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

Laura Roszyk, Sebastian Kollenda, Sven Hennig

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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.

Original languageEnglish
Pages (from-to)2958-2964
Number of pages7
JournalACS chemical biology
Volume12
Issue number12
Early online date8 Nov 2017
DOIs
Publication statusPublished - 15 Dec 2017

Funding

L.R., S.K. and S.H. are grateful for support from AstraZeneca, Bayer CropScience, Bayer HealthCare, Boehringer Ingelheim, Merck KGaA, and the Max Planck Society. We thank D. Schade and D. Langlë for providing DFHBI and T. N. Grossmann for critical reading of the manuscript. We also thank S. Koch for technical support.

FundersFunder number
AstraZeneca
Boehringer Ingelheim
Bayer CropScience
Merck KGaA
Bayer HealthCare
Max-Planck-Gesellschaft

    Keywords

    • Journal Article

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