Anomalously Rapid Hydration Water Diffusion Dynamics Near DNA Surfaces

The emerging Overhauser effect dynamic nuclear polarization (ODNP) technique measures the translational mobility of water within the vicinity (5–15 Å) of preselected sites. The work presented here expands the capabilities of the ODNP technique and illuminates an important, previously unseen, property of the translational diffusion dynamics of water at the surface of DNA duplexes. We attach nitroxide radicals (i.e., spin labels) to multiple phosphate backbone positions of DNA duplexes, allowing ODNP to measure the hydration dynamics at select positions along the DNA surface. With a novel approach to ODNP analysis, we isolate the contributions of water molecules at these sites that undergo free translational diffusion from water molecules that either loosely bind to or exchange protons with the DNA. The results reveal that a significant population of water in a localized volume adjacent to the DNA surface exhibits fast, bulk-like characteristics and moves unusually rapidly compared to water found in similar probe volumes near protein and membrane surfaces. Control studies show that the observation of these characteristics are upheld even when the DNA duplex is tethered to streptavidin or the mobility of the nitroxides is altered. This implies that, as compared to protein or lipid surfaces, it is an intrinsic feature of the DNA duplex surface that it interacts only weakly with a significant fraction of the surface hydration water network. The displacement of this translationally mobile water is energetically less costly than that of more strongly bound water by up to several <i>k</i>_B<i>T</i> and thus can lower the activation barrier for interactions involving the DNA surface

Investigating Functional DNA Grafted on Nanodiamond Surface Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

Nanodiamonds (NDs) are a new and attractive class of materials for sensing and delivery in biological systems. Methods for functionalizing ND surfaces are highly valuable in these applications, yet reported approaches for covalent modification with biological macromolecules are still limited, and characterizing behaviors of ND-tethered biomolecules is difficult. Here we demonstrated the use of copper-free click chemistry to covalently attach DNA strands at ND surfaces. Using site-directed spin labeling and electron paramagnetic resonance spectroscopy, we demonstrated that the tethered DNA strands maintain the ability to undergo repetitive hybridizations and behave similarly to those in solutions, maintaining a large degree of mobility with respect to the ND. The work established a method to prepare and characterize an easily addressable identity tag for NDs. This will open up future applications such as targeted ND delivery and developing sensors for investigating biomolecules

Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling

The condensation of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shaped protein/RNA complex, and its function requires an RNA component called packaging RNA (pRNA). Current structural information on pRNA is limited, which hinders studies of motor function. Here, we used site-directed spin labeling to map the conformation of a pRNA three-way junction that bridges binding sites for the motor ATPase and the procapsid. The studies were carried out on a pRNA dimer, which is the simplest ring-shaped pRNA complex and serves as a functional intermediate during motor assembly. Using a nucleotide-independent labeling scheme, stable nitroxide radicals were attached to eight specific pRNA sites without perturbing RNA folding and dimer formation, and a total of 17 internitroxide distances spanning the three-way junction were measured using Double Electron–Electron Resonance spectroscopy. The measured distances, together with steric chemical constraints, were used to select 3662 viable three-way junction models from a pool of 65 billion. The results reveal a similar conformation among the viable models, with two of the helices (H_T and H_L) adopting an acute bend. This is in contrast to a recently reported pRNA tetramer crystal structure, in which H_T and H_L stack onto each other linearly. The studies establish a new method for mapping global structures of complex RNA molecules, and provide information on pRNA conformation that aids investigations of phi29 packaging motor and developments of pRNA-based nanomedicine and nanomaterial

Nitroxide Sensing of a DNA Microenvironment: Mechanistic Insights from EPR Spectroscopy and Molecular Dynamics Simulations

The behavior of the nitroxide spin labels 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a) and 1-oxyl-2,2,5,5-tetramethylpyrroline (R5) attached at a phosphorothioate-substituted site in a DNA duplex is modulated by the DNA in a site- and stereospecific manner. A better understanding of the mechanisms of R5a/R5 sensing of the DNA microenvironment will enhance our capability to relate information from nitroxide spectra to sequence-dependent properties of DNA. Toward this goal, electron paramagnetic resonance (EPR) spectroscopy and molecular dynamics (MD) simulations were used to investigate R5 and R5a attached as R_<i>p</i> and S_<i>p</i> diastereomers at phosphorothioate _pSC₇ of d­(CTACTG_pSC₇Y₈TTAG). d­(CTAAAGCAGTAG) (Y = T or U). X-band continuous-wave EPR spectra revealed that the dT₈ to dU₈ change alters nanosecond rotational motions of R_<i>p</i>-R5a but produces no detectable differences for S_<i>p</i>-R5a, R_<i>p</i>-R5, and S_<i>p</i>-R5. MD simulations were able to qualitatively account for these spectral variations and provide a plausible physical basis for the R5/R5a behavior. The simulations also revealed a correlation between DNA backbone B_I/B_II conformations and R5/R5a rotational diffusion, thus suggesting a direct connection between DNA local backbone dynamics and EPR-detectable R5/R5a motion. These results advance our understanding of how a DNA microenvironment influences nitroxide motion and the observed EPR spectra. This may enable use of R5/R5a for a quantitative description of the sequence-dependent properties of large biologically relevant DNA molecules

CRISPR–Cas9 Mediated DNA Unwinding Detected Using Site-Directed Spin Labeling

The RNA-guided CRISPR–Cas9 nuclease has revolutionized genome engineering, yet its mechanism for DNA target selection is not fully understood. A crucial step in Cas9 target recognition involves unwinding of the DNA duplex to form a three-stranded R-loop structure. Work reported here demonstrates direct detection of Cas9-mediated DNA unwinding by a combination of site-directed spin labeling and molecular dynamics simulations. The results support a model in which the unwound nontarget strand is stabilized by a positively charged patch located between the two nuclease domains of Cas9 and reveal uneven increases in flexibility along the unwound nontarget strand upon scissions of the DNA backbone. This work establishes the synergistic combination of spin-labeling and molecular dynamics to directly monitor Cas9-mediated DNA conformational changes and yields information on the target DNA in different stages of Cas9 function, thus advancing mechanistic understanding of CRISPR–Cas9 and aiding future technological development