Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Frequency-Chirped Magic Angle Spinning Dynamic Nuclear Polarization Combined with Electron Decoupling.

Magic angle spinning (MAS) dynamic nuclear polarization (DNP) increases the signal intensity of solid-state nuclear magnetic resonance. DNP typically uses continuous wave (CW) microwave irradiation close to the resonance frequency of unpaired electron spins. In this study, we demonstrate that frequency-chirped microwaves improve DNP performance under MAS. By modulating the gyrotron anode potential, we generate a train of microwave chirps with a maximum bandwidth of 310 MHz and a maximum incident power on the spinning sample of 18 W. We characterize the efficiency of chirped DNP using the following polarizing agents: TEMTriPol-1, AsymPolPOK, AMUPol, and Finland trityl. The effects of different chirp widths and periods are analyzed at different MAS frequencies and microwave powers. Furthermore, we show that chirped DNP can be combined with electron decoupling to improve signal intensity by 59%, compared to CW DNP without electron decoupling, using Finland trityl as a polarizing agent.

Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

Structure and Internal Dynamics of Short RNA Duplexes Determined by a Combination of Pulsed EPR Methods and MD Simulations.

We used EPR spectroscopy to characterize the structure of RNA duplexes and their internal twist, stretch and bending motions. We prepared eight 20-base-pair-long RNA duplexes containing the rigid spin-label Çm, a cytidine analogue, at two positions and acquired orientation-selective PELDOR/DEER data. By using different frequency bands (X-, Q-, G-band), detailed information about the distance and orientation of the labels was obtained and provided insights into the global conformational dynamics of the RNA duplex. We used 19F Mims ENDOR experiments on three singly Çm- and singly fluorine-labeled RNA duplexes to determine the exact position of the Çm spin label in the helix. In a quantitative comparison to MD simulations of RNA with and without Çm spin labels, we found that state-of-the-art force fields with explicit parameterization of the spin label were able to describe the conformational ensemble present in our experiments. The MD simulations further confirmed that the Çm spin labels are excellent mimics of cytidine inducing only small local changes in the RNA structure. Çm spin labels are thus ideally suited for high-precision EPR experiments to probe the structure and, in conjunction with MD simulations, motions of RNA.

Förster resonance energy transfer within the neomycin aptamer.

Förster resonance energy transfer (FRET) measurements between two dyes is a powerful method to interrogate both structure and dynamics of biopolymers. The intensity of a fluorescence signal in a FRET measurement is dependent on both the distance and the relative orientation of the dyes. The latter can at the same time both complicate the analysis and give more detailed information. Here we present a detailed spectroscopic study of the energy transfer between the rigid FRET labels Çmf (donor) and tCnitro (quencher/acceptor) within the neomycin aptamer N1. The energy transfer originates from multiple emitting states of the donor and occurs on a low picosecond to nanosecond time-scale. To fully characterize the energy transfer, ultrafast transient absorption measurements were performed in conjunction with static fluorescence and time-correlated single photon counting (TCSPC) measurements, showing a clear distance dependence of both signal intensity and lifetime. Using a known NMR structure of the ligand-bound neomycin aptamer, the distance between the two labels was used to estimate κ2 and, therefore, make qualitative statements about the change in orientation after ligand binding with unprecedented temporal and spatial resolution. The advantages and potential applications of absorption-based methods using rigid labels for the characterization of FRET processes are discussed.

Role of Protons in and around Strongly Coupled Nitroxide Biradicals for Cross-Effect Dynamic Nuclear Polarization.

In magic angle spinning dynamic nuclear polarization (DNP), biradicals such as bis-nitroxides are used to hyperpolarize protons under microwave irradiation through the cross-effect mechanism. This mechanism relies on electron-electron spin interactions (dipolar coupling and exchange interaction) and electron-nuclear spin interactions (hyperfine coupling) to hyperpolarize the protons surrounding the biradical. This hyperpolarization is then transferred to the bulk sample via nuclear spin diffusion. However, the involvement of the protons in the biradical in the cross-effect DNP process has been under debate. In this work, we address this question by exploring the hyperpolarization pathways in and around bis-nitroxides. We demonstrate that for biradicals with strong electron-electron interactions, as in the case of the AsymPols, the protons on the biradical may not be necessary to quickly generate hyperpolarization. Instead, such biradicals can efficiently, and directly, polarize the surrounding protons of the solvent. The findings should impact the design of the next generation of biradicals.

AsymPol-TEKs as efficient polarizing agents for MAS-DNP in glass matrices of non-aqueous solvents.

