A quantitative Raman spectroscopic signal for metal-phosphodiester interactions in solution

Accurate identification and quantification of metal ion-phosphodiester interactions are essential for understanding the role of metal ions as determinants of three-dimensional folding of large RNAs and as cofactors in the active sites of both RNA and protein phosphodiesterases. Accomplishing this goal is difficult due to the dynamic and complex mixture of direct and indirect interactions formed with nucleic acids and other phosphodiesters in solution. To address this issue, Raman spectroscopy has been used to measure changes in bond vibrational energies due to metal interactions. However, the contributions of inner-sphere, H-bonding, and electrostatic interactions to the Raman spectrum of phosphoryl oxygens have not been analyzed quantitatively. Here, we report that all three forms of metal ion interaction result in attenuation of the Raman signal for the symmetric vibration of the nonbridging phosphate oxygens (nu(s)PO(2)(-)), while only inner-sphere coordination gives rise to an apparent shift of nu(s)PO(2)(-) to higher wavenumbers (nu(s)PO(2)(-)M) in solution. Formation of nu(s)PO(2)(-)M is shown to be both dependent on metal ion identity and an accurate measure of site-specific metal ion binding. In addition, the spectroscopic parameter reflecting the energetic difference between nu(s)PO(2)(-) and nu(s)PO(2)(-)M (DeltanuM) is largely insensitive to changes in phosphodiester structure but strongly dependent on the absolute electronegativity and hardness of the interacting metal ion. Together, these studies provide strong experimental support for the use of nu(s)PO(2)(-)M and DeltanuM as general spectroscopic features for the quantitative analysis of metal binding affinity and the identification of metal ions associated with phosphodiesters in solution.