EPR methods to study specific metal-ion binding sites in RNA.

The properties of metal-ion interactions with RNA can be explored by spectroscopic methods. In this chapter, we describe the use of paramagnetic Mn(2+) ions and electron paramagnetic resonance (EPR)-based techniques to monitor the association of Mn(2+) with RNA and related nucleotides. Solution EPR methods are used to determine the numbers of Mn(2+) ions associating with RNA. For RNA poised with a single-bound Mn(2+), low-temperature EPR characteristics provide information about the asymmetry of the Mn(2+) coordination site. To identify the RNA groups coordinating to the Mn(2+) ion, ENDOR (electron nuclear double resonance) and ESEEM (electron spin echo envelope modulation) methods are applied. Both continuous-wave (CW) and electron spin echo (ESE)-detected ENDOR methods are described. This chapter includes practical details for RNA sample preparation, including isotope substitution and cryoprotection, and an overview of data acquisition and analysis methods used in these techniques, as well as examples from the current literature.