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.

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…Site-specific installation of FRET reporters was demonstrated on small and large RNAs (5S, 16S and 23S rRNA) in the context of total cellular RNA. 304,305 These studies pave the way for future evolution of catalytic RNA labelling tools in cells.…”

“…In this respect, ribozymes for a comparable labelling strategy of RNA were recently described. 304,305 These RNA catalysts resulted from a targeted in vitro selection strategy that used a structured RNA library to guide the ribozyme to the predetermined labelling site, and were directly selected with ATP derivatives as small-molecule labelling reagents. 305 To enhance the bioorthogonality of this RNA labelling strategy, a new generation of ribozymes was developed that used antiviral nucleotide analogues as substrates.…”

Site-Specific Fluorescent Labeling of RNA Interior Positions

“…This procedure has been applied to simple RNA sequences, giving yields of incorporated fluorophores of 50–80%. Substitution of tenofovir diphosphate in place of ATP resulted in formation of a more stable reaction product [ 75 ]. More complex RNAs, such as bacterial 5S rRNA and 23S rRNA, have been labeled site-specifically, although the yields of the labeling reactions were not indicated.…”

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.…”