Using a specific RNA-protein interaction to quench the fluorescent RNA Spinach

Laura Roszyk, Sebastian Kollenda, Sven Hennig

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet, their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labelled. 'Spinach' is a genetically encodable RNA aptamer, which starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyse RNA-protein interactions by quenching the spinach aptamer.

LanguageEnglish
JournalAcs chemical biology
Early online date23 Oct 2017
DOIs
Publication statusPublished - 15 Dec 2017

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Spinacia oleracea
RNA
Proteins
Energy Transfer
Energy transfer
Quenching
Pseudomonas Phages
Nucleotide Aptamers
Molecules
Bacteriophages
Fluorophores
Capsid Proteins
Biomolecules
Pseudomonas aeruginosa
Coloring Agents
DNA

Keywords

  • Journal Article

Cite this

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title = "Using a specific RNA-protein interaction to quench the fluorescent RNA Spinach",
abstract = "RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet, their biochemical analysis remains challenging. For F{\"o}rster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labelled. 'Spinach' is a genetically encodable RNA aptamer, which starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyse RNA-protein interactions by quenching the spinach aptamer.",
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Using a specific RNA-protein interaction to quench the fluorescent RNA Spinach. / Roszyk, Laura; Kollenda, Sebastian; Hennig, Sven.

In: Acs chemical biology, 15.12.2017.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Using a specific RNA-protein interaction to quench the fluorescent RNA Spinach

AU - Roszyk, Laura

AU - Kollenda, Sebastian

AU - Hennig, Sven

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N2 - RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet, their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labelled. 'Spinach' is a genetically encodable RNA aptamer, which starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyse RNA-protein interactions by quenching the spinach aptamer.

AB - RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet, their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labelled. 'Spinach' is a genetically encodable RNA aptamer, which starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyse RNA-protein interactions by quenching the spinach aptamer.

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