Expanding the Available RNA Labeling Toolbox With CutA Nucleotidyltransferase for Efficient Transcript Labeling with Purine and Pyrimidine Nucleotide Analogs.

Abstract

RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates. Here, we report the use of another NTase, CutA from the fungus Thielavia terrestris. Using this enzyme, we were able to incorporate into the 3' end of RNA not only purine analogs, but also pyrimidine analogs. We engaged strain-promoted azide-alkyl cycloaddition (SPAAC) to obtain fluorescently labeled or biotinylated transcripts from RNAs extended with azide analogs by CutA. Importantly, modified transcripts retained their biological properties. Furthermore, fluorescently labeled mRNAs were suitable for visualization in cultured mammalian cells. Finally, we demonstrate that either affinity studies or molecular dynamic (MD) simulations allow for rapid screening of NTase substrates, what opens up new avenues in the search for the optimal substrates for this class of enzymes.