Repurposing Antiviral Drugs for Orthogonal RNA‐Catalyzed Labeling of RNA

Abstract

In vitro selected ribozymes are promising tools for site-specific labeling of RNA. Previously known nucleic acid catalysts attached fluorescently labeled adenosine or guanosine derivatives through 2',5'-branched phosphodiester bonds to the RNA of interest. Herein, we report new ribozymes that use orthogonal substrates, derived from the antiviral drug tenofovir, and attach bioorthogonal functional groups, as well as affinity handles and fluorescent reporter units through a hydrolytically more stable phosphonate ester linkage. The tenofovir transferase ribozymes were identified by in vitro selection and are orthogonal to nucleotide transferase ribozymes. As genetically encodable functional RNAs, these ribozymes may be developed for potential cellular applications. The orthogonal ribozymes addressed desired target sites in large RNAs in vitro, as shown by fluorescent labeling of E. coli 16S and 23S rRNAs in total cellular RNA.

Site-specific RNA methylation by a methyltransferase ribozyme

“…After precipitation by ethanol, the biotinylated RNA were captured using either neutravidin- or streptavidin-coated magnetic beads (Dynabeads, Thermo Fisher Scientific, ca 1 nmol RNA per mg of beads), eluted with formamide, and amplified by RT-PCR, following established procedures. 29 , 30 In vitro transcription was performed (total volume of 100 μL), followed by PAGE purification to prepare the enriched RNA library for the next selection round. After 11 rounds of selection, the library was cloned (TOPO-TA cloning), and ribozymes generated from randomly picked colonies were examined for catalytic activity (by streptavidin gel shift assay on native PAGE) and sequenced.…”

“…coli RNA was extracted as previously reported. 30 A fraction (0.5 μg) was analysed by 10% denaturing PAGE, that was stained with a solution of 20 μM DFHBI in 100 mM KCl, 5 mM Mg 2+ , 40 mM HEPES, pH 7.5, for 15 min, and imaged on a ChemiDoc imager. Afterwards the gel was stained with Sybr gold and imaged again to visualize all RNA and the size marker.…”

“…We used a structured RNA pool containing 40 random nucleotides that was designed according to our previously used strategy to direct RNA-catalysed labeling of a specific adenosine in a target RNA. 29 , 30 RNA methylation would most likely occur at an O or N nucleobase heteroatom, on the 2'-OH group or on the phosphate backbone. In either case, attachment of a single methyl group would hardly enable physical separation of the active sequences based on size or charge.…”

New Deoxyribozymes for the Native Ligation of RNA

“…As an additional confirmation, we checked if the ligation product could serve as a substrate for RNA-catalyzed labeling at the ligation junction. The recently reported FH14 ribozyme uses the 2′-OH group of an internal adenosine for labeling with fluorescent N 6 -aminohexyl-ATP via the formation of a 2′,5′-branched phosphodiester linkage [ 31 , 32 ]. Fluorescein-labeled R2 was ligated with R1 by incubation with SC9, and the resulting product was then treated with FH14 and Atto550-ATP.…”

Covalent labeling of nucleic acids

“…Labeling of 16S and 23S rRNAs with fluorescent dyes and attachment of functional groups was successful in total cellular RNA. 177 Furthermore, they developed a labeling strategy called DEoxyribozyme-CAtalyzed Labeling (DECAL). To this end, a tagging RNA with a 5-aminoallylcytidine was first labeled with either a fluorescein or TAMRA fluorophore via an NHS esterification.…”