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Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

We report the synthesis of atom-specifically (13)C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-(13)C-modified pyrimidine and 8-(13)C purine DNA phosphoramidites...

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Autores principales: Nußbaumer, Felix, Juen, Michael Andreas, Gasser, Catherina, Kremser, Johannes, Müller, Thomas, Tollinger, Martin, Kreutz, Christoph
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587810/
https://www.ncbi.nlm.nih.gov/pubmed/28911104
http://dx.doi.org/10.1093/nar/gkx592
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author Nußbaumer, Felix
Juen, Michael Andreas
Gasser, Catherina
Kremser, Johannes
Müller, Thomas
Tollinger, Martin
Kreutz, Christoph
author_facet Nußbaumer, Felix
Juen, Michael Andreas
Gasser, Catherina
Kremser, Johannes
Müller, Thomas
Tollinger, Martin
Kreutz, Christoph
author_sort Nußbaumer, Felix
collection PubMed
description We report the synthesis of atom-specifically (13)C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-(13)C-modified pyrimidine and 8-(13)C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-(13)C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min(−1). Finally, we used (13)C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s(−1) and with a backward rate constant of 10.6 s(−1).
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spelling pubmed-55878102017-09-11 Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR Nußbaumer, Felix Juen, Michael Andreas Gasser, Catherina Kremser, Johannes Müller, Thomas Tollinger, Martin Kreutz, Christoph Nucleic Acids Res Structural Biology We report the synthesis of atom-specifically (13)C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-(13)C-modified pyrimidine and 8-(13)C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-(13)C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min(−1). Finally, we used (13)C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s(−1) and with a backward rate constant of 10.6 s(−1). Oxford University Press 2017-09-06 2017-07-07 /pmc/articles/PMC5587810/ /pubmed/28911104 http://dx.doi.org/10.1093/nar/gkx592 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Structural Biology
Nußbaumer, Felix
Juen, Michael Andreas
Gasser, Catherina
Kremser, Johannes
Müller, Thomas
Tollinger, Martin
Kreutz, Christoph
Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR
title Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR
title_full Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR
title_fullStr Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR
title_full_unstemmed Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR
title_short Synthesis and incorporation of (13)C-labeled DNA building blocks to probe structural dynamics of DNA by NMR
title_sort synthesis and incorporation of (13)c-labeled dna building blocks to probe structural dynamics of dna by nmr
topic Structural Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587810/
https://www.ncbi.nlm.nih.gov/pubmed/28911104
http://dx.doi.org/10.1093/nar/gkx592
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