From Aptamer to Riboswitch: High-throughput Screens and Selections for the Identification and Creation of Synthetic Riboswitches Pubblico
Lynch, Sean (2009)
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 an "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……………………………………………………………….……...........18Chapter 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……………………………………………………………………………86Chapter 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 ………………………………………………………….………1498.1 Summary……………………………………………………………………………149
8.2 References…………………………………………………………………………..154
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