Date of Award

1-1-2009

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Content Description

1 online resource (viii, 154 pages) : illustrations (some color)

Dissertation/Thesis Chair

Li Niu

Committee Members

Paul Toscano, Carla Theimer, Alexander Shekhtman, Hua Shi

Keywords

Enzymatic probing, footprinting, purification of rna, RNA aptamer, secondary structure, UV melting, Receptor-ligand complexes, RNA, Propionic acid, Enzymatic analysis

Subject Categories

Biochemistry

Abstract

Using systematic evolution of ligands by exponential enrichment (SELEX), our lab previously selected a class of competitive RNA aptamers against the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. One aptamer in this class was found to have a minimal, functional sequence of 58 nucleotides. In vitro transcription generates two RNA transcripts with the same sequence, which we named M1 and M2, both of which are required to work together to inhibit the AMPA receptors. Although the M1 and M2 species have the same sequence, they cannot be interconverted through unfolding by denaturation/refolding by renaturation. To probe the secondary structures and the thermodynamic properties of these species, I carried out enzymatic probing, RNA footprinting and UV melting experiments. In these experiments, I used chemically synthesized AN58 (SynAN58) as a control. SynAN58 is a potent inhibitor by itself and possesses a different structure from either M1 or M2 although it shares the same sequence. Specifically, using a combination of ribonucleases, including those that are specific to single-stranded and double-stranded RNAs, I found that each aptamer species has its own unique digestion pattern. These data were used as constraints to predict secondary structures for each of the three aptamers using structure prediction programs that are based on free energy minimization algorithms. Indeed the three species that share the same sequence exhibit different secondary structures. RNA footprinting experiments with each of the aptamer species bound to the extracellular ligand binding domain of the GluR2 AMPA receptor revealed that a unique set of nucleotides from each aptamer species is involved in interaction with the receptor. Using UV melting experiments, I further examined the effects of aptamer concentration, monovalent salt concentration and urea on the unfolding and refolding profiles of the aptamers. All three RNA species had clearly distinct melting profiles with multiple melting transitions. M1, for instance, is found to have the highest melting temperature and the largest number of transitions. On the other hand, the melting profile of M2 is more similar to the SynAN58 profile. These results suggest that all three RNA species have different structures. The thermodynamic parameters obtained from the melting studies were used to characterize unfolding transitions and to correlate with the predicted secondary structures, relating unfolding transitions to structural motifs. A complete unfolding pathway for SynAN58 and a partial unfolding pathway for M2 were determined. Furthermore, the studies indicate that M1 forms a dimer, and that the dimer transition dominates the melting profile. As a result, it is difficult to establish an unfolding mechanism for the monomeric M1. The NMR spectra suggested that M2 appears to have some base pairs in common with SynAN58 and M1. The broad spectra seen for the aptamers suggest that all three species form multiple conformations and have flexible or unstructured regions in their structures, particularly for M1.

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