Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy.
A new approach is described for the insertion of nitroxide spin-labels at specific positions within DNA oligomers. The latter bioconjugaison strategy is based on a click chemistry 1,3-dipolar cycloaddition between a spin-labeling reagent, namely the 4-azido-TEMPO, and alkyne modified uridine-containing oligonucleotides. This highly efficient labeling method was applied for site-specific incorporation of two TEMPO units within a set of double-stranded DNA constructs. Then the determination of the inter-nitroxide distances was achieved by using a four-pulses DEER technique that successfully validates the new site-directed spin labeling strategy.
Flaender
M
M
Laboratoire des Lésions des Acides Nucléiques, SCIB/UMR E3 CEA-UJF, INAC, CEA-Grenoble, F-38054 Grenoble Cedex 9, France.
Sicoli
G
G
Fontecave
Th
T
Mathis
G
G
Saint-Pierre
C
C
Boulard
Y
Y
Gambarelli
S
S
Gasparutto
D
D
D001386:Azides / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D003497:Cyclic N-Oxides / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D013113:Spin Labels; D014529:Uridine / Q000031:analogs & derivatives / Q000737:chemistry
D001386:Azides; D003497:Cyclic N-Oxides; D015342:DNA Probes; D013113:Spin Labels; D014529:Uridine