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Non‐radioactive RNA labeling and detection of RNA:protein interactions using a modified ribonucleotide
Author(s) -
Opperman Kay,
Etienne Chris,
Schultz JeanSamuel,
Meier Scott,
Hommema Eric,
Kaboord Barbara
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.lb53
Subject(s) - rna , biotin , chemistry , streptavidin , cytidine , rna binding protein , biotinylation , biochemistry , messenger rna , computational biology , microbiology and biotechnology , biology , gene , enzyme
The regulation of cellular processes is highly dependent on the interactions of RNA with proteins; however, these interactions are challenging to isolate, frequently use radioactivity for detection, and are highly dependent on the integrity of the RNA and the protein. We have developed non‐radioactive tools for end‐labeling RNA for in vitro detection of RNA:protein interactions. To achieve non‐radioactive RNA labeling, we modified cytidine to contain a 3′5′ bisphosphate for RNA ligation and a linker that can accommodate a variety of detectors. We used a biotin detector and achieved greater than 75% efficiency using either synthetic RNA or in vitro‐transcribed RNA. The biotin‐labeled RNA was able to enrich for associated proteins as assessed electrophoretic mobility shift assays (EMSA) and as bait for RNA‐binding proteins using an immobilized streptavidin sensor chip or streptavidin affinity resin. The flexibility and robustness of the labeling method, in combination with the adaptability of the chemical synthetic method, allow modification of additional nucleotides for downstream applications. These tools will enable RNA researchers to isolate RNA:protein complexes critical to cellular function, especially in the emerging field of miRNA.