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Base-specific spin-labeling of RNA for structure determination

To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base...

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Autores principales: Piton, Nelly, Mu, Yuguang, Stock, Gerhard, Prisner, Thomas F., Schiemann, Olav, Engels, Joachim W.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2007
Materias:
RNA
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891445/
https://www.ncbi.nlm.nih.gov/pubmed/17452362
http://dx.doi.org/10.1093/nar/gkm169
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author Piton, Nelly
Mu, Yuguang
Stock, Gerhard
Prisner, Thomas F.
Schiemann, Olav
Engels, Joachim W.
author_facet Piton, Nelly
Mu, Yuguang
Stock, Gerhard
Prisner, Thomas F.
Schiemann, Olav
Engels, Joachim W.
author_sort Piton, Nelly
collection PubMed
description To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed cross-coupling on column. For this purpose 5-iodo-uridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE® chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed.
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spelling pubmed-18914452007-06-22 Base-specific spin-labeling of RNA for structure determination Piton, Nelly Mu, Yuguang Stock, Gerhard Prisner, Thomas F. Schiemann, Olav Engels, Joachim W. Nucleic Acids Res RNA To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed cross-coupling on column. For this purpose 5-iodo-uridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE® chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed. Oxford University Press 2007-05 2007-04-22 /pmc/articles/PMC1891445/ /pubmed/17452362 http://dx.doi.org/10.1093/nar/gkm169 Text en © 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle RNA
Piton, Nelly
Mu, Yuguang
Stock, Gerhard
Prisner, Thomas F.
Schiemann, Olav
Engels, Joachim W.
Base-specific spin-labeling of RNA for structure determination
title Base-specific spin-labeling of RNA for structure determination
title_full Base-specific spin-labeling of RNA for structure determination
title_fullStr Base-specific spin-labeling of RNA for structure determination
title_full_unstemmed Base-specific spin-labeling of RNA for structure determination
title_short Base-specific spin-labeling of RNA for structure determination
title_sort base-specific spin-labeling of rna for structure determination
topic RNA
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891445/
https://www.ncbi.nlm.nih.gov/pubmed/17452362
http://dx.doi.org/10.1093/nar/gkm169
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