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

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 approximately 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 approximately 80 bases in length and PELDOR yields reliably spin-spin distances up to approximately 6.5 nm. The synthesis of TPA takes approximately 5 days and spin labeling together with purification approximately 4 days. The PELDOR measurements usually take approximately 16 h and data analysis from an hour up to several days depending on the extent of analysis.