Applying Thymine Isostere 2,4-Difluoro-5-methylbenzene as an Assignment Tool and Probe of Purine-rich DNA Dynamics Using Proton NMR Spectroscopy

Published

June 30, 2015

Author(s)

Robert G. Brinson, Jennifer T. Miller, Kahn Jason, Stuart F. Le Grice, John P. Marino

Abstract

Proton assignment of NMR spectra of oligonucleotide duplexes can become difficult with increasing helical length due to cross peak overlap. Here we describe an alternative to 15N and 13C labeling that facilitates assignment via single site substitution with the thymine isostere 2,4-difluoro-5-methylbenzene (dF) into highly redundant DNA sequences. The impact of the method in assigning intractable spectra and analyzing structure and dynamics is demonstrated using A-Tract and TATA Box DNA and two polypurine tract-containing RNA:DNA hybrids derived from HIV-1 and Saccharomyces cerevisiae retrotransposon. Only resonances proximal to the site of dF substitution exhibit sizable chemical shift changes, allowing chemical shift mapping of resonances from unaffected residues distal to the site of modification back to the unmodified sequence. Furthermore, we illustrate that incorporation of dF substitution subtly alters the global helical dynamics on the Ty3 PPT and the TATA Box DNA duplexes. We demonstrate these effects with an orthogonal functional assay with the binding of the TATA Box binding protein to the TATA Box DNA duplex, suggesting that dF stabilizes an alternative DNA conformation.

Citation

Methods In Enzymology

Volume

30

Issue

566

Pub Type

Journals

Keywords

Polypurine tract, A-tracts, TATA Box, TBP, 2, 4-difluoro-5-methylbenzene (dF), high-resolution NMR

Citation

Brinson, R. , Miller, J. , Jason, K. , Le, S. and Marino, J. (2015), Applying Thymine Isostere 2,4-Difluoro-5-methylbenzene as an Assignment Tool and Probe of Purine-rich DNA Dynamics Using Proton NMR Spectroscopy, Methods In Enzymology (Accessed October 17, 2025)