Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances

Olav Schiemann, N. Piton, J. Plackmeyer, Bela Ernest Bode, TF. Prisner, J.W. Engels

Research output: Contribution to journalArticlepeer-review

116 Citations (Scopus)

Abstract

In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of similar to 10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to similar to 80 bases in length and PELDOR yields reliably spin-spin distances up to similar to 6.5 nm. The synthesis of TPA takes similar to 5 days and spin labeling together with purification similar to 4 days. The PELDOR measurements usually take similar to 16 h and data analysis from an hour up to several days depending on the extent of analysis.

Original languageEnglish
Pages (from-to)904-923
Number of pages20
JournalNature Protocols
Volume2
Issue number4
DOIs
Publication statusPublished - Apr 2007

Keywords

  • ELECTRON DOUBLE-RESONANCE
  • TRANSFER RIBONUCLEIC-ACID
  • COLI TRANSFER-RNA
  • DUPLEX DNA
  • PARAMAGNETIC-RESONANCE
  • DEPENDENT DYNAMICS
  • SITE
  • SPECTROSCOPY
  • SEQUENCE
  • PROBE

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