Abstract
The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.
Highlights
Electron paramagnetic resonance (EPR) spectroscopy can offer unique insight into the structure and dynamics of biomolecules such as DNA by observation of the behaviour of free radicals incorporated into the target of interest [1,2,3]
We show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2 position offers a robust and minimally perturbing tool for obtaining such measurements
Our results show that EPR spectroscopy with 2 -triazole spin-labelled nucleotides can be used as a tool for accurate and precise distance measurements in DNA, providing a sound basis for the exploration of nucleic acid conformation and dynamics
Summary
Electron paramagnetic resonance (EPR) spectroscopy can offer unique insight into the structure and dynamics of biomolecules such as DNA by observation of the behaviour of free radicals incorporated into the target of interest [1,2,3]. The pulsed technique double electron–electron resonance (DEER) is useful for the measurement of distances between two paramagnetic centres (typically 1.5–8 nm), termed ‘spin labels’ (Figure 1) [4,5,6,7,8,9,10] Provided these labels adopt well-defined positions, DEER can afford distance distributions with sub-nanometre precision, as well as information on their relative orientation [8,11]. Such accuracy depends on the rigidity of the tether between the spin label and the biomolecule, balanced against the inevitable, but ideally minimal, structural perturbation imparted by the chemical modification itself [12]. Improved understanding of the positioning of molecular probes on biomolecules would bring significant benefits in structural biology
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