A Nucleotide-Independent Nitroxide Probe Reports on Site-Specific Stereomeric Environment in DNA

作者：Popova Anna M; Qin Peter Z

来源：Biophysical Journal, 2010, 99(7): 2180-2189.

DOI：10.1016/j.bpj.2010.08.005

In this report, stereospecific structural and dynamic features in DNA are studied using the site-directed spin labeling technique. A stable nitroxide radical, 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), was attached postsynthetically to phosphorothioates that were chemically introduced, one at a time, at five sites of a DNA duplex. The two phosphorothioate diastereomers (R(p) or S(p)) were separated, and nitroxide rotational motions were monitored using electron paramagnetic resonance spectroscopy. The resulting spectra vary according to diastereomer identity and location of the labeling site, with R(p)-R5a spectra effectively reporting on local DNA structural features and S(p)-R5a spectra sensing variations in local DNA motions. This establishes R(p)- and S(p)-R5a as unique probes for investigating nucleic acids in a site- and stereospecific manner, which may aid studies of stereospecific DNA/protein interactions. In addition, weighted averages of individual R(p) and S(p) spectra match those of R5a attached to mixed diastereomers. This suggests that R5a linked to mixed diastereomers reports on the composite behaviors of R(p)- and S(p)-R5a and is useful in initial probing of the DNA local environment. This work advances understanding of R5a/DNA coupling, and is a key step forward in developing a nucleotide-independent spectroscopic probe for studying nucleic acids.

• 出版日期2010-10-6