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

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

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Figure 1: Examples of EPR pulse sequences for distance measurements.
Figure 2: Schematic representation of the PELDOR data processing.
Figure 3: Reaction scheme for the synthesis of TPA 5.
Figure 4: Modified DNA cycle.
Figure 5
Figure 6: Setup as used for the Sonogashira cross-coupling on column.
Figure 7
Figure 8: As an example, this figure shows (a) the PELDOR time trace after time adjustment and normalization, (b) the Fourier transformation of a after background subtraction and (c) the Tikhonov regularization of a.

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Acknowledgements

We thank D. Margraf for helpful advice on this manuscript. This work was supported by the DFG within the SFB 579.

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Correspondence to Olav Schiemann or Joachim W Engels.

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Schiemann, O., Piton, N., Plackmeyer, J. et al. Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat Protoc 2, 904–923 (2007). https://doi.org/10.1038/nprot.2007.97

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