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Site‐Specific Covalent Labeling of RNA with RNA‐TAG: A Platform for the Elucidation of RNA Function
Author(s) -
Busby Kayla N,
Alexander Seth C,
Devaraj Neal K
Publication year - 2016
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.30.1_supplement.lb178
Subject(s) - rna , biotinylation , riboswitch , computational biology , transfer rna , messenger rna , non coding rna , biology , biochemistry , chemistry , microbiology and biotechnology , gene
The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Here, we demonstrate the site‐specific covalent labeling of RNA transcripts using the RNA‐TAG ( t ransglycosylation a t g uanosine) methodology. This technology takes advantage of a bacterial tRNA transglycosylase (TGT) capable of incorporating nucleobase derivatives bearing functional probes into an encodable RNA stem loop recognition motif. Using this methodology, we have successfully labeled mRNA transcripts with a wide variety of probes including biotin, BODIPY, and Cy7 in a single enzymatic step. Endogenously transcribed and processed RNAs containing the RNA‐TAG recognition hairpin have been successfully visualized in cell imaging studies. Furthermore, we have demonstrated that an RNA of interest can be selectively biotinylated and affinity purified from a complex mixture of RNAs and cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient platform for the elucidation of RNA function via cellular localization studies and affinity purification of RNA‐protein complexes. Support or Funding Information This work was supported by the Department of Defense Army Research Office Contract W911NF‐13‐1‐0383. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1144086 (fellowship to K.N.B.).