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Laney Graduate School

Emory College

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From Aptamer to Riboswitch: High-throughput Screens and Selections for the Identification and Creation of Synthetic Riboswitches

Lynch, Sean (2009)
Dissertation (169 pages)
Committee Chair / Thesis Adviser: Gallivan, Justin
Committee Members: Lynn, David ; Lutz, Stefan
Research Fields: Chemistry, General
Keywords: Riboswitch; Synthetic Biology
Program: Laney Graduate School, Chemistry
Permanent url: http://pid.emory.edu/ark:/25593/50s1p

Abstract

The discovery of metabolite responsive riboswitches in bacteria supported the
notion that ligand-binding RNA molecules could be used to control bacterial gene
expression. Riboswitches are comprised of small-molecule binding sequences of RNA,
known as "aptamers", linked to "expression platforms", which translate the binding of
a small molecule into a change in gene expression. While the number of engineered
systems described in the literature utilizing RNA-small molecule interactions to control
gene expression is limited, the principles driving their function are straightforward.
Therefore, it is tempting to believe that in vitro selected aptamers could be readily used in
the development of synthetic riboswitches that respond to new ligands.

In this thesis we describe a series of high-throughput screens and selections for
the identification and creation of dynamic synthetic riboswitches in bacteria that respond
to the small molecule, theophylline. We present a high-throughput, enzymatic assay that
successfully identifies synthetic riboswitches with improved dynamic ranges. These new
switches display essentially no background translation in the absence of their
small-molecule effector, and large increases in its presence. Sequence data, coupled with
in vitro and in vivo studies, enabled the development of a model describing their function
where a transcript's secondary structure influences the translation of the downstream
genes.

A second high-throughput screen capable of rapidly identifying synthetic
riboswitches using fluorescent-activated cell sorting (FACS) is also described. The
throughput of this screen approaches that of a genetic selection and was successfully used
to identify riboswitches with capabilities that match or exceed those of most natural
riboswitches. Characterization of these switches indicated the capacity of the ribosome
binding site to dramatically alter the dynamic behavior of a synthetic riboswitch.

Using what we had learned about the function of theophylline riboswitches, we
endeavored to select an RNA aptamers that binds the antibiotic erythromycin and
subsequently attempted to integrate these aptamers into erythromycin-sensitive
riboswitches using a genetic selection scheme. Despite limited success, the analysis of a
single aptamer has revealed features that may guide the semi-rational design of a new
erythromycin riboswitch and future efforts to select RNA aptamers for use in engineered
riboswitches.

Table of Contents

Contents
Chapter 1: Introduction…………………………………………………………………1

1.1 Regulating Gene Expression through Small Molecule-Protein Interactions………….1

1.2 Regulating Gene Expression through Small Molecule-RNA Interactions …………...3

1.3 Engineering Small-Molecule Control of Gene Expression………………...………..10

1.4 References……………………………………………………………….……...........18

Chapter 2: A High-Throughput Screen for Synthetic Riboswitches …………………………………………………………………………….22

2.1 Introduction…………………………………………………………………………..22

2.2 Results and Discussion………………………………………………………………25

2.2.1 Creation of Library of Randomized Mutants………………………………………25

2.2.2 A High-Throughput Screen for Optimally Functioning Riboswitches……………26

2.3 Conclusion…………………………………………………………………………...30

2.4 Experimental………………………………………………………………………....31

2.5 References……………………………………………………………………………33

Chapter 3: Investigations into the Mechanisms of Synthetic Riboswitches ………………………………………………………………...…………..35

3.1 Introduction………………………………………………………………………….35

3.2 Results and Discussion………………………………………………………………37

3.2.1 Sequencing Suggests a Possible Mechanism of Action for Synthetic Riboswitches……………………………………………………………………………..37

3.2.2 The Benefits of N-Terminal Fusions in the Design of Synthetic Riboswitches…...44

3.2.3 A Model for Synthetic Riboswitch Function………………………………………45

3.2.4 Possible Design Implications for Synthetic Riboswitches…………………………52

3.2.5 Possible Evolutionary Implications for Natural Riboswitches…………...………..53

3.3 Experimental…………………………………………………………………………54

3.4 References……………………………………………………………………………60

Chapter 4: A Flow Cytometry Based Screen for Synthetic Riboswitches ………………………………………………………………………….…62

4.1 Introduction…………………………………………………………………………..62

4.2 Results………………………………………………………………………………..65

4.2.1 Screening of an N8 Library…………………………………………………….......65

4.2.2 Screening of an N12 Library………………………………………………………..68

4.2.3 Exploring the Mechanism of Improved Switches………………………………….73

4.3 Discussion……………………………………………………………………………78

4.4 Conclusion…………………………………………………………………………...80

4.5 Experimental…………………………………………………………………………81

4.6 References……………………………………………………………………………86

Chapter 5: Selection of RNA Aptamers that Bind Erythromycin ………………………………………………………………..………….89

5.1 Introduction…………………………………………………………………………..89

5.2 Results and Discussion………………………………………………………………97

5.2.1 SELEX……………………………………………………………………………..97

5.2.2 Analysis of Putative Aptamers……………………………………………………100

5.3 Conclusion………………………………………………………………………….106

5.4 Experimental………………………………………………………………………..107

5.5 References…………………………………………………………………………..112

Chapter 6: Using a Dual-Selection Strategy to Identify Erythromycin Riboswitches …………………………………………………………………………...116

6.1 Introduction…………………………………………………………………………116

6.2 Results and Discussion...…………………………………………………………...119

6.2.1 Selection to Identify Erythromycin Riboswitch………………………………….119

6.2.2 Determining the Parameters of a Dual Selection System………………………..122

6.3 Conclusion …………………………………………………………………………127

6.4 Future Directions …………………………………………………………………..129

6.5 Experimental ……………………………………………………………………….129

6.6 References…………………………………………………………………………..132

Chapter 7: Controlling Gene Expression with Visible Light …………………………......................................................................................134

7.1 Introduction…………………………………………………………………………133

7.2 Results and Discussion……………………………………………………………..139

7.3 Conclusion and Future Directions………………………………………………….144

7.4 Experimental………………………………………………………………………..145

7.5 References…………………………………………………………………………..147

Chapter 8: Summary ………………………………………………………….………149

8.1 Summary……………………………………………………………………………149

8.2 References…………………………………………………………………………..154

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