Phosphorothioates oligonucleotides, strands of DNA in which a non-bridging oxygen atom in the phosphate backbone is replaced with a sulfur atom, have many applications to both drug development and spectroscopy: 1) Since phosphothioate oligonucleotides resist degradation by cellular mechanisms, they hold much potential to be used as an anti-sense anti-viral drug. 2) In the field of spectroscopy, phosphorothioate oligonucleotides are used as a site for the attachment of a paramagnetic molecular tag for EPR studies. This method of site-directed spin labeling has been used to monitor conformational changes in the DNA backbone. Using a phosphorothioate, however, introduces a chiral center on the phosphorous of the DNA backbone that was not previously there, and thus creates two distinct diastereomers: Rp and Sp. Here we use a combination of one-dimensional and two-dimensional NMR techniques to study the effects of the two different diastereomers on the dodecamer ACACACsGTGTGT which contains the Cre binding site.  We quantified the structure and dynamics of the DNA backbone, specifically %BII values and transition barriers, in both a sequence containing a phosphorothioate and a native sequence. Structurally, we have shown that a sample with a racemic mixture of the Sp and Rp diastereomers behaves as two distinct samples, especially in the region of the phosphorothioate substitution, as indicated in the chemical shifts of both the phosphorus and  H3’ protons on the dioxyribose adjacent to the phosophorthioate. Dynamically we can draw conclusions about the relative stability and dynamics of the two diastereomers using variable temperature NMR and complete lineshape analysis to provide the barrier for BI/BII transitions.  The results of this study should better inform the use of phosphorothioates in spectroscopic and pharmaceutical applications.

Difficulties with diastereomers: The effect of a chiral phosphorothioate on the backbone conformations and dynamics of DNA: A preliminary study of the Cre Sequence

251st National Meeting (2016)

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