Two polarizing agents from the AsymPol family, AsymPol-TEK and cAsymPol-TEK (methyl-free version) are introduced for MAS-DNP applications in non-aqueous solvents. The performance of these new biradicals is rationalized in detail using a combination of electron paramagnetic resonance spectroscopy, density functional theory, molecular dynamics and quantitative MAS-DNP spin dynamics simulations. By slightly modifying the experimental protocol to keep the sample temperature low at insertion, we are able to obtain reproducable DNP-NMR data with 1,1,2,2-tetrachloroethane (TCE) at 100 K, which facilitates optimization and comparison of different polarizing agents. At intermediate magnetic fields, AsymPol-TEK and cAsymPol-TEK provide 1.5 to 3-fold improvement in sensitivity compared to TEKPol, one of the most widely used polarizing agents for organic solvents, with significantly shorter DNP build-up times of ∼1 s and ∼2 s at 9.4 and 14.1 T respectively. In the course of the work, we also isolated and characterized two diastereoisomers that can form during the synthesis of AsymPol-TEK; their difference in performance is described and discussed. Finally, the advantages of the AsymPol-TEKs are demonstrated by recording 2D 13C-13C correlation experiments at natural 13C-abundance of proton-dense microcrystals and by polarizing the surface of ZnO nanocrystals (NCs) coated with diphenyl phosphate ligands. For those experiments, cAsymPol-TEK yielded a three-fold increase in sensitivity compared to TEKPol, corresponding to a nine-fold time saving.

Simultaneous Localization of Two High Affinity Divalent Metal Ion Binding Sites in the Tetracycline RNA Aptamer with Mn2+-Based Pulsed Dipolar EPR Spectroscopy.

Mg2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg2+ was replaced with paramagnetic Mn2+, which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.

Probing the Conformational Space of the Cannabinoid Receptor 2 and a Systematic Investigation of DNP-Enhanced MAS NMR Spectroscopy of Proteins in Detergent Micelles.

Tremendous progress has been made in determining the structures of G-protein coupled receptors (GPCR) and their complexes in recent years. However, understanding activation and signaling in GPCRs is still challenging due to the role of protein dynamics in these processes. Here, we show how dynamic nuclear polarization (DNP)-enhanced magic angle spinning nuclear magnetic resonance in combination with a unique pair labeling approach can be used to study the conformational ensemble at specific sites of the cannabinoid receptor 2. To improve the signal-to-noise, we carefully optimized the DNP sample conditions and utilized the recently introduced AsymPol-POK as a polarizing agent. We could show qualitatively that the conformational space available to the protein backbone is different in different parts of the receptor and that a site in TM7 is sensitive to the nature of the ligand, whereas a site in ICL3 always showed large conformational freedom.

Frequency-swept dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) improves the sensitivity of NMR spectroscopy by the transfer of electron polarization to nuclei via irradiation of electron-nuclear transitions with microwaves at the appropriate frequency. For fields > 5 T and using g ∼ 2 electrons as polarizing agents, this requires the availability of microwave sources operating at >140 GHz. Therefore, microwave sources for DNP have generally been continuous-wave (CW) gyrotrons, and more recently solid state, oscillators operating at a fixed frequency and power. This constraint has limited the DNP mechanisms which can be exploited, and stymied the development of new time domain mechanisms. We report here the incorporation of a microwave source enabling facile modulation of frequency, amplitude, and phase at 9 T (250 GHz microwave frequency), and we have used the source for magic-angle spinning (MAS) NMR experiments. The experiments include investigations of CW DNP mechanisms, the advantage of frequency-chirped irradiation, and a demonstration of an Overhauser enhancement of ∼25 with a recently reported water-soluble BDPA radical, highlighting the potential for affordable and compact microwave sources to achieve significant enhancement in aqueous samples, including biological macromolecules. With the development of suitable microwave amplifiers, it should permit exploration of multiple new avenues involving time domain experiments.

PyrroTriPol: a semi-rigid trityl-nitroxide for high field dynamic nuclear polarization.

