[18] Isotope labeling for 13C relaxation measurements on RNA

Publisher Summary This chapter discusses isotope labeling for 13 C relaxation measurements on RNA. Three technical advances have opened way to examine the local atomic dynamics of RNAs in unprecedented detail: the ability to produce large quantities of RNA sequences by in vitro transcription, the development of multidimensional inverse-detected pulse sequences, and the introduction of efficient isotope labeling methods. While relatively large-scale dynamic processes, such as the formation of new tertiary interactions, are known to be important in the function of some RNAs, the nature and significance of rapid local motions remain uncertain. Most information on these motions comes from molecular dynamics simulations, the reliability of which is unclear in the absence of confirmatory experimental measurements. NMR relaxation studies that are sensitive to the frequency and amplitude of motions in the ps-ns time scale should provide insight into the dynamic properties of RNA in solution and permit the results of molecular dynamics simulations to be tested. The general experimental procedure for 13 C relaxation studies consists of four basic components: (1) preparation of 3 C-labeled RNA, (2) two-dimensional NMR data acquisition, (3) extraction of relaxation parameters, and (4) motional analysis.