PELDOR study of conformations of double-spin-labeled single- and double-stranded DNA with non-nucleotide inserts

Abstract

DNA fragments were synthesized consisting of 12 nucleotides and containing non-nucleotide inserts of different length in the middle. Two nitroxide spin labels 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl were attached at the two ends of the molecules. Single-stranded DNAs and double-stranded DNAs (DNA duplexes) in frozen at 77 K glassy water/glycerol solutions were studied using pulsed electron-electron double resonance (PELDOR). The distance distributions between two spin labels in molecules were obtained from PELDOR data using Tikhonov regularization algorithm, and were found to be close to the Gaussian functions. Experimental PELDOR data were fitted by adjusting precisely the maximum position and the width of these functions. The obtained results show that duplexes possess a substantially narrower distribution, as compared to the single-stranded DNAs. Introduction of a non-nucleotide insert 2-hydroxymethyl-3-hydroxy-tetrahydrofuran leads to a slight but nevertheless detectable decrease of the mean distance between two spin labels. This decrease may be attributed to bending of the molecule around the insert site, by an angle of approximately 20 degrees . An introduction of a non-nucleotide insert bis-(di-ethyleneglycol)-phosphate results in a remarkable broadening of the distance distribution. The results evidence that PELDOR of spin-labeled DNA molecules may be used as a "molecular ruler" for studying the influence of local damages on the DNA conformations.

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

“…Using the mean distances obtained from the orientation-averaged PELDOR time traces for the bound and unbound duplexes and trigonometry (41) yields a bending angle of 42°, which is in good agreement with a bending angle of ∼45° observed from crystal structure (39) and the 48.5° obtained from PELDOR measurements on the covalently spin-labeled DNA (Supplementary Information, Supplementary Figure S4). Although the change in the observed interspin distance corresponds nicely to a 42° bending of the DNA, this approach does not take into account the fact that the spin labels stick out from the DNA helix and change their relative orientation when LacI bends the DNA.…”

Physiological-Temperature Distance Measurement in Nucleic Acid using Triarylmethyl-Based Spin Labels and Pulsed Dipolar EPR Spectroscopy

“…1,2 For this purpose, two spin labels are introduced at selected sites of biological system and the value of dipolar interaction between them is measured by Double Electron-Electron Resonance (DEER or PELDOR) [3][4][5] or Double Quantum Coherence (DQC) 6,7 techniques. [12][13][14][15][16][17] As a rule, PELDOR/DEER experiments with nitroxide spin labels are carried out in frozen solutions at low temperatures ~50-80 K. 9 The freezing allows the two following requirements of pulsed dipolar EPR to be simultaneously fulfilled. 8,9 Most often, SDSL uses stable nitroxide radicals attached to proteins 10,11 , DNA and RNA.…”

Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

“…Since the time axis t for the inversion pulse can be chosen to start before the Hahn echo, one actually records negative times for the dipolar evolution, but more importantly one has no dead time for the dipolar evolution. In the three-pulse PELDOR version ( Figure 1A), a dead-time occurs because the inversion pulse cannot usually be applied on the π-pulse of the detection sequence [46] without introducing too strong artefacts at the beginning of the time trace.…”

“…In the field of nucleic acids, PELDOR has been used to follow the conversion of two complementary hairpin RNAs into a duplex RNA [25], ligand binding to the neomycin riboswitch [64] and magnesium(II)-ion-induced folding of the tertiary stabilized hammerhead ribozyme [65]. Structures of damaged nucleic acids yielding bended DNA double helices have also been studied [46,66].…”