It has been long accepted that detailed structural information is necessary in order to fully understand the means by which duplex DNA interacts with transcription factors, and in general performs the full range of its biological functions. Accordingly, our view of the structure of DNA has evolved through the years, as high resolution structural models of DNA obtained by X-ray diffraction and solution NMR display sequence specific variations from the canonical B form. It is reasonable to ask how an equally detailed picture of the internal dynamics of DNA would modify our view of these structural variations and thus our understanding of how DNA performs its biological functions. Solid state NMR is an ideal technique for probing the localized dynamics of biopolymers from near solution conditions to the crystalline environment. In addition, a number of novel solid state NMR techniques have been developed in recent years that enable the definition of biomolecular structure with high accuracy and precision over the same broad range of sample conditions, with no molecular weight limit. In the next five years we plan to use solid state NMR structural and dynamical techniques to define the relationship(s) between structure, dynamics and function in DNA and RNA. Specifically we propose the following five stage program: 1) Use solid state NMR to explore the sequence specificity of localized dynamics in DNA oligomers containing binding sites for restriction enzymes, transcription factors and methyltransferases; 2) Use solid state NMR to investigate the dynamic and structural impact of DNA methylation, especially in CpG-rich DNA sequences; 3) Use solid state NMR techniques to investigate the dynamics and structure of the catalytic sites of the hammerhead and hairpin ribozymes; 4) Continue to develop and optimize solid state NMR techniques for structure elucidation in high molecular weight nucleic acids. Extend these NMR techniques to 17.6 Tesla by completing construction of a multi-resonant CPMAS probe, thus aiding in the completion of specific goals 1-4; 5) Synthesize all isotopically labeled DNA and RNA phosphoramidites required to achieve specific goals 1-4.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058914-04
Application #
6498754
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Wehrle, Janna P
Project Start
1999-02-01
Project End
2003-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
4
Fiscal Year
2002
Total Cost
$309,605
Indirect Cost
Name
University of Washington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Shajani, Zahra; Varani, Gabriele (2007) NMR studies of dynamics in RNA and DNA by 13C relaxation. Biopolymers 86:348-59
Miller, Paul A; Shajani, Zahra; Meints, Gary A et al. (2006) Contrasting views of the internal dynamics of the HhaI methyltransferase target DNA reported by solution and solid-state NMR spectroscopy. J Am Chem Soc 128:15970-1
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Olsen, Greg L; Louie, Elizabeth A; Drobny, Gary P et al. (2003) Determination of DNA minor groove width in distamycin-DNA complexes by solid-state NMR. Nucleic Acids Res 31:5084-9
Karlsson, T; Popham, J M; Long, J R et al. (2003) A study of homonuclear dipolar recoupling pulse sequences in solid-state nuclear magnetic resonance. J Am Chem Soc 125:7394-407
Markley, J C; Chirakul, P; Sologub, D et al. (2001) Incorporation of 2'-deoxy-5-(trifluoromethyl)uridine and 5-cyano-2'-deoxyuridine into DNA. Bioorg Med Chem Lett 11:2453-5
Chirakul, P; Litzer, J R; Sigurdsson STh (2001) Preparation of base-deuterated 2'-deoxyadenosine nucleosides and their site-specific incorporation into DNA. Nucleosides Nucleotides Nucleic Acids 20:1903-13
Meints, G A; Karlsson, T; Drobny, G P (2001) Modeling furanose ring dynamics in DNA. J Am Chem Soc 123:10030-8
Meints, G A; Drobny, G P (2001) Dynamic impact of methylation at the M. Hhai target site: a solid-state deuterium NMR study. Biochemistry 40:12436-43

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