Base-specific spin labeling of RNA for structure determination

N. Piton, Y. Mu, G. Stock, TF. Prisner, Olav Schiemann, J.W. Engels

Research output: Contribution to journalArticlepeer-review

122 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)3128-3143
Number of pages16
JournalNucleic Acids Research
Volume35
Issue number9
DOIs
Publication statusPublished - May 2007

Keywords

  • PAIR DISTANCE DISTRIBUTIONS
  • EPR SPECTROSCOPY
  • NUCLEIC-ACIDS
  • TIKHONOV REGULARIZATION
  • PARAMAGNETIC-RESONANCE
  • HAMMERHEAD RIBOZYME
  • PROTECTING GROUPS
  • PULSED ESR

Fingerprint

Dive into the research topics of 'Base-specific spin labeling of RNA for structure determination'. Together they form a unique fingerprint.

Cite this