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General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direc in vitro selection, characterization, and application of versatile rans -acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for in vitro evolution of ribozymes that modify a predetermined nucleotide in cis (i.e., intramolecular reaction) and can then be easily engineered for applications in rans (i.e., in an intermolecular setup). The resulting ribozymes are readily designed for specific target sites in small and large RNAs and accept a wide variety of N  6 -modified ATP analogues as small-molecule substrates. The most efficient new ribozyme (FH14) shows excellent specificity toward its target sequence also in the context of total cellular RNA.

Direct in Vitro Selection of Trans-Acting Ribozymes for Posttranscriptional, Site-Specific, and Covalent Fluorescent Labeling of RNA