Determining RNA solution structure by segmental isotopic labeling and NMR: Application to Caenorhabditis elegans spliced leader RNA 1

作者：Xu J^*; Lapham J; Crothers DM

来源：Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(1): 44-48.

DOI：10.1073/pnas.93.1.44

Recent developments in multidimensional heteronuclear NMR spectroscopy and large-scale synthesis of uniformly C-13- and N-15-labeled oligonucleotides have greatly improved the prospects for determination of the solution structure of RNA, However, there are circumstances in which it may be advantageous to label only a segment of the entire RNA chain. For example, in a larger RNA molecule the structural question of interest may reside in a localized domain. Labeling only the corresponding nucleotides simplifies the spectrum and resonance assignments because one can filter proton spectra for coupling to C-13 and N-15. Another example is in resolving alternative secondary structure models that are indistinguishable in imino proton connectivities. Here ne report a general method for enzymatic synthesis of quantities of segmentally labeled RNA molecules required for NMR spectroscopy. me use the method to distinguish definitively two competing secondary structure models for the 5' half of Caenorhabditis elegans spliced leader RNA by comparison of the two-dimensional {N-15}H-1 heteronuclear multiple quantum correlation spectrum of the uniformly labeled sample with that of a segmentally labeled sample. The method requires relatively small samples; solutions in the 200-300 mu M concentration range, with a total of 30 nmol or approximate to 40 mu g of RNA in approximate to 150 mu l, give strong NMR signals in a short accumulation time, The method can be adapted to label an internal segment of a larger RNA chain for study of localized structural problems, This definitive approach provides an alternative to the more common enzymatic and chemical footprinting methods for determination of RNA secondary structure.

• 出版日期1996-1-9