title: Applying Thymine Isostere 2,4-Difluoro-5-Methylbenzene as a NMR Assignment Tool and Probe of Homopyrimidine/Homopurine Tract Structural Dynamics.
abstract: Proton assignment of nuclear magnetic resonance (NMR) spectra of homopyrimidine/homopurine tract oligonucleotides becomes extremely challenging with increasing helical length due to severe cross-peak overlap. As an alternative to the more standard practice of (15)N and (13)C labeling of oligonucleotides, here, we describe a method for assignment of highly redundant DNA sequences that uses single-site substitution of the thymine isostere 2,4-difluoro-5-methylbenzene (dF). The impact of this approach in facilitating the assignment of intractable spectra and analyzing oligonucleotide 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 the Saccharomyces cerevisiae long-terminal repeat-containing retrotransposon Ty3. Only resonances proximal to the site of dF substitution exhibit sizable chemical shift changes, providing spectral dispersion while still allowing chemical shift mapping of resonances from unaffected residues distal to the site of modification directly back to the unmodified sequence. It is further illustrated that dF incorporation can subtly alter the conformation and dynamics of homopyrimidine/homopurine tract oligonucleotides, and how these NMR observations can be correlated, in the cases of the TATA box DNA, with modulation in the TATA box-binding protein interaction using an orthogonal gel assay.
authors.affiliation: Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and the University of Maryland, Rockville, Maryland, USA. Electronic address: brinson@ibbr.umd.edu.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Brinson
authors.affiliation: Basic Research Laboratory, NCI-Frederick National Institutes of Health, Frederick, Maryland, USA.
authors.forename: Jennifer T
authors.initials: JT
authors.lastname: Miller
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Kahn
authors.affiliation: Basic Research Laboratory, NCI-Frederick National Institutes of Health, Frederick, Maryland, USA.
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.affiliation: Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and the University of Maryland, Rockville, Maryland, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Marino
mesh_terms: D001483:Base Sequence / Q000235:genetics; D008279:Magnetic Resonance Imaging / Q000379:methods*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D016385:TATA Box / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000737:chemistry*
chemical_list: D009841:Oligonucleotides; D011687:Purines; D011743:Pyrimidines; D035181:TATA-Box Binding Protein; D012313:RNA; C030986:pyrimidine; C030985:purine
keywords: 2,4-Difluoro-5-methylbenzene; A-tracts; High-resolution NMR; Polypurine tract; TATA box; TATA box-binding protein