Direct in Vitro Selection of Trans-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 in vitro selection, characterization, and application of versatile transacting 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 were then easily engineered for applications in trans (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 towards its target sequence also in the context of total cellular RNA.

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

“…Nucleic acid catalysts, that is, ribozymes and deoxyribozymes can be engineered for any particular sequence and have successfully been used to address internal target sites for various in vitro applications . Recently, we reported the FH14 ribozyme to attach N 6 ‐labeled adenosine to specific internal 2′‐OH groups through 2′,5′‐phosphodiester bonds . This ribozyme was identified by direct in vitro selection with N 6 ‐modified ATP as substrate and a structured RNA library with a bulged adenosine as target site for labeling.…”

“…The previously identified adenylyl‐transferase ribozyme FH14 was tested with tenofovir diphosphate, but it was not able to use the acyclic adenosine derivative as a substrate for RNA labeling (Figure S1). Therefore, we used in vitro selection from a structured RNA pool containing 40 random nucleotides to identify new ribozymes for site‐specific labeling of RNA with bioorthogonal derivatives of antiviral nucleoside analogues (the in vitro selection scheme is shown in the Supporting Information, Figure S2).…”

Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

“…[7] Recently, we reported the FH14 ribozyme to attach N 6 -labeled adenosine to specific internal 2'-OH groups through 2',5'-phosphodiester bonds. [8] This ribozyme was identified by direct in vitro selection with N 6 -modified ATP as substrate and a structured RNA library with a bulged adenosine as target site for labeling. The resulting ribozymes proved specific, efficient and generally applicable even in the context of cellular RNA.…”

“…The previously identified adenylyl-transferase ribozyme FH14 [8] was tested with tenofovir diphosphate, but it was not able to use the acyclic adenosine derivative as a substrate for RNA labeling ( Figure S1). Therefore, we used in vitro selection from a structured RNA pool containing 40 random nucleotides to identify new ribozymes for site-specific labeling of RNA with bioorthogonal derivatives of antiviral nucleoside analogues (the in vitro selection scheme is shown in the Supporting Information, Figure S2).…”

“…A biotinylated tenofovir substrate was used for a bead-based in vitro selection strategy through streptavidin-coated magnetic beads, in analogy to the previously established procedure (with N 6 -biotin-ATP that resulted in FH ribozymes). [8] The N 6 -biotinylated tenofovir diphosphate (biotin-Ten-DP) was synthesized from 6-chloropurine in 7 steps (Scheme S1), in analogy to other N 6substituted [N-(2-phosphonomethoxy)alkyl adenines, [16] and was used at a concentration of 200 mm with 20 mm Mg 2+ at pH 7.4. The RNA library was partially labeled with Lucifer yellow at its 3'-terminal ribose, and the progress of the in vitro selection was monitored by measuring fluorescence of eluted RNA after each selection round (Figure 2 a).…”