ion atmosphere consists of positive ions separated by at least one layer of water molecules from the phosphate groups. The functional role of the different geometries and the underlying interactions are far from being understood. A deeper insight at the molecular level requires highly sensitive probes which allow for discerning the different ion geometries without disturbing them, and for mapping their dynamics on the ultrafast time scale of molecular motions.

In a recent publication, researchers from the Max-Born-Institute demonstrate that vibrations of phosphate groups represent sensitive and noninvasive probes of ion geometries in a water environment. Dimethylphosphate (DMP, (CH₃O)₂PO₂^-), an established model system for the DNA and RNA backbone, was prepared in liquid water with an excess of Mg²⁺ ions (Figure 2, top) and studied by nonlinear vibrational spectroscopy in the femtosecond time domain (1 fs = 10^-15 s). The experiments make use of two-dimensional infrared (2D-IR) spectroscopy, a most sophisticated method for analyzing the ionic interactions and structures on the intrinsic time scale of fluctuating molecular motions.

Fig. 2: Top: Molecular structure of a contact ion pair consisting of dimethylphosphate (DMP) and a magnesium ion Mg²⁺ embedded in water. The arrows mark the elongations of the phosphorus-oxygen bonds in the asymmetric PO₂^- stretching vibration. Bottom: Two-dimensional infrared (2D-IR) spectra of the asymmetric PO₂^- stretching vibration measured at a waiting time T=500 fs after vibrational excitation. The vibrational response is shown as a function of the infrared excitation and the detection frequencies and consists of a component P1 from DMP molecules without a magnesium ion in the neighborhood and the contribution P2 from contact ion pairs. The latter is shifted to higher frequencies due to the interaction between PO₂^- and Mg²⁺.

The experiments map Mg²⁺ ions in direct contact with a PO₂^- group via a distinct feature in the 2D-IR spectrum (Figure 2, bottom). The interaction with the Mg²⁺ ion shifts the asymmetric PO₂^- stretching vibration to a frequency which is higher than in absence of Mg²⁺ ions. The lineshape and the time evolution of this new feature reveal fluctuations of the contact ion pair geometry and the embedding water shell on a time scale of hundreds of femtoseconds while the contact pair itself exists for much longer times (∼10^-6 s). An in-depth theoretical analysis shows that the subtle balance of attractive electrostatic (Coulomb) forces and repulsive forces due to the quantum-mechanical exchange interaction govern the frequency position of the phosphate vibration.

The ability of 2D-IR spectroscopy to characterize the short-ranged phosphate-ion interaction in solution provides a novel analytical tool that complements currently available structural techniques. An extension of this new approach to DNA and RNA and their ionic environment is most promising and expected to provide new insight in the forces stabilizing equilibrium structures and driving folding processes.