FRET Analysis of RNA –Protein Interactions Using Spinach Aptamers

The method development to analyze direct RNA–protein interaction is of high importance as not many homogeneous assay formats are available. The discovery of fluorescent light-up aptamers (FLAPs), short RNA aptamers that switch the fluorescence of small, cell-permeable, and nontoxic organic chromophores on, paved the road for their utilization in direct RNA–protein interactions. The combination with fluorescent proteins as biological fluorophores enabled the development of homogeneous assays that are in principle even encodable on genomic level. Here the rules and methods to design a homogeneous in vitro assay for the detection and quantification of a direct RNA–protein interaction will be described. The design and application of a homogeneous assay to observe and quantify the interaction of the Pseudomonas aeruginosa bacteriophage coat protein 7 (PP7) with its binding RNA sequence (pp7-RNA) will be shown. For this, the Spinach-DFHBI aptamer as RNA fusion and the red fluorescent mCherry as protein fusion was used. The methods presented here do not require any chemical modification of proteins or RNAs which make them relatively easy to use and to adopt on other systems. As all fluorophores are fusion tags to the according biomolecules, standard cloning strategies and molecular biology technologies are sufficient and make this method available for a broad community of researchers.