Nucleic Acids Research Advance Access originally published online on April 22, 2007
Nucleic Acids Research 2007 35(9):3128-3143; doi:10.1093/nar/gkm169
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Nucleic Acids Research, 2007, Vol. 35, No. 9 3128-3143
© 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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Base-specific spin-labeling of RNA for structure determination
1Institute of Organic Chemistry and Chemical Biology, J. W. Goethe-University, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany, 2Institute of Physical and Theoretical Chemistry, J. W. Goethe-University, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany and 3Center of Biological Magnetic Resonance, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
*To whom correspondence should be addressed.Tel: +49-69-798-29150; Fax: +49-69-798-29148; Email: joachim.engels{at}chemie.uni-frankfurt.de Correspondence may also be addressed to Olav Schiemann. Tel: +49-69-798-29786; Fax: +49-69-798-29404; Email: o.schiemann{at}prisner.de
Received January 30, 2007. Revised March 6, 2007. Accepted March 6, 2007.
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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