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

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.

SUBMITTER: Boussebayle A

PROVIDER: S-EPMC6511860 | biostudies-other | 2019 May

REPOSITORIES: biostudies-other

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

Boussebayle Adrien A Torka Daniel D Ollivaud Sandra S Braun Johannes J Bofill-Bosch Cristina C Dombrowski Max M Groher Florian F Hamacher Kay K Suess Beatrix B

Nucleic acids research 20190501 9

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 r ...[more]

PMID: 30957848

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Project description:BackgroundThe detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples.MethodsA virus panel based on the principle of biotinylated RNA baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2-positive samples. Libraries generated from complex samples were sequenced via generic HTS (without enrichment) and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted.ResultsThe VirBaits set consists of 177,471 RNA baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also points to the risk of cross-contamination between samples.ConclusionsThe VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector, or research question. The risk of cross-contamination needs to be taken into consideration. The application of the RNA-baits principle turned out to be user friendly, and even non-experts can easily use the VirBaits workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible as shown for SARS-CoV-2. Video abstract.

Project description:BackgroundIn healthcare and allied healthcare settings, leadership that supports effective implementation of evidenced-based practices (EBPs) is a critical concern. However, there are no empirically validated measures to assess implementation leadership. This paper describes the development, factor structure, and initial reliability and convergent and discriminant validity of a very brief measure of implementation leadership: the Implementation Leadership Scale (ILS).MethodsParticipants were 459 mental health clinicians working in 93 different outpatient mental health programs in Southern California, USA. Initial item development was supported as part of a two United States National Institutes of Health (NIH) studies focused on developing implementation leadership training and implementation measure development. Clinician work group/team-level data were randomly assigned to be utilized for an exploratory factor analysis (n?=?229; k?=?46 teams) or for a confirmatory factor analysis (n?=?230; k?=?47 teams). The confirmatory factor analysis controlled for the multilevel, nested data structure. Reliability and validity analyses were then conducted with the full sample.ResultsThe exploratory factor analysis resulted in a 12-item scale with four subscales representing proactive leadership, knowledgeable leadership, supportive leadership, and perseverant leadership. Confirmatory factor analysis supported an a priori higher order factor structure with subscales contributing to a single higher order implementation leadership factor. The scale demonstrated excellent internal consistency reliability as well as convergent and discriminant validity.ConclusionsThe ILS is a brief and efficient measure of unit level leadership for EBP implementation. The availability of the ILS will allow researchers to assess strategic leadership for implementation in order to advance understanding of leadership as a predictor of organizational context for implementation. The ILS also holds promise as a tool for leader and organizational development to improve EBP implementation.

Dataset Information

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

Publications

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

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