Open AccessA facile method for attaching nitroxide spin labels at the 5′ terminus of nucleic acids†
Author(s) -
Gian Paola G. Grant,
Peter Z. Qin
Publication year - 2007
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkm240
Subject(s) - nucleic acid , nitroxide mediated radical polymerization , site directed spin labeling , electron paramagnetic resonance , moiety , covalent bond , spin label , polynucleotide , nucleic acid structure , macromolecule , biochemistry , rna , combinatorial chemistry , chemistry , biology , stereochemistry , organic chemistry , nuclear magnetic resonance , polymerization , membrane , gene , polymer , physics , radical polymerization
In site-directed spin labeling (SDSL), a nitroxide moiety containing a stable, unpaired electron is covalently attached to a specific site within a macromolecule, and structural and dynamic information at the labeling site is obtained via electron paramagnetic resonance (EPR) spectroscopy. Successful SDSL requires efficient site-specific incorporation of nitroxides. Work reported here presents a new method for facile nitroxide labeling at the 50 terminus of nucleic acids of arbitrary sizes. T4-polynucleotide kinase was used to enzymatically substitute a phosphorothio- ate group at the 50 terminus of a nucleic acid, and the resulting phosphorothioate was then reacted with an iodomethyl derivative of a nitroxide. The method was successfully demonstrated on both chemically synthesized and naturally occurring nucleic acids. The attached nitroxides reported duplex formation as well as tertiary folding of nucleic acids, indicating that they serve as a valid probe in nucleic acid studies.