# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 3 Diversity of Synthetic Cap Structures
(**a**) propagyl group; (**b**) ally group; (**c**) 2\({}^{\prime}\)-OH to Fluor substitution; (**d**) isopropylidene group; (**e**) locked nucleic acid (LNA) modification; (**f**) anthraniolyl (R: -H) and methylanthraniolyl (R: -CH\({}_{3}\)) fluorescent moieties; (**g**) 2\({}^{\prime}\) amino group; (**h**) example of carbamate modification, possible label moieties include: trifluoromethyl aladirine (**i**), 6-nitrovertryl alcohol (NVA) (**ii**), fluorescein or biotin; (**i**) example of modification with azide group. gistic effect that increase the inhibitory potency of synthesized compounds was shown for simultaneous modification at both N\({}^{7}\) and N\({}^{2}\) positions of m\({}^{7}\)G of mono- and dinucleotide analogs [41]. Photolabile groups put at the N\({}^{2}\) (such as ortho-nitrobenzyl [94; 95; 96]) or at the N\({}^{7}\) position (e.g., bezophenone or coumarin [97; 98], represent yet another series of 7-methyl-guanosine modified cap analogues that can be incorporated into mRNA transcripts. Upon irradiation and photodetprotection of the cap structure, these analogues trigger the cap-dependent translation. [PERSON] et al. [96] also synthesized cap analogues where the nitrobenzyl photoleavable tag was introduced at 2\({}^{\prime}\)-OH or 3\({}^{\prime}\)-OH position of m\({}^{7}\)G. Additionally, the m7G nucleobase of the cap structure can be modified at the C8-position, as demonstrated by [PERSON] et al. [99] (Figure 7). Further development of synthetic cap analogues has led to the creation of trinucleotide and tetranucleotide compounds. Those, from one point of view, facilitated in vitro capping of mRNA transcripts in a correct orientation (such as ARCA analogues [9]; from another perspective they allowed the acquisition of analogues that reproduce the Cap-1-type and the Cap-2-type structures, that are present on mRNA transcripts in higher eukaryotes, including mammals [8; 37], that are involved in suppression of the innate response activation trough IFIT1 and RIG-I [100; 101; 102] and confer resistance towards DXO activity [103]. Following the report of a series of m\({}^{7}\)GppApG trinucleotide cap analogues (with differentially methylated adenosine: A, A\({}_{\text{m}}\), m\({}^{6}\)A, m\({}^{6}\)A\({}_{\text{m}}\)) [18] and commercially available CleanCap trinucleotides [104], the trinucleotide analogues with the remaining nucleobases (in relation adenosine) were synthesized (m\({}^{7}\)GppNpG, where N = C,C\({}_{\text{m}}\), U, U\({}_{\text{m}}\), G, G\({}_{\text{m}}\)) [105], as were  
Figure 7: Examples of the modifications of N\({}^{7}\), N\({}^{2}\), and C8 sites of m\({}^{7}\)Guanine. (**A**) N\({}^{7}\) substitutions include: (**a**) benzyl, (**b**) 4-nitrobenzyl, (**c**) napthalene, (**d**) 2\(A\)-difluorobenzyl, (**e**) 4-chlorophenyloxyethyl, (**f**) 7-(diethylamino)-4-methyl-coumarin, (**g**) benzophenone. (**B**) N\({}^{2}\) substitutions include: (**a**) methyl, (**b**) benzyl, (**c**) 4-methoxybenzyl, (**d**) triazole, (**e**) 1-phenyl-triazole, (**f**) 2,6-dimethoxyphenyl triazole, (**g**) 3-phenyl-isoxazole, (**h**) 4-methyl-triazole. (**C**) **C8** substitutions include: (**a**) methyl, (**b**) 1-pyrene, (**c**) benzyl, (**d**) 4-dimethylaminophenyl, (**e**) 4-cyanophenyl. also the tetranucleotide cap analogues (with Cap 2 signature) [103]. Additionally, other tri- and tetranucleotide cap analogues with modifications within the triphosphate bridge and/or m\({}^{7}\)G have been described [65; 69; 74; 93; 96; 100; 107]. Following the discovery of non-canonical cap structures, the synthesis of their analogs (both di- and trinucleotide series, with additional modification within the phosphate linkage) represents yet another series of compounds that facilitate the in vitro synthesis of NAD-, FAD-, or UDP-glucose-capped RNA transcripts [108; 109; 110].