Structure and Flexibility of Copper‐Modified DNA G‐Quadruplexes Investigated by ¹⁹F ENDOR Experiments at 34 GHz**

Abstract

DNA G‐quadruplexes (GQs) are of great interest due to their involvement in crucial biological processes such as telomerase maintenance and gene expression. Furthermore, they are reported as catalytically active DNAzymes and building blocks in bio‐nanotechnology. GQs exhibit remarkable structural diversity and conformational heterogeneity, necessitating precise and reliable tools to unravel their structure‐function relationships. Here, we present insights into the structure and conformational flexibility of a unimolecular GQ with high spatial resolution via electron‐nuclear double resonance (ENDOR) experiments combined with Cu(II) and fluorine labeling. These findings showcase the successful application of the 19F‐ENDOR methodology at 34 GHz, overcoming the limitations posed by the complexity and scarcity of higher‐frequency spectrometers. Importantly, our approach retains both sensitivity and orientational resolution. This integrated study not only enhances our understanding of GQs but also expands the methodological toolbox for studying other macromolecules.

Extending the Range of Distances Accessible by 19F Electron–Nuclear Double Resonance in Proteins Using High-Spin Gd(III) Labels

“…It is also possible to analyze the set of spectra in Figure individually, which is usually done when the anisotropy is determined by g -anisotropy. , In this case, information regarding the orientation of the electron–nuclear dipolar vector in the framework of the g -tensor is obtained and, in turn, provides additional important geometrical information . This approach, however, comes with several caveats for cases when the anisotropy is dominated by the Gd­(III) ZFS.…”

“…Finally, performing these measurements at the Q-band, taking advantage of currently easily accessible pulsed EPR spectrometers, is possible, and 19 F ENDOR distance measurements at the Q-band have already been reported for trityl- and Cu­(II)-labeled biomolecules. , However, at lower frequencies, interference between 19 F and 1 H ENDOR lines may occur, especially for high-spin electron transitions, since the corresponding 1 H ENDOR lines can extend far out from the Larmor frequency according to eq . If this is the case, subtraction of spectra obtained in the absence of 19 F is necessary, as recently shown for Cu­(II)– 19 F ENDOR . In addition, the smaller thermal spin polarization at the Q-band may be limiting.…”

“…In ENDOR, hyperfine interactions between the electron spin in a paramagnetic center and the surrounding nuclei are measured. Traditionally, it has been applied to extract the local spatial and electronic structure of intrinsic paramagnetic metal ions or metal clusters in proteins, − while, at present, ENDOR experiments for distance determination on spin-labeled biomolecules are being developed. − For measuring weak hyperfine interactions, which are dipolar in nature, Mims ENDOR is the technique of choice, as demonstrated for a nitroxide label situated 1 nm away from a 31 P nucleus in a membrane bilayer …”

“…Trityl , and Gd­(III) , labels have been also used for distance determination by 19 F ENDOR, with the advantage of not requiring a set of orientation selection measurements owing to the isotropic nature of their EPR spectrum. Recently, phenoxyl and Cu­(II) have also been used for distance measurements. Going beyond in vitro measurements, in-cell Gd­(III)– 19 F ENDOR significantly expanded the scope of this technique …”

“…If this is the case, subtraction of spectra obtained in the absence of 19 F is necessary, as recently shown for Cu(II)− 19 F ENDOR. 26 In addition, the smaller thermal spin polarization at the Q-band may be limiting. To achieve the same thermal occupancy of the low-lying levels at the Q-band, an ∼3 times lower temperature has to be employed compared to the W-band.…”