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 ∼20°. 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.