Diastereomer characterizations of nitroxide-labeled nucleic acids.
Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.
Grant
Gian Paola G
GP
Department of Chemistry, University of Southern California, LJS-251, 840 Downey Way, Los Angeles, CA 90089-0744, USA.
Popova
Anna
A
Qin
Peter Z
PZ
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry*; D054735:Phosphorothioate Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*; D012313:RNA / Q000737:chemistry*; D013113:Spin Labels; D013237:Stereoisomerism
D054735:Phosphorothioate Oligonucleotides; D013113:Spin Labels; D009569:Nitric Oxide; D012313:RNA; D004247:DNA