# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 5 RNA Decapping with Mammalian Nudt2, Nudt5, Nudt12, Nudt16, and Dcp2  
In the case of the in-vitro-transcribed mRNA (IVT-mRNA), the presence of the cap structure at the 5\({}^{\prime}\) end is one of the key features that enhances its translational activity and stability, thus underpinning the development of mRNA-based therapeutics. As the Dcp2 (Nudt20) is the major decapping enzyme in the 5\({}^{\prime}\)- to -3\({}^{\prime}\) mRNA degradation pathway [122], recombinant hDcp2 is used in the in vitro assays to test stability of RNA transcripts capped with modified cap analogues or with analogues of the Cap 1 and Cap 2 structures (e.g., [55; 56; 83; 103; 105]). Dcp2 interacts with several regulatory proteins that act as decapping activators, including Dcp1 (S. _pombe_ Dcp1 enhanced the Dcp2 catalytic activity 10-fold in vitro [123] or PNRC2 that, together with Dcp1, synergistically stimulates decapping activity of hDcp2 [124]. Both mentioned Dcp2 protein complexes were used in decapping susceptibility analysis of in vitro capped RNA: with N2-modified caps (here _Sp_Dcp1/Dcp2 complex [89] or with N6-benzylated cap analogues (here: PNRC2-hDcp1/Dcp2 complex [107]. With regards to in-vitro-designed therapeutic (m)RNAs, next to the principal confirmation of enzymatic resistance of modified-cap -RNAs, a few additional aspects regarding Dcp2-mediated decapping should be taken into account. First, Dcp2 is not ubiquitously present throughout different human tissues [119]. Second, it is not involved in bulk mRNA decay in cells but rather is important for specific degradation pathways (e.g., nonsense-mediated decay, microRNA-mediated mRNA decay, or degradation in response to interferon [125; 126]. Third, methylation status of adenosine adjacent to 7-methylguanosine in the cap structure (m\({}^{7}\)Gpppm\({}^{6}\)A\({}_{\text{m}}\)) was shown to be involved in decreased susceptibility to Dcp2 decapping [19] and that this phenomenon is related to selected transcripts rather than to general mRNA stabilization [105; 127] (Table 2). hNudt16 is another NUDIX enzyme used in analysis of modified-cap -RNA transcripts. It was initially identified as U8 snoRNA binding protein with decapping activity, which was shown for in vitro m\({}^{7}\)G- and m\({}_{3}\)\({}^{2,2,7}\)G- capped RNA [15]. Further studies revealed that it is ubiquitously detected in all analysed human tissues, is responsible for decapping at least of a subset of mRNA in cells [119] and is able to hydrolyse m\({}^{7}\)G-capped RNA transcripts in vitro [119; 128]. Recombinant hNudt16 appeared as a potent tool to test triphosphate bridge modification of trimethylated m\({}^{7}\)G cap analogues and capped RNAs in a study on m\({}_{3}\)\({}^{2,2,7}\)G cap as a nuclear import signal and m\({}_{3}\)\({}^{2,2,7}\)G-mediated transport of therapeutics [121; 129]. A direct comparative analysis pointed out to non-methylated cap structure at the 5\({}^{7}\) RNA transcript end (Gpppp-G, GpppA-) as preferred substrate for hNudt16 decapping [120] and that decapping of RNAs with mono- and trimethylated cap structures is influenced by the nucleotide type adjacent to m\({}^{7}\)G (or m\({}_{3}\)\({}^{2,2,7}\)G)--those containing adenosine (or 2\({}^{7}\)-O adenosine) are very poor substrates to hNudt16 [120; 121] (Table 2). This is in line with hNudt16 substrate specificity towards m\({}^{7}\)G dinucleotide cap analogues, where m\({}^{7}\)GpppA, m\({}^{7}\)GpppA, and m\({}^{7}\)Gpppm\({}^{6}\)A\({}_{\text{m}}\) appeared resistant to hNudt16 hydrolysis [130]. Additionally, modification of guanosine moiety (but not m\({}^{7}\)G) at the C8-position in the dinucleotide compounds resulted in their resistance to hNudt16 (except for the pyrene substituent) [99]. hNudt16 preference to the exposed unmethylated guanosine in those C8-modified cap analogues was shown also for dinucleotide diphosphate compounds, where the methylated m\({}^{7}\)GppG was hydrolys around eight-fold slower in comparison to GppG [131].