Magic angle spinning (MAS) dynamic nuclear polarization (DNP) has significantly broadened the scope of solid-state NMR to study biomolecular systems and materials. In recent years, the advent of very high field DNP combined with fast MAS has brought new challenges in the design of polarizing agents (PA) used to enhance nuclear spin polarization. Here, we present a trityl-nitroxide PA family based on a piperazine linker, named PyrroTriPol, for both aqueous and organic solutions. These new radicals have similar properties to that of TEMTriPol-I and can be readily synthesized, and purified in large quantities thereby ensuring widespread application. The family relies on a rigid bridge connecting the trityl and the nitroxide offering a better control of the electron spin-spin interactions thus providing improved performance across a broad range of magnetic fields and MAS frequencies while requiring reduced microwave power compared to bis-nitroxides. We demonstrate the efficiency of the PyrroTriPol family under a magnetic field of 9.4, 14.1 and 18.8 T with respect to TEMTriPol-I. In particular, the superiority of PyrroTriPol was demonstrated on γ-Al2O3 nanoparticles which enabled the acquisition of a high signal-to-noise surface-selective 27Al multiple-quantum MAS experiment at 18.8 T and 40 kHz MAS frequency.

Solid-Effect Dynamic Nuclear Polarization in Viscous Liquids at 9.4 T Using Narrow-Line Polarizing Agents.

Dynamic nuclear polarization (DNP) is a hyperpolarization method that is widely used for increasing the sensitivity of nuclear magnetic resonance (NMR) experiments. DNP is efficient in solid-state and liquid-state NMR, but its implementation in the intermediate state, namely, viscous media, is still less explored. Here, we show that a 1H DNP enhancement of over 50 can be obtained in viscous liquids at a magnetic field of 9.4 T and a temperature of 315 K. This was accomplished by using narrow-line polarizing agents in glycerol, both the water-soluble α,γ-bisdiphenylen-ß-phenylallyl (BDPA) and triarylmethyl radicals, and a microwave/RF double-resonance probehead. We observed DNP enhancements with a field profile indicative of the solid effect and investigated the influence of microwave power, temperature, and concentration on the 1H NMR results. To demonstrate potential applications of this new DNP approach for chemistry and biology, we show hyperpolarized 1H NMR spectra of tripeptides, triglycine, and glypromate, in glycerol-d8.

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by 13C-13C correlation DNP-enhanced NMR.

We show that multidimensional solid-state NMR 13C-13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Highly Efficient Polarizing Agents for MAS-DNP of Proton-Dense Molecular Solids.

Efficiently hyperpolarizing proton-dense molecular solids through dynamic nuclear polarization (DNP) solid-state NMR is still an unmet challenge. Polarizing agents (PAs) developed so far do not perform well on proton-rich systems, such as organic microcrystals and biomolecular assemblies. Herein we introduce a new PA, cAsymPol-POK, and report outstanding hyperpolarization efficiency on 12.76 kDa U-13 C,15 N-labeled LecA protein and pharmaceutical drugs at high magnetic fields (up to 18.8 T) and fast magic angle spinning (MAS) frequencies (up to 40 kHz). The performance of cAsymPol-POK is rationalized by MAS-DNP simulations combined with electron paramagnetic resonance (EPR), density functional theory (DFT) and molecular dynamics (MD). This work shows that this new biradical is compatible with challenging biomolecular applications and unlocks the rapid acquisition of 13 C-13 C and 15 N-13 C correlations of pharmaceutical drugs at natural isotopic abundance, which are key experiments for structure determination.

A Carbazole-Derived Nitroxide That Is an Analogue of Cytidine: A Rigid Spin Label for DNA and RNA.

A variety of semirigid and rigid spin labels comprise a valuable arsenal for measurements of biomolecular structures and dynamics by electron paramagnetic resonance (EPR) spectroscopy. Here, we report the synthesis and characterization of rigid spin labels C and Cm for DNA and RNA, respectively, that are carbazole-derived nitroxides and analogues of cytidine. C and Cm were converted to their phosphoramidites and used for their incorporation into oligonucleotides by solid-phase synthesis. Analysis of C and Cm by single-crystal X-ray crystallography verified their identity and showed little deviation from planarity of the nucleobase. Analysis of the continuous-wave (CW) EPR spectra of the spin-labeled DNA and RNA duplexes confirmed their incorporation into the nucleic acids and the line-shape was characteristic of rigid spin labels. Circular dichroism (CD) and thermal denaturation studies of the C-labeled DNAs and Cm-labeled RNAs indicated that the labels are nonperturbing of duplex structure.

The distance between g-tensors of nitroxide biradicals governs MAS-DNP performance: The case of the bTurea family.

