This project focuses on the unusual nucleic acid conformation of quadruplexes, built upon the guanine quartet. Evidence for the presence of these structures in biological systems, and their functional roles, has been mounting over the past five years: for example, quadruplex structures have been proposed to play a role in regulating telomerase activity, in initiation of transcription, in translational repression, and in binding specifically to a substantial number of proteins. Furthermore, applications have been described for quadruplexes as a folding motif for protein recognition, and as diagnostic agents, in addition to antiviral and anticancer chemotherapeutic agents. Structures based on guanine quartets are unique among nucleic acids in their dependence on specific binding of certain stabilizing cations. Our long term goal is an enhanced understanding of the thermodynamics, structure and interactions of this class of nucleic acids, in particular, in terms of the specific nature of cation binding, the properties of folded RNA quadruplexes and their differences with respect to DNA quadruplexes, and their interactions with other molecules of biological interest. In particular, we propose thermodynamic analyses of a series of biologically significant guanine-rich sequences, both in DNA and RNA forms, structural analyses of these sequences, development of methods to assess the molecularity of quadruplex forms and a detailed investigation of a catalytic DNA construct and its interactions with hemin. Experimental techniques to be employed in this goal include UV/CD spectroscopy, isothermal titration calorimetry, NMR, and fluorescence, and equilibrium sedimentation. This combination of approaches will afford a detailed examination of the conformation and interaction properties of the very important nucleic acid species. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067607-04
Application #
7071879
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2003-06-01
Project End
2009-05-31
Budget Start
2006-06-01
Budget End
2009-05-31
Support Year
4
Fiscal Year
2006
Total Cost
$260,800
Indirect Cost
Name
University of California San Francisco
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
Majhi, Pinaki R; Qi, Jianying; Tang, Chung-Fei et al. (2008) Heat capacity changes associated with guanine quadruplex formation: an isothermal titration calorimetry study. Biopolymers 89:302-9
Qi, Jianying; Shafer, Richard H (2007) Human telomere quadruplex: refolding and selection of individual conformers via RNA/DNA chimeric editing. Biochemistry 46:7599-606
Smirnov, Ivan V; Shafer, Richard H (2007) Electrostatics dominate quadruplex stability. Biopolymers 85:91-101
Majhi, Pinaki R; Shafer, Richard H (2006) Characterization of an unusual folding pattern in a catalytically active guanine quadruplex structure. Biopolymers 82:558-69
Tang, Chung-Fei; Shafer, Richard H (2006) Engineering the quadruplex fold: nucleoside conformation determines both folding topology and molecularity in guanine quadruplexes. J Am Chem Soc 128:5966-73
Qi, Jianying; Shafer, Richard H (2005) Covalent ligation studies on the human telomere quadruplex. Nucleic Acids Res 33:3185-92
Mondragon-Sanchez, J A; Liquier, J; Shafer, R H et al. (2004) Tetraplex structure formation in the thrombin-binding DNA aptamer by metal cations measured by vibrational spectroscopy. J Biomol Struct Dyn 22:365-73