In Vitro Selections for Imidazole-Modified RNA Catalysts

Florance, Cara Lena

Graduate Thesis Or Dissertation

In Vitro Selections for Imidazole-Modified RNA Catalysts Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/sq87bt940

Abstract

In vitro selection from a random RNA library allows for the identification of single sequences capable of performing a desired task. This process has yielded RNA sequences that function as specific and efficient ligands and catalysts for many targets and reactions. This thesis details the development and application of advanced methods for in vitro selection of complex RNA catalysts. Imidazole modifications were used to impart additional functionality to the RNA scaffold in the experiments described herein. The first chapter details a new type of selection that can be used to identify aptamer-like sequences that can not only bind a protein, but also catalyze the addition of a tethered reactive molecule to target residues, forming a covalently bound RNA/protein complex. These sequences are termed reactamers. This method has isolated a sequence that forms a target protein-dependent, denaturing-resistant complex. The presence of a covalent bond, however, has yet to be confirmed. Recommended optimization strategies for future reactamer selections are discussed. The second chapter describes a selection to identify RNA capable of cleaving an amide bond. This experiment did not yield catalysts with the intended activity, however an improved scheme prompted by results during the selection is described. The last chapter details experiments performed on an RNA sequence that can self-circularize which was discovered during the amide cleavage selection. In addition, it appears this sequence forms a four-way junction in the middle of the circle, creating a pinched figure-eight-like structure. It is hypothesized that this junction is a phosphotriester bond. Together, these three reports highlight not only the power of in vitro selections, but also the catalytic versatility of modified RNA.

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