1. Technical: This study explores the folded structures of RNAs and their interactions with other molecules. Aptamers isolated from random sequence pools, including RNAs that specifically bind the enzymatic cofactors cyanocobalamin and biotin and the chromophore malachite green, will serve as model systems. Focus is placed on biochemical characterization of these molecules and on the preparation of crystals and solution samples that will enable their analysis by x-ray diffraction and NMR spectroscopy. The structures of cyanocobalamin and biotin aptamer crystals will be determined by X-ray crystallography and the isotopically-labeled malachite green aptamer RNA by solution NMR spectroscopy. This is to understand the specific atomic interactions that stabilize RNA folding, the variety of structural folds available to RNA, the involvement of metals and waters in RNA function, and the nature of conformational changes in RNA that result from its interaction with other molecules. The educational component of this project aims to use structural biology for introducing students to the physical sciences through a combination of both classroom and laboratory instruction. New and modified curricula in eukaryotic molecular biology and structural biology, supplemented by the development of computer-based educational materials, will be developed to establish the relationship between macromolecular structure and function. Selected number of students will be encouraged to engage in the research.
2. Non-technical: The capacity for ribonucleic acids (RNAs) to fold into specific conformations and thereby obtain functional properties (e.g. the ability to bind molecules or to catalyze reactions) has been increasingly appreciated, in part through experiments in which RNAs have been evolved de novo from pools of random sequence molecules. This recognition has fueled speculation about the involvement of RNAs in many biological processes (e.g. ribosome-directed protein synthesis) and supported theories favoring an RNA-based biology as a precursor to modern life. Our understanding of the structural basis for RNA function, critical for addressing these issues, remains poor in part because few RNA structures have been determined at atomic resolution. In vitro-evolved ligand-binding RNAs will serve as model systems for understanding RNA structure and its relationship to RNA function. X-ray crystallography and NMR spectroscopy will be used in this study to determine high resolution models for three different RNA-ligand complexes. Analysis of these structures will reveal the mechanisms by which specific RNA sequences adopt unique structures and how these structures enable specific interactions with other molecules. Integrating these studies with teaching, this study provides undergraduate students with basic skills in structural biology. Enhancements to existing eukaryotic molecular biology curricula will emphasize the concept that biological processes are driven by molecular interactions specified by the folded conformations of macromolecules. A new course in structural biology will expand upon this theme with the additional goal of providing interdisciplinary training in applied physical and computational sciences.
