High-resolution EPR distance measurements on RNA and DNA with the non-covalent Ǵ spin label.

Marcel Heinz,Nicole Erlenbach,Nilesh R Kamble,Snorri Th Sigurdsson,Lukas S Stelzl,Thomas F Prisner,Gerhard Hummer,Grace Thierolf

doi:10.1093/nar/gkz1096

Marcel Heinz, Nicole Erlenbach + Show 6 more

Open Access

https://doi.org/10.1093/nar/gkz1096

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Abstract

Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed n}{}bfacute{G} (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize n}{}bfacute{G}, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled n}{}bfacute{G} bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. n}{}bfacute{G} remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound n}{}bfacute{G} labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. n}{}bfacute{G} and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

Highlights

Pulsed electron paramagnetic resonance (EPR) experiments can be used to probe the global structure and flexibility of nucleic acids with high resolution [1]
In molecular dynamics (MD) simulations of 2 ␮s, the two Glabels remained in the abasic sites of the double-stranded DNA (dsDNA)
The atom position root-mean-square fluctuation (RMSF) of the labeled dsDNA trajectory, aligned onto the averaged structure, revealed base flexibility at the 5 -ends and 3 -ends of each strand, as expected (Figure 2B and Supplementary Figure S4)

Summary

Introduction

Pulsed EPR experiments can be used to probe the global structure and flexibility of nucleic acids with high resolution [1]. PELDOR provides detailed information on distances even in highly dynamic systems, where traditional structure determination is not possible, reporting on the conformational flexibility of proteins, nucleic acids and protein-nucleic acid complexes [10,11]. PELDOR can even provide angular information [12]. PELDOR experiments require the introduction of a pair of spin labels. Flexible spin labels complicate the determination of high-resolution distances [13] and do not permit the extraction of angular information. The synthesis of the Citself and especially of C -labeled nucleic acids remains difficult, limiting the wide applicability of high-resolution pulsed EPR experiments on nucleic acids

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Results

Conclusion