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

Abstract

General and efficient tools for site-specific fluorescent or bioorthogonal labeling of RNA are in high demand. Here, we report direct <i>in vitro</i> selection, characterization, and application of versatile <i>trans</i>-acting 2'-5' adenylyl transferase ribozymes for covalent and site-specific RNA labeling. The design of our partially structured RNA pool allowed for <i>in vitro</i> evolution of ribozymes that modify a predetermined nucleotide in <i>cis</i> (i.e., intramolecular reaction) and can then be easily engineered for applications in <i>trans</i> (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 <i>N</i>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.