Next-level riboswitch development-implementation of Capture-SELEX facilitates identification of a new synthetic riboswitch.
Nucleic Acids Res
; 47(9): 4883-4895, 2019 05 21.
Article
en En
| MEDLINE
| ID: mdl-30957848
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ARN
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Aptámeros de Nucleótidos
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Técnica SELEX de Producción de Aptámeros
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Riboswitch
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2019
Tipo del documento:
Article
País de afiliación:
Alemania