Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly13C-labeled deoxyribose ring
✍ Scribed by Akira Ono; Shin-ichi Tate; Yoshiharu Ishido; Masatsune Kainosho
- Publisher
- Springer Netherlands
- Year
- 1994
- Tongue
- English
- Weight
- 405 KB
- Volume
- 4
- Category
- Article
- ISSN
- 0925-2738
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✦ Synopsis
13Cs]-2-Deoxy-D-ribose , synthesized from [13C6]-o-glucose (98% ~3C), was coupled with thymine to give [l',2',3',4',-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3'-phosphoramidite derivative and was then incorporated into a dodecamer 5'-d(CGCGAATTCGCG)-3' by solid-phase DNA synthesis. Preparation of 0.24 gmole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the 13C labels, all of the ~H-IH vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.
Conformational diversity of the sugar moieties in DNA has been considered to be an important issue in elucidation of sequence-specific DNA-protein recognition processes (Saenger, 1984). Structural information on DNA in solution, however, can only be obtained for small oligomers, because of the difficulties in analyzing the 1H NMR spectra of larger DNAs. Heteronuclear multidimensional NMR methods, which have been successfully used for 13C/15N-labeled proteins (