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. 1997 Dec 1;25(23):4758–4763. doi: 10.1093/nar/25.23.4758

An efficient and economic site-specific deuteration strategy for NMR studies of homologous oligonucleotide repeat sequences.

X Huang 1, P Yu 1, E LeProust 1, X Gao 1
PMCID: PMC147113  PMID: 9365253

Abstract

We describe herein the use of a 2H-labeling strategy to achieve specific assignments of considerably overlapped cross peaks in the 1H-NMR spectrum of a DNA trinucleotide repeat sequence. Our strategy focuses on site-specific 2H-labeling of base moieties to simplify the NMR spectral regions which contain the major portion of the structural information. To achieve efficient preparation of 2H8- or 2H6-labeled DNA and RNA nucleosides and nucleotides, the existing synthetic and purification procedures were significantly improved. Our experiments demonstrate that pyrimidine H6 deuteration reactions may be carried out using non-deuterated base reagents with DMSO-d6 as a 2H donor. These reactions are simple and economic to perform and produce base deuterated nucleosides and nucleotides in high yield. The 2H-labeled residues have been incorporated into oligonucleotides with minor modifications of the existing reaction conditions. Using the homologous CGG repeat sequence, d(CGG)5, as an example, the effectiveness of the site-specific base deuteration strategy is demonstrated. In the otherwise extensively overlapped spectra of d(CGG)5, 2H-labeling has permitted unambiguous identification of a sequential connectivity at a central CG step and confirmation of several other NOE assignments. This information is critical for elucidation of the structure and the folding of the CGG repeat sequences and will contribute to the intensive effort to understand the mechanisms of triplet expansion, which has been implicated in the development of a number of hereditary neurodegenerative diseases. In addition to the two dimensional spectral simplification in a key spectral region using site-specific 2H8/2H6-labeling, the potential applications of the prescribed strategy in homonuclear three dimensional experiments are also discussed.

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Selected References

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