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Base-specific spin labeling of RNA for structure determination

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Date
05/2007
Author
Piton, N.
Mu, Y.
Stock, G.
Prisner, TF.
Schiemann, Olav
Engels, J.W.
Keywords
PAIR DISTANCE DISTRIBUTIONS
EPR SPECTROSCOPY
NUCLEIC-ACIDS
TIKHONOV REGULARIZATION
PARAMAGNETIC-RESONANCE
HAMMERHEAD RIBOZYME
PROTECTING GROUPS
PULSED ESR
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Abstract
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- tetramethylpyrrolin- 1- yloxyl- 3- acetylene ( TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed crosscoupling on column. For this purpose 5- iodouridine, 5- iodo- cytidine and 2- iodo- adenosine phosphoramidites were synthesized and incorporated into RNA- sequences. Application of the recently developed ACE (R) 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.
Citation
Piton , N , Mu , Y , Stock , G , Prisner , TF , Schiemann , O & Engels , J W 2007 , ' Base-specific spin labeling of RNA for structure determination ' , Nucleic Acids Research , vol. 35 , no. 9 , pp. 3128-3143 . https://doi.org/10.1093/nar/gkm169
Publication
Nucleic Acids Research
Status
Peer reviewed
DOI
https://doi.org/10.1093/nar/gkm169
ISSN
0305-1048
Type
Journal article
Rights
© 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.
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  • University of St Andrews Research
URL
http://www.scopus.com/inward/record.url?scp=34250688400&partnerID=8YFLogxK
URI
http://hdl.handle.net/10023/4274

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