# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 3 Diversity of Synthetic Cap Structures
Figure 3: Anti-reverse cap analogue (ARCA). within the oligophosphate chain (with accompanying oxygen substitutions mentioned above in the text) were obtained, expanding the library of diverse substituents for the triphosphate bride modification [63; 64; 65] (Figure 5). Further modifications that utilize chemical modifications of either the 2\({}^{\prime}\)-OH or/and 3\({}^{\prime}\)-OH on m\({}^{7}\)G, similar to ARCA cap analogues, have also been reported. These include substitution of 2\({}^{\prime}\)-OH with a fluorine or allyl moiety [66; 67], 2\({}^{\prime}\)-propagyl substituted di- and tri-nucleotide cap analogues [68; 69], isopropylene analogues [70; 71], anthraniolyl and methylanthraniolyl fluorescent cap analogues [72], and locked nucleic acid (LNA) di- and tri-nucleotide cap analogues [73; 74]. Analogues with 2\({}^{\prime}\)-amino groups instead of 2\({}^{\prime}\)-OH of m\({}^{7}\)G were next subject for biotin attachment, facilitating the capture of mRNA-protein complexes [75; 76]. Further development of this series of cap analogues resulted in compounds carrying amino-functionalized linkers at 2\({}^{\prime}\)-OH or 3\({}^{\prime}\)-OH of m\({}^{7}\)G, allowing subsequent attachment of fluorescent moieties (e.g., fluorescein), biotin [77], or photoreactive tags such as trifluoromethyl diazirine, and 6-nitrovartyl alcohol, as very recently reported by [PERSON] et al. [78]. Similarly, azido-functionalized analogues with Cy3 or Cy5 fluorescent dyes, were reported [79]. The 2\({}^{\prime}\)-propagyl substituted cap analogue was also the subject of fluorescent labelling (with Alexa Fluor) [69] (Figure 6). Figure 5: Examples of the modifications introduced into triphosphate bridge of mRNA cap dinucleotide. (**a**) X\({}_{1}\), X\({}_{2}\) indicate sites of bridging oxygen substitution with -NH, -CH\({}_{2}\), -CCl\({}_{2}\), or -CF\({}_{2}\), and Y\({}_{1}\), Y\({}_{2}\), and Y\({}_{3}\) indicate sites of non-bridging oxygen substitution with -S, -Se, or-BH\({}_{3}\); (**b**) representative phosphorothiolate modifications after O-to-S substitution within 5\({}^{\prime}\)-phosphoester bond in the triphosphate chain; (**c**) examples of triazole moiety introduced within cap oligophosphate linkage. Modifications of the cap structure at the N\({}^{7}\), N\({}^{2}\) or C8-position of the nucleobase of m\({}^{7}\)G have also been widely described. Initial focus was on the substitution of the N\({}^{7}\) methyl moiety, as it is one of the structural features of the cap required for specific recognition by the eIF4E translation initiation factor, as revealed by crystallographic and biophysical data [80; 81; 82]. In turn, the N\({}^{7}\)-substituted compounds with diverse alkyl and aryl groups were synthesized [49; 83; 84; 85]. Among those, N\({}^{7}\)-benzyl substitution increased inhibitory properties of mononucleotide analogues, and its pronucleotide counterpart appeared promising for targeting lung and breast cancer [39; 49; 86]. Furthermore, mRNA transcripts bearing N\({}^{7}\)-benzyl (or its derivatives) dinucleotide cap analogues showed higher translational activity [83; 85; 87]. Another favourable modification site was the N\({}^{2}\) position of m\({}^{7}\)G, where the presence of a single methyl group has a positive effect on inhibition and translation [49; 83]. Several other N\({}^{2}\)-alkyl- and -aryl-substituted analogues, including N\({}^{2}\)-benzyl and N\({}^{2}\)-(4-metoxybenzyl) ARCA, were next reported [88; 89]. Further N\({}^{2}\)-modified analogues included triazole-, isoxazole-, and thiazole-ring-based substituents [10; 90; 91; 92]. Combined modifications at N\({}^{2}\) position of m\({}^{7}\)G and triphosphate bridge of dinucleotide and trinucleotide cap analogues (Cap 1-type) have also been described [93]. Recently, the syner  
Figure 6: Examples of the modifications introduced at 2\({}^{\prime}\)-OH or 3\({}^{\prime}\)-OH of m\({}^{7}\)Guanine.