12] Systems for the NMR study of modified nucleoside-dependent, metal-ion induced conformational changes in nucleic acids

Abstract

This chapter discusses systems for the nuclear magnetic resonance (NMR) study of modified nucleoside-dependent, metal-ion induced conformational changes in nucleic acids. RNAs have more than 90 nucleoside modifications and function through a large variety of interactions with other macromolecules. The structure–function interrelationships of modified nucleosides and their interactions with the ribose 2'OH and metal ions are largely unknown. Modified nucleosides and stable isotope-labeled nucleosides are chemically synthesized and introduced into RNA by solid phase manual synthesis or by automated chemical synthesis. The latter requires production of the protected phosphoramidites. Modified nucleoside-containing stem-loop domains and smaller sequences of RNAs and their DNA analogs are produced, and their physical, chemical, and biological properties are studied. Modified nucleosides have evolved to alter the RNA chemistry and structure and the potential to accomplish this is as varied as are the chemistries and structures of the modifications. There are three methods of incorporation of modified nucleosides and stable isotope-enriched nucleosides into nucleic acids. From the in vivo incorporation of modified nucleosides and stable isotope-enriched nucleosides, a fully mature, completely modified, and isotopically enriched RNA can be isolated from the cells grown in culture and purified by chromatography.