The primary goals of this proposal are to develop improved methods for probing the structure and dynamics of RNAs by nuclear magnetic resonance (NMR) spectroscopy and the application of NMR and biochemical methods to understand the molecular determinants that RNA aptamers use to recognize their protein targets with high affinity and specificity. State-of-the-art NMR techniques will be used to study an in vitro selected RNA aptamer that is a potent and specific inhibitor of angiogenesis.
Aim 1 will develop improved methods for NMR solution structure determinations of RNA. One focus will be development of novel alignment techniques for measurement of residual dipolar couplings (RDCs) data. These RDCs provide long-range structural information which is critical for structure determinations of extended molecules such as RNAs. Paramagnetic tags for RNA will be developed and used to improve the global and local structures of RNAs. Additionally, recently developed methods for rapid acquisition of high-resolution four-dimensional NMR will be applied to resonance assignments of RNA. These methods have the potential to greatly facilitate NMR structure determinations of RNA by allowing acquisition of high-resolution high-dimensional spectra in a fraction of the time of current methods.
Aim 2 will study the molecular mechanism of inhibition of the RNA aptamer drug, Macugen, for its physiological target, vascular endothelial growth factor (VEGF). Macugen was recently approved by the FDA for treatment of the wet-form of Age Related Macular Degeneration, which is the leading cause of blindness in the elderly. Macugen binds with high affinity and specificity to VEGF;thereby blocking binding of VEGF to cell surface receptors. Multi-dimensional heteronuclear NMR will be used to determine the solution structure of Macugen bound to the heparin-binding domain (HBD) of VEGF. These structural studies will be complemented with biochemical studies of HBD mutants to better understand the specific interactions that lead to high affinity of the VEGF-Macugen complex.
Aim 3 will study a series of RNA aptamers that all bind with high affinity to the HBD. The goal is to identify aptamers that recognize different surfaces of the HBD. This study will identify different mechanisms that aptamers employ to recognize their target protein and will lead to a better understanding of the molecular determinants that cellular RNAs use to recognize their protein partners.
This proposal will study the recently FDA-approved RNA aptamer drug, Macugen, interacting with its cellular target, vascular endothelial growth factor. Macugen is used to treat the wet-form of Age- Related Macular Degeneration, the leading cause of blindness in people over age 50.
|Dodevski, Igor; Nucci, Nathaniel V; Valentine, Kathleen G et al. (2014) Optimized reverse micelle surfactant system for high-resolution NMR spectroscopy of encapsulated proteins and nucleic acids dissolved in low viscosity fluids. J Am Chem Soc 136:3465-74|
|Dallmann, Andre; Simon, Bernd; Duszczyk, Malgorzata M et al. (2013) Efficient detection of hydrogen bonds in dynamic regions of RNA by sensitivity-optimized NMR pulse sequences. Angew Chem Int Ed Engl 52:10487-90|
|Lee, Sang Won; Zhao, Liang; Pardi, Arthur et al. (2010) Ultrafast dynamics show that the theophylline and 3-methylxanthine aptamers employ a conformational capture mechanism for binding their ligands. Biochemistry 49:2943-51|