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Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.

Boussebayle, Adrien ; Torka, Daniel ; Ollivaud, Sandra ; Braun, Johannes ; Bofill-Bosch, Cristina ; Dombrowski, Max ; Groher, Florian ; Hamacher, Kay ; Suess, Beatrix (2019):
Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.
In: Nucleic acids research, 47 (9), pp. 4883-4895. ISSN 1362-4962,
DOI: 10.1093/nar/gkz216,
[Article]

Abstract

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

Item Type: Article
Erschienen: 2019
Creators: Boussebayle, Adrien ; Torka, Daniel ; Ollivaud, Sandra ; Braun, Johannes ; Bofill-Bosch, Cristina ; Dombrowski, Max ; Groher, Florian ; Hamacher, Kay ; Suess, Beatrix
Title: Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.
Language: English
Abstract:

The development of synthetic riboswitches has always been a challenge. Although a number of interesting proof-of-concept studies have been published, almost all of these were performed with the theophylline aptamer. There is no shortage of small molecule-binding aptamers; however, only a small fraction of them are suitable for RNA engineering since a classical SELEX protocol selects only for high-affinity binding but not for conformational switching. We now implemented RNA Capture-SELEX in our riboswitch developmental pipeline to integrate the required selection for high-affinity binding with the equally necessary RNA conformational switching. Thus, we successfully developed a new paromomycin-binding synthetic riboswitch. It binds paromomycin with a KD of 20 nM and can discriminate between closely related molecules both in vitro and in vivo. A detailed structure-function analysis confirmed the predicted secondary structure and identified nucleotides involved in ligand binding. The riboswitch was further engineered in combination with the neomycin riboswitch for the assembly of an orthogonal Boolean NOR logic gate. In sum, our work not only broadens the spectrum of existing RNA regulators, but also signifies a breakthrough in riboswitch development, as the effort required for the design of sensor domains for RNA-based devices will in many cases be much reduced.

Journal or Publication Title: Nucleic acids research
Journal volume: 47
Number: 9
Divisions: 10 Department of Biology
10 Department of Biology > Computational Biology and Simulation
10 Department of Biology > Synthetic Genetic Circuits
Date Deposited: 15 Apr 2019 10:44
DOI: 10.1093/nar/gkz216
Identification Number: pmid:30957848
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