# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 5 RNA Decapping with Mammalian Nudt2, Nudt5, Nudt12, Nudt16, and Dcp2
This specific hNudt16 feature was used in developing of the \"cap-orientation\" test in analysis of RNA transcripts with N2-susbstituted caps [92]. Here, the GppG-capped transcript underwent complete decapping with hNudt16, whereas m\({}^{7}\)GppG-RNA SP6 polymerase transcript (that is a mixture of correctly and in-reverse-orientation capped products, m\({}^{7}\)GpppG-RNA and GppppGm\({}^{7}\)-RNA) was decapped at around 45%. In the correct orientation, ARCA-capped transcript was decapped at around 8% in the same experimental conditions, which is attributed to the lower activity of hNudt16 towards m\({}^{7}\)G-capped RNA [92; 120]. Functionality of that test was shown for mRNA transcripts capped in vitro with a series of N\({}^{2}\)-modified dinucleotide cap analogues (triazole, isoxazole or thiazole substituted) [92; 93]. RNA transcripts bearing cap structure with the attached FAM moiety at 2\({}^{7}\)-OH or 3\({}^{\prime}\)-OH of m\({}^{7}\)G were also subject to decapping with hNudt16 [77]. Next to the aforementioned analogues of the canonical cap structure, hNudt16 showed enzymatic activity towards RNA capped with non-canonical metabolite 5\({}^{\prime}\) caps (NAD, dpCoA and FAD) [132]. Such wide activity of hNudt16 towards different 5\({}^{\prime}\) cap structures on in vitro prepared RNA transcripts points out to it as a convenient, single enzyme that can be widely used in testing functionality of introduced chemical modifications within a polyphosphate linkage or within a nucleotide part of cap structure, and in test of cap-orientation and capping efficiency of non-ARCA cap analogues. It can be also considered in removing of the incorrectly (reversely) capped RNA transcripts after transcription in vitro with RNA polymerase. In such a case, hNudt16 treatment would expose monophosphate at the 5\({}^{\prime}\) end of RNA (e.g., pGm\({}^{7}\)-RNA, after GppppGm\({}^{7}\)-RNA decapping), which is then degraded by Xrn1 processesive 5' to 3' exoribonuclease (similarly to removing of 5' triphosphorylated RNA from the primary transcripts after 5'RNA polyphosphatase treatment, e.g., [107]). Mammalian Nudt12 NUDIX enzyme was also shown to possess decapping activity of m7 GpppG- and GpppG-capped transcripts in vitro [116]. Subsequent reports identified human and murine Nudt12 as a cytosolic RNA deNADing enzyme [133; 134] and its decapping activity towards dpCoA-capped RNA [132]. Trimmethylated cap structure at the 5' end of RNA has been also effectively hydrolyzed with Nudt12, similarly to unmethylated an monomethylated caps [130]. Studies on differentially methylated dinucleotide cap analogs pointed out towards compounds containing adenosine (with different methylation status, e.g., A, Am or m6 Am) as preferred substrates for Nudt12, which is different in comparison to Nudt16 activity. For example, in the case of trimethylated analogues (m3\({}^{2,2,7}\)GpppG and m3\({}^{2,2,7}\)GpppA), hNudt12 efficiently hydrolysed dinucleotides with adenine but not guanine, whereas hNudt16 showed the opposite effect [130]. Thus, it seems reasonable to replace Nudt16 with Nudt12 in functional decapping studies of RNA transcripts modified cap analogues with adenosine as a nucleoside adjacent to initial m7G, as deNADing enzyme Nudt12 was already used in decapping test of RNAs with synthetic NAD analogues modified within diphosphate bridge [108]. Nudt2 was among other mammalian NUDIX enzymes with decapping activity towards canonical cap and metabolite FAD-cap and dpCoA cap [116; 132]. In a recent report, following the discovery of diadenosine tetraphosphate capped RNAs, Nudt2 was shown to be responsible for the hydrolysis of AppppA-RNA [33]. Extended data for in vitro Nudt2-mediated decapping of Ap\({}_{n}\)A-RNA, Gp\({}_{n}\)G-RNA, AP\({}_{n}\)G-RNA, and Gp\({}_{n}\)A-RNA showed its primary preference towards Ap\({}_{n}\)A-capped substrates (Table 2). Among other dinucleotide structures those with tetraphosphate bridge were also effectively hydrolyzed.