Site-directed spin-labeling of nucleic acids by click chemistry: detection of abasic sites in duplex DNA by EPR spectroscopy.
This paper describes a spin label that can detect and identify local structural deformations in duplex DNA, in particular abasic sites. The spin label was incorporated into DNA by a new postsynthetic approach using click-chemistry on a solid support, which simplified both the synthesis and purification of the spin-labeled oligonucleotides. A nitroxide-functionalized azide, prepared by a short synthetic route, was reacted with an oligomer containing 5-ethynyl-2'-dU. The conjugation proceeded in quantitative yield and resulted in a fairly rigid linker between the modified nucleotide and the nitroxide spin label. The spin label was used to detect, for the first time, abasic sites in duplex DNA by X-band CW-EPR spectroscopy and give information about other structural deformations as well as local conformational changes in DNA. For example, reduced mobility of the spin label in a mismatched pair with T was consistent with the spin label displacing the T from the duplex. Addition of mercury(II) to this mispair resulted in a substantial increase in the motion of the spin label, consistent with formation of a metallopair between the T and the spin-labeled base that results in movement of the spin label out of the duplex and toward the solution. Thus, reposition of the spin label, when acting as a mercury(II)-controlled mechanical lever, can be readily detected by EPR spectroscopy. The ease of incorporation and properties of the new spin label make it attractive for EPR studies of nucleic acids and other macromolecules.
Jakobsen
Ulla
U
Department of Physics and Chemistry, University of Southern Denmark, Nucleic Acid Center Campusvej 55, 5230 Odense, Denmark.
Shelke
Sandip A
SA
Vogel
Stefan
S
Sigurdsson
Snorri Th
ST
D004247:DNA / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D013113:Spin Labels / Q000138:chemical synthesis
D009841:Oligonucleotides; D013113:Spin Labels; D004247:DNA