Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling
In this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RN...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | Journal of Magnetic Resonance Open |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666441022000474 |
| _version_ | 1811294127523889152 |
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| author | David Klingler Matthias Huber Martin Tollinger Christoph Kreutz |
| author_facet | David Klingler Matthias Huber Martin Tollinger Christoph Kreutz |
| author_sort | David Klingler |
| collection | DOAJ |
| description | In this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RNA comprising 66 nucleotides. The assignment strategy is based on position specific labeling by chemical solid phase synthesis and dilute stable isotope 13C/15N-labeling by mixing labeled and commercially available unlabeled RNA phosphoramidites. The assignment process is further facilitated by nucleotide specific labeling using T7 RNA polymerase in vitro transcription with in house produced atom specific 13C labeled ribonucleotide triphosphates. The approach is fast with a total NMR measurement time of only 22 h and also competitive in terms of costs as compared to the standard methodology relying on in vitro transcription using 2H, 15N and 13C/15N uniformly labeled ribonucleotide triphosphates. Furthermore, the assignment procedure revealed a slow exchange process on the NMR chemical shift time scale in the 66 nt non-coding RNA with possible biological implications in the regulation of bacterial competence. |
| first_indexed | 2024-04-13T05:12:52Z |
| format | Article |
| id | doaj.art-5f8f55f29ec24d0cb0b098740b0b931f |
| institution | Directory Open Access Journal |
| issn | 2666-4410 |
| language | English |
| last_indexed | 2024-04-13T05:12:52Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Magnetic Resonance Open |
| spelling | doaj.art-5f8f55f29ec24d0cb0b098740b0b931f2022-12-22T03:00:59ZengElsevierJournal of Magnetic Resonance Open2666-44102022-12-0112100077Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labelingDavid Klingler0Matthias Huber1Martin Tollinger2Christoph Kreutz3Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, AustriaInstitute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, AustriaInstitute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, AustriaCorresponding author.; Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020 Innsbruck, AustriaIn this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RNA comprising 66 nucleotides. The assignment strategy is based on position specific labeling by chemical solid phase synthesis and dilute stable isotope 13C/15N-labeling by mixing labeled and commercially available unlabeled RNA phosphoramidites. The assignment process is further facilitated by nucleotide specific labeling using T7 RNA polymerase in vitro transcription with in house produced atom specific 13C labeled ribonucleotide triphosphates. The approach is fast with a total NMR measurement time of only 22 h and also competitive in terms of costs as compared to the standard methodology relying on in vitro transcription using 2H, 15N and 13C/15N uniformly labeled ribonucleotide triphosphates. Furthermore, the assignment procedure revealed a slow exchange process on the NMR chemical shift time scale in the 66 nt non-coding RNA with possible biological implications in the regulation of bacterial competence.http://www.sciencedirect.com/science/article/pii/S2666441022000474RNA NMRResonance assignmentStable isotope labelingSolid phase RNA synthesis |
| spellingShingle | David Klingler Matthias Huber Martin Tollinger Christoph Kreutz Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling Journal of Magnetic Resonance Open RNA NMR Resonance assignment Stable isotope labeling Solid phase RNA synthesis |
| title | Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling |
| title_full | Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling |
| title_fullStr | Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling |
| title_full_unstemmed | Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling |
| title_short | Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling |
| title_sort | rapid and reliable rna resonance assignment by combining chemical and enzymatic stable isotope labeling |
| topic | RNA NMR Resonance assignment Stable isotope labeling Solid phase RNA synthesis |
| url | http://www.sciencedirect.com/science/article/pii/S2666441022000474 |
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