Bis-nitroxide radicals are common polarizing agents (PA), used to enhance the sensitivity of solid-state NMR experiments via Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP). These biradicals can increase the proton spin polarization through the Cross-Effect (CE) mechanism, which requires PAs with at least two unpaired electrons. The relative orientation of the bis-nitroxide moieties is critical to ensure efficient polarization transfer. Recently, we have defined a new quantity, the distance between g-tensors, that correlates the relative orientation of the nitroxides with the ability to polarize the surrounding nuclei. Here we analyse experimentally and theoretically a series of biradicals belonging to the bTurea family, namely bcTol, AMUPol and bcTol-M. They differ by the degree of substitution on the urea bridge that connects the two nitroxides. Using quantitative simulations developed for moderate MAS frequencies, we show that these modifications mostly affect the relative orientations of the nitroxide, i.e. the length and distribution of the distance between the g-tensors, that in turn impacts both the steady state nuclear polarization/depolarization as well as the build-up times. The doubly substituted urea bridge favours a large distance between the g-tensors, which enables bcTol-M to provide ∊on/off>200 at 14.1 T/600 MHz/395 GHz with build-up times of 3.8 s using a standard homogenous solution. The methodology described herein was used to show how the conformation of the spirocyclic rings flanking the nitroxide function in the recently described c- and o-HydrOPol affects the distance between the g-tensors and thereby polarization performance.

Water-soluble BDPA radicals with improved persistence.

1,3-Bis(diphenylene)-2-phenylallyl (BDPA) radicals are promising polarizing agents for increasing the sensitivity of NMR spectroscopy through dynamic nuclear polarization (DNP), but have low persistence and solubility in aqueous media. New tetraalkyl/aryl-ammonium derivatives of BDPA are soluble in polar solvents and are highly persistent, with 5-20-fold lower initial rates of degradation than BDPA.

On the Limited Stability of BDPA Radicals.

1,3-Bis(diphenylene)-2-phenylallyl (BDPA)-based radicals are of interest as polarizing agents for dynamic nuclear polarization (DNP). For this purpose, a BDPA-nitroxide biradical, employing a phosphodiester linkage, was synthesized. Contrary to what is commonly assumed, BDPA-derived radicals were observed to have limited stability. Hence, the effects of various factors on the stability of BDPA radicals were investigated. Solvent polarity was found to play a significant role on degradation; a polar BDPA radical was observed to degrade faster in a non-polar solvent, whereas non-polar radicals were more unstable in polar solvents. The rate of decomposition was found to increase non-linearly with increasing radical concentration; a 2-fold increase in concentration led to a 3-fold increase in the rate of degradation. Collectively, these results indicate that the dimerization is a significant degradation pathway for BDPA radicals and indeed, a dimer of one BDPA radical was detected by mass spectrometry.

Nitroxide-Derived N-Oxide Phenazines for Noncovalent Spin-Labeling of DNA.

Two o-benzoquinone derivatives of isoindoline were synthesized for use as building blocks to incorporate isoindoline nitroxides into different compounds and materials. These o-quinones were condensed with a number of o-phenylenediamines to form isoindoline-phenazines in high yields. Subsequent oxidation gave phenazine-di-N-oxide isoindoline nitroxides that were evaluated for noncovalent and site-directed spin-labeling of duplex DNA and RNA that contained abasic sites. Although only minor binding was observed for RNA, the unsubstituted phenazine-N,N-dioxide tetramethyl isoindoline nitroxide showed high binding affinity and selectivity towards abasic sites in duplex DNA that contained cytosine as the orphan base.

Characterization of frequency-chirped dynamic nuclear polarization in rotating solids.

Continuous wave (CW) dynamic nuclear polarization (DNP) is used with magic angle spinning (MAS) to enhance the typically poor sensitivity of nuclear magnetic resonance (NMR) by orders of magnitude. In a recent publication we show that further enhancement is obtained by using a frequency-agile gyrotron to chirp incident microwave frequency through the electron resonance frequency during DNP transfer. Here we characterize the effect of chirped MAS DNP by investigating the sweep time, sweep width, center-frequency, and electron Rabi frequency of the chirps. We show the advantages of chirped DNP with a trityl-nitroxide biradical, and a lack of improvement with chirped DNP using AMUPol, a nitroxide biradical. Frequency-chirped DNP on a model system of urea in a cryoprotecting matrix yields an enhancement of 142, 21% greater than that obtained with CW DNP. We then go beyond this model system and apply chirped DNP to intact human cells. In human Jurkat cells, frequency-chirped DNP improves enhancement by 24% over CW DNP. The characterization of the chirped DNP effect reveals instrument limitations on sweep time and sweep width, promising even greater increases in sensitivity with further technology development. These improvements in gyrotron technology, frequency-agile methods, and in-cell applications are expected to play a significant role in the advancement of MAS DNP.