Cargando…
Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA
We present the access to [5‐(19)F, 5‐(13)C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐(19)F, 5‐(13)C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans ac...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540360/ https://www.ncbi.nlm.nih.gov/pubmed/32558232 http://dx.doi.org/10.1002/anie.202006577 |
_version_ | 1783591190345547776 |
---|---|
author | Nußbaumer, Felix Plangger, Raphael Roeck, Manuel Kreutz, Christoph |
author_facet | Nußbaumer, Felix Plangger, Raphael Roeck, Manuel Kreutz, Christoph |
author_sort | Nußbaumer, Felix |
collection | PubMed |
description | We present the access to [5‐(19)F, 5‐(13)C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐(19)F, 5‐(13)C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic (19)F–(13)C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic (13)C–(19)F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐(19)F, 5‐(13)C]/[5‐(19)F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial (19)F–(13)C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [(19)F, (13)C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting. |
format | Online Article Text |
id | pubmed-7540360 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-75403602020-10-09 Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA Nußbaumer, Felix Plangger, Raphael Roeck, Manuel Kreutz, Christoph Angew Chem Int Ed Engl Research Articles We present the access to [5‐(19)F, 5‐(13)C]‐uridine and ‐cytidine phosphoramidites for the production of site‐specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5‐(19)F, 5‐(13)C]‐pyrimidine labels into five RNAs—the 30 nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus ϵ (hHBV ϵ) RNA, the 49 nt SAM VI riboswitch aptamer domain from B. angulatum, the 29 nt apical stem loop of the pre‐microRNA (miRNA) 21 and the 59 nt full length pre‐miRNA 21. The main stimulus to introduce the aromatic (19)F–(13)C‐spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole‐dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic (13)C–(19)F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5‐(19)F, 5‐(13)C]/[5‐(19)F] pyrimidine labeling. For the 61 nt hHBV ϵ we find a beneficial (19)F–(13)C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [(19)F, (13)C]‐labeling of the SAM VI aptamer domain and the pre‐miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting. John Wiley and Sons Inc. 2020-07-29 2020-09-21 /pmc/articles/PMC7540360/ /pubmed/32558232 http://dx.doi.org/10.1002/anie.202006577 Text en © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Nußbaumer, Felix Plangger, Raphael Roeck, Manuel Kreutz, Christoph Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA |
title | Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA |
title_full | Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA |
title_fullStr | Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA |
title_full_unstemmed | Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA |
title_short | Aromatic (19)F–(13)C TROSY—[(19)F, (13)C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA |
title_sort | aromatic (19)f–(13)c trosy—[(19)f, (13)c]‐pyrimidine labeling for nmr spectroscopy of rna |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540360/ https://www.ncbi.nlm.nih.gov/pubmed/32558232 http://dx.doi.org/10.1002/anie.202006577 |
work_keys_str_mv | AT nußbaumerfelix aromatic19f13ctrosy19f13cpyrimidinelabelingfornmrspectroscopyofrna AT planggerraphael aromatic19f13ctrosy19f13cpyrimidinelabelingfornmrspectroscopyofrna AT roeckmanuel aromatic19f13ctrosy19f13cpyrimidinelabelingfornmrspectroscopyofrna AT kreutzchristoph aromatic19f13ctrosy19f13cpyrimidinelabelingfornmrspectroscopyofrna |