Publication details
Strategy for automated NMR resonance assignment of RNA: application to 48-nucleotide K10
| Authors | |
|---|---|
| Year of publication | 2014 |
| Type | Article in Periodical |
| Magazine / Source | Journal of Biomolecular NMR |
| MU Faculty or unit | |
| Citation | |
| web | http://link.springer.com/article/10.1007%2Fs10858-014-9841-3 |
| Doi | http://dx.doi.org/10.1007/s10858-014-9841-3 |
| Field | Biochemistry |
| Keywords | Nucleic acids; NMR; Projection spectroscopy; APSY; Automated assignment; FLYA; Novel sampling methods |
| Description | A procedure is presented for automated sequence-specific assignment of NMR resonances of uniformly [C-13, N-15]-labeled RNA. The method is based on a suite of four through-bond and two through-space high-dimensional automated projection spectroscopy (APSY) experiments. The approach is exemplified with a 0.3 mM sample of an RNA stem-loop with 48 nucleotides, K10, which is responsible for dynein-mediated localization of Drosophila fs(1)K10 mRNA transcripts. The automated analysis of the APSY data led to highly accurate and precise 3- to 4-dimensional peak lists. They provided a reliable basis for the subsequent sequence-specific resonance assignment with the algorithm FLYA and resulted in the fully automated resonance assignment of more than 80 % of the resonances of the C-13-H-1 moieties at the 1', 2', 5, 6, and 8 positions in the nucleotides. The procedure was robust with respect to numerous impurity peaks, low concentration of this for NMR comparably large RNA, and structural features such as a loop, single-nucleotide bulges and a non-Watson-Crick wobble base pairs. Currently, there is no precise chemical shift statistics (as used by FLYA) for RNA regions which deviate from the regular A-form helical structure. Reliable and precise peak lists are thus required for automated sequence-specific assignment, as provided by APSY. |