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

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 $\bf\acute{G}$ (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 $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ 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. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

Precise Distance Measurements in DNA G‐Quadruplex Dimers and Sandwich Complexes by Pulsed Dipolar EPR Spectroscopy

“…Mean Cu 2+ –Cu 2+ distances and corresponding distributions for all dimers are listed in Table 2. The distance distributions achieved in this work were approximately 5 to 10 times narrower than those obtained for DNA and RNA structures labeled either with nitroxide‐ [53, 55, 57–59] or other, less structurally confined Cu 2+ ‐based [51, 63] spin labels. Such narrow distance distributions not only highlight the pronounced rigidity of our Cu(pyridine) 4 spin label within its G‐quadruplex environment, but also demonstrate the overall defined structure adopted by the G‐quadruplex dimers investigated in this work.…”

“…[41][42][43][44][45][46][47][48][49][50][51] While PDEPR is most commonly used to determine structural constraints in protein systems, its application to distance measurements within nucleic acids has been steadily expanding. [51][52][53][54][55][56][57][58] So far, few reports describe PDEPR-based investigations on G-quadruplexes, [59][60][61] including in-cell spin-labeled G-quadruplexes [62] and quadruplex-metal complex adducts. [63,64] Recently, we incorporated new Cu 2+ -based spin labels into tetramolecular DNA G-quadruplexes of varying Gtetrad count, which allowed us to determine intramolecular distances within the secondary structure with unprecedented accuracy.…”

“…The potential of Cu 2+ ions as spin labels for PDEPR‐based studies has been well documented in the literature over the past years [41–51] . While PDEPR is most commonly used to determine structural constraints in protein systems, its application to distance measurements within nucleic acids has been steadily expanding [51–58] . So far, few reports describe PDEPR‐based investigations on G‐quadruplexes, [59–61] including in‐cell spin‐labeled G‐quadruplexes [62] and quadruplex‐metal complex adducts [63, 64] …”

Noncovalent Spin‐Labeling of DNA and RNA Triplexes

“…[19 -23] There are examples of noncovalent spin-labeling of nucleic acids, such as the malachite green aptamer [24] and designed abasic sites in nucleic acid duplexes. [25][26][27][28][29] Of several purine-nitroxide derivatives that were recently prepared for noncovalent labeling (Figure 1, 1-5 and Ǵ), Ǵ (G-spin) was found to have high affinity to abasic sites in RNA opposite C, with an equilibrium dissociation constant (K d ) of 1.46 · 10 À 7 M at 20°C. [26] Here, we report the evaluation of purinenitroxides as spin labels for rationally designed purinebinding triple helices containing nucleotide gaps (I -III, Figure 1).…”

“…Nitroxide spin‐labels are good reporter groups for electron paramagnetic resonance (EPR) spectroscopy, which is a useful spectroscopic technique for the study of the structure and dynamics of biomolecules . There are examples of noncovalent spin‐labeling of nucleic acids, such as the malachite green aptamer and designed abasic sites in nucleic acid duplexes . Of several purine‐nitroxide derivatives that were recently prepared for noncovalent labeling ( Figure , 1–5 and Ǵ ), Ǵ (G‐spin) was found to have high affinity to abasic sites in RNA opposite C, with an equilibrium dissociation constant (K d ) of 1.46 ⋅ 10 −7 m at 20 °C .…”