Date of Award

Spring 1-1-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Robert T. Batey

Second Advisor

James A. Goodrich

Third Advisor

Deborah S. Wuttke

Fourth Advisor

Roy Parker

Fifth Advisor

Norman R. Pace

Abstract

The utility of RNA as a sensor for in vivo enzyme evolution, metabolomics monitoring, and bacterial control modules is slowly being realized, but is met with some outstanding challenges. Current work to date on genetically encoded RNA-biosensors has focused on independently developing modular sensors or adaptors. The engineering of adaptor domains has been generally successful in their broad application and modularity, with small molecule fluorescent activators and several strategies for gene regulation now at our disposal. The sensor domains primarily consist of naturally occurring riboswitches and synthetic aptamers generated by in vitro selection. The riboswitch aptamer domains have seen wide application due to their modularity and high affinity, yet their diversity is limited to natural availability. In contrast, hundreds of synthetic aptamers have been created, yet their modularity and application has been met with limited success. Several studies have now shown the faults in assuming that the transition from aptamer to device sensor would be trivial. In this work, a recurrent and model RNA motif is used to introduce robust folding and complexity to a limited library that is selected to bind 5-hydroxytryptophan. The introduced peripheral motif benefits the binding and structure of the RNA, producing an aptamer with higher affinity and specificity than sequences lacking the greater fold. The developed workflow relies on deep sequencing and bioinformatics for the identification of robust elements, reducing the need for tedious validation and allowing the immediate screening of candidate sequences. Additionally, when multiple scaffolds are employed to host limited libraries and parallel selections are carried out, a suite of high affinity aptamers are obtained that are functionally related, yet distant in sequence space. This allows for an aptameric screening strategy, where a desired adaptor platform may be selected and candidate sensors screened for function, an ability not readily afforded by traditional selection techniques. These strategies ease constraints on and expedite the development of high affinity RNA devices, adding yet another robust component to the synthetic biologist's toolbox.

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