Enzymatic Site‐Specific Labeling of RNA for Affinity Isolation of RNA‐Protein Complexes

Abstract

The role of RNA in regulating cellular processes has generated tremendous interest in methods to image RNAs and profile their interactions within cells. Building on our recently developed enzymatic technique, RNA‐TAG (Transglycosylation At Guanosine), we demonstrate the site‐specific labeling and affinity purification of RNA transcripts. This technology takes advantage of a bacterial tRNA guanine transglycosylase (TGT) for the incorporation of nucleobase derivatives bearing functional probes, such as biotin, into an encodable RNA stem loop recognition motif. Affinity purification enables high levels of enrichment of target RNA complexes and analysis of bound proteins. Using this methodology, we have demonstrated that an RNA of interest can be selectively biotinylated in live mammalian cells or cell lysate. Through the development of RNA‐TAG, we aim to create a robust, convenient methodology for the elucidation of RNA function via affinity purification of cellular RNA‐protein complexes.Support or Funding InformationThis work is supported by the NIH under grant R01 GM123285. K.N.B. is supported by the Chemical Biology Interfaces Training Grant, NIH Grant T32 GM112584.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.