The overall goal of the work described in this proposal is aimed at understanding the range of conformations that DNA can adopt, both in vivo and in vitro, and the principles underlying the folding of DNA into these non-B-DNA conformations. To this end, we propose to investigate two classes of folded DNA molecules: (1) DNA triplexes and (2) DNA aptamers. Multidimensional proton and heteronuclear NMR methods will be used to study the conformation and dynamics of the DNA oligonucleotides. DNA triplexes: In order to elucidate the factors governing sequence specific recognition of DNA by third strand binding to form triplexes, we propose to investigate the structures and cation dependence of several different triplexes and related structures. These include DNA triplexes with modified bases, ribose and 2'Omethyl ribose third strands, purine third strands, and crossover triplexes. We also propose to determine optimal loop sequences for intramolecular triplexes. Studies of a novel triplex-guanine 'clamp' are also planned. DNA aptamers: Aptamers are RNA or DNA molecules which have been selected, from a large pool of oligonucleotides containing a region of random nucleotide sequence, for binding to a specific target molecule. In collaboration with the Szostak laboratory, we will determine the structures of a variety of DNA aptamers which have been selected for binding to the cofactors ATP. GTP, biotin, and riboflavin. These will provide insight into the tertiary structure of nucleic acid binding pockets for protein cofactors. Ultimately, we also plan to determine the structures of DNA catalysts which contain cofactor binding sites. Since many of the folded oligonucleotides which we propose to study are too large to obtain well-defined structures using 1H spectroscopy alone, we plan to develop and optimize methods for synthesizing uniformly 15N and/or 13C labeled DNA oligonucleotides. Once the labeled DNA oligonucleotides are obtained, optimal methods for assignment and structure determination using double and triple-resonance NMR experiments will be developed, similar to those already widely used in protein NMR spectroscopy and more recently applied to labeled RNA oligonucleotides. Where possible, complete three-dimensional structures will be refined from starting structures generated by metric matrix distance geometry calculations. An understanding of the three-dimensional non-B-DNA structures that DNA can adopt and the factors that stabilize these conformations is important for a number of reasons. The solution of new DNA structures should lead to a better understanding of the principles of nucleic acid folding, stability, and sequence specific recognition by ligands including other nucleic acids. These studies should also provide a structural basis for potential applications of nucleic acids as pharmaceuticals, biosensors, and diagnostics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037254-11
Application #
2392015
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1986-07-01
Project End
1999-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
11
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Hartman, Elon; Wang, Zhonghua; Zhang, Qi et al. (2013) Intrinsic dynamics of an extended hydrophobic core in the S. cerevisiae RNase III dsRBD contributes to recognition of specific RNA binding sites. J Mol Biol 425:546-62
Wang, Zhonghua; Hartman, Elon; Roy, Kevin et al. (2011) Structure of a yeast RNase III dsRBD complex with a noncanonical RNA substrate provides new insights into binding specificity of dsRBDs. Structure 19:999-1010
Koo, Bon-Kyung; Park, Chin-Ju; Fernandez, Cesar F et al. (2011) Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface. J Mol Biol 411:927-42
Kim, Nak-Kyoon; Theimer, Carla A; Mitchell, James R et al. (2010) Effect of pseudouridylation on the structure and activity of the catalytically essential P6.1 hairpin in human telomerase RNA. Nucleic Acids Res 38:6746-56
Singh, Mahavir; Gonzales, Fernando A; Cascio, Duilio et al. (2009) Structure and functional studies of the CS domain of the essential H/ACA ribonucleoparticle assembly protein SHQ1. J Biol Chem 284:1906-16
Kim, Nak-Kyoon; Zhang, Qi; Zhou, Jing et al. (2008) Solution structure and dynamics of the wild-type pseudoknot of human telomerase RNA. J Mol Biol 384:1249-61
Wu, Haihong; Feigon, Juli (2007) H/ACA small nucleolar RNA pseudouridylation pockets bind substrate RNA to form three-way junctions that position the target U for modification. Proc Natl Acad Sci U S A 104:6655-60
Khanna, May; Wu, Haihong; Johansson, Carina et al. (2006) Structural study of the H/ACA snoRNP components Nop10p and the 3' hairpin of U65 snoRNA. RNA 12:40-52
Qin, Peter Z; Feigon, Juli; Hubbell, Wayne L (2005) Site-directed spin labeling studies reveal solution conformational changes in a GAAA tetraloop receptor upon Mg(2+)-dependent docking of a GAAA tetraloop. J Mol Biol 351:1-8
Wu, Haihong; Finger, L David; Feigon, Juli (2005) Structure determination of protein/RNA complexes by NMR. Methods Enzymol 394:525-45

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