Apr 9, 2019

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

Nucleic Acids Research
Adrien BoussebayleBeatrix Suess

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 ri...Continue Reading

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Mentioned in this Paper

Aptamers, Nucleotide
Study
In Vivo
Mucocutaneous Lymph Node Syndrome
Ligand Binding
Neomycin Palmitate
Riboswitch
Structure-Activity Relationship
Macromolecular Alteration
Selex Aptamer Technique

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