Sequence-Specific Stereomeric Environment in a DNA Duplex Revealed by a Nucleotide-Independent Nitroxide Probe

Abstract

In site-directed spin labeling, structural and dynamic information on a parent macromolecule is obtained by monitoring a covalently linked nitroxide radical using electron paramagnetic resonance (EPR) spectroscopy. Our group have developed a method of attaching nitroxide species, such as 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), to a specific nucleotide position within a target DNA or RNA sequence. The method relies on site-specific introduction of a phosphorothiate during the solid phase chemical synthesis of nucleic acids, and at each given labeling site the nitroxide is attached to one of two phosphorothioate diastereomers (Rp or Sp) in an approximately 50/50 ratio. We have recently reported that variations in DNA structural and dynamic features at the level of an individual nucleotide can be detected using R5a attached to mixed phosphorothioate diastereomers, in which an observed EPR spectrum is presumably a sum of those obtained from either diastereomer (Popova et al., Biochemistry, 2009, 48, 8540-50). In this work, we report X-band EPR spectra of R5a attached to purified Rp and Sp diastereomers at different sites within a B-form DNA duplex. Results are compared to those obtained with mixed nitroxide diastereomers, and advantages and limitations are discussed regarding the necessity of diastereomer separation when probing DNA local environment. Our work is a further step forward in developing a SDSL methodology that may provide a mean for studying structure and dynamics in large DNA molecules.