In eukaryotes, the DXO/Rai1 enzymes can eliminate most of the incomplete and non-canonical NAD caps through their decapping, deNADding and pyrophosphohydrolase activities. Here, we report that these enzymes can also remove FAD and dephospho-CoA (dpCoA) non-canonical caps from RNA, and we have named these activities deFADding and deCoAping. The crystal structures of mammalian DXO with 3′-FADP or CoA and fission yeast Rai1 with 3′-FADP provide elegant insight to these activities. FAD and CoA are accommodated in the DXO/Rai1 active site by adopting folded conformations. The flavin of FAD and the pantetheine group of CoA contact the same region at the bottom of the active site tunnel, which undergoes conformational changes to accommodate the different cap moieties. We have developed FAD-capQ to detect and quantify FAD-capped RNAs and determined that FAD caps are present on short RNAs (with less than ∼200 nucleotides) in human cells and that these RNAs are stabilized in the absence of DXO.

We report the chemical synthesis of a set of nicotinamide adenine dinucleotide (NAD) cap analogues containing chemical modifications that reduce their susceptibility to NAD-RNA-degrading enzymes. These analogues can be incorporated into transcripts in a similar way as NAD. Biochemical characterization of RNAs carrying these caps with DXO, NudC, and Nudt12 enzymes led to the identification of compounds that can be instrumental in unraveling so far unaddressed biological aspects of NAD-RNAs.

Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3′‐end labeling of in vitro transcribed RNAs. We synthesized novel adenosine 3′,5′‐bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide‐containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain‐promoted alkyne‐azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD‐capped RNAs, and 5′‐fluorescently labeled m7Gp3Am‐capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence‐based monitoring of decapping. This method will facilitate the use of various functionalized mRNA‐based probes.

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Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO 3 ) transfer. 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is the universal ST cofactor, serving as the “active sulfate” source in cells. Herein, we report the synthesis of three fluorinated PAPS analogues that bear fluorine or trifluoromethyl substituents at the C2 or C8 positions of adenine and their evaluation as substitute cofactors that enable ST activity to be quantified and real-time-monitored by fluorine-19 nuclear magnetic resonance ( 19 F NMR) spectroscopy. Using plant AtSOT18 and human SULT1A3 as two model enzymes, we reveal that the fluorinated PAPS analogues show complementary properties with regard to recognition by enzymes and the working 19 F NMR pH range and are attractive versatile tools for studying STs. Finally, we developed an 19 F NMR assay for screening potential inhibitors against SULT1A3, thereby highlighting the possible use of fluorinated PAPS analogues for the discovery of drugs for ST-related diseases.