# Application of Mammalian Nudix Enzymes to Capped RNA Analysis
## 3 Diversity of Synthetic Cap Structures  
Synthetic cap analogues, and synthetic capped (m)RNAs have proven to be invaluable tools for studying various cellular processes where the cap plays a pivotal role, including mRNA translation, splicing, intracellular transport, and RNA decay [38]. They are also important in studying cap-binding proteins and enzymes, including potential inhibitors of translation initiation factor eIF4E overexpressed in certain human malignancies and cancers (e.g., breast, colon, prostate, neck cancers) [39; 40; 41]. Now, following pioneering work demonstrating the first successful example of protein expression in muscle tissue after injection of in-vitro-transcribed capped mRNA [3] and the recent launch of anti-COVID-19 vaccines [1; 2], capped RNAs have emerged as potent therapeutic agents with almost unlimited potential. In vitro transcription (IVT) methodology is a convenient approach for producing mRNA transcripts from a provided DNA template, allowing for co-transcriptional capping with synthetic dinucleotide cap analogues, resulting in capped-mRNA within a single reaction [42; 43; 44; 45]. One of the problems with the direct using IVT-capped transcripts in therapeutic applications is the heterogeneity of the 5\({}^{\prime}\) ends of the transcription reaction products, resulting in both m\({}^{7}\)GpppG-capped and reversely oriented Gppp(m\({}^{7}\)G)-capped transcripts due to priming of the polymerisation reaction by bacteriophage RNA polymerases (T7, SP6) from both ends of the m\({}^{7}\)GpppG cap dinucleotide [46]. The breakthrough in this issue was the development of anti-reverse cap analogues (ARCAs) by the [PERSON] lab, where the 3\({}^{\prime}\)-OH group of 7-methyl-guanosine (m\({}^{7}\)G) was either replaced by methyl group (m\({}^{7,3^{\prime}}\)GpppG) (Figure 3) or removed (in 7-methyl-(3\({}^{\prime}\)deoxy)GpppG analogue) [47]. Such modifications prevent RNA polymerase from initiating from m\({}^{7}\)G moiety, resulting in the synthesis of transcripts capped exclusively in correct orientation, and thus in biologically active form. Similar ARCA-type properties have also been shown for dinucleotide cap analogues with 2\({}^{\prime}\)-OH methylated 7-methyl-guanosine [48]. A natural choice for further chemical modifications was the triphosphate bridge of the dinucleotide cap structure (Figure 4), as it may protect the resulting analogues against decapping enzyme (e.g., Dcp2, [11; 12; 13; 14]). Additionally, elongation of the triphosphate bridge to tetra- or pentaphosphate increases binding affinity towards eIF4E and has a positive effect on cap-dependent translation in vitro [48; 49; 50]. Figure 2: Structures of non-canonical RNA caps. Numerous different substituents have been used in synthetic cap analogs where at least one oxygen atom in the triphosphate bridge was replaced, either the non-bridging \(\alpha\), \(\beta\) or \(\gamma\) oxygen (e.g., O-to-S, O-to-Se, O-to-BH\({}_{3}\)) [51; 52; 53; 54; 55; 56], or bridging \(\alpha\)-\(\beta\) and \(\beta\)-\(\gamma\) oxygen (e.g., O to NH, O to CH\({}_{2}\), and O to CCl\({}_{2}\), to CH\({}_{2}\) or to CF\({}_{2}\) in tetrapphosphate analogs) [57; 58; 59; 56]. In addition to cap analogues with a single replacement within the tri- or tetra-phosphate bridge, a series of multiple-modified compounds were synthesized, e.g., with double thiophosphate or double boronophosphate moieties, with or without O to CH\({}_{2}\) exchange [60; 61]. Another site of O-to-S substitution was the 5'-phosphoester bond in the triphosphate chain, resulting in a cap dinucleotide analogues with a phosphorotholate moiety [62]. By applying a \"click-chemistry\" approach, a series of cap analogues containing the triazole moiety  
Figure 4: Sites of chemical modifications of the canonical cap structures. (**1**) 2’OH and 3’OH of m7’G, (**2**) the triphosphate bridge, (**3–5**) substitutions at the N7, N2, and C8 position of guanine, respectively, (**6**) modifications at N6 position of adenine adjacent to the m7’G in the cap nucleotide structure.