RNA modifying enzymes

Numerous examples of methyltransferase enzymes have been applied for labeling of various target molecules such as proteins and DNA and more recently RNA (Holstein and Rentmeister, 2016; Lukinavičius et al., 2007; Muthmann et al., 2020). The common feature among these enzymes is the use of S-adenosyl-methionine as the methyl group donor. A number of these enzymes have been reported in literature, which can transfer extended alkyl chains from the various synthetic analogs of SAM to their target molecule. The most notable examples of such extensions include bioorthogonal functionalities and chemical probes (Muthmann et al., 2020). Motorin et al published the earliest report on application of such enzymes for RNA labeling. They reported that the RNA methyltransferases Trm1 and Trm11 could transfer beta-unsaturated carbon chains with terminal alkynyl functionality. Trm1and Trm11 modify N²-groups of tRNA^Phe at positions 26 and 10, respectively (Figure 1-14 B &

C). Alexa fluor 594 azide was then subsequently conjugated to these modified positions using CuAAC reaction (Motorin et al., 2011).

Protein engineering techniques can sometimes be employed to improve the reactivity of RNA methyltransferases towards the unnatural SAM analogs. Schulz et al, for example engineered Giardia lamblia trimethyl guanine synthase 2 (Gla-Tgs2) for efficient labeling of mRNA cap-structures (Schulz et al., 2013). The engineered enzyme Gla-TGs2-Var1 demonstrates three times higher turnover number, Km and kcat for S-AdoPropen compared to the wild type. Such improvement was achieved with only one single amino acid substitution (V34A). Gla-Tgs2-Var1 was then applied for functionalization of the N²-position in the m⁷G of the mRNA cap structure. The groups transferred using this enzyme include alkynyl

functionalities. The functionalized cap can subsequently be derivatized with chemical probes using CuAAC, SPAAC, or iEDDA (Inverse electron-demand Diels-Alder) reactions (Figure 1-14).

Ecm1 is another cap methyltransferase with an inherent ability to transfer bulky moieties from their corresponding SAM-analogs. This enzyme has been exploited by Rentmeister’s research group, to transfer a wide variety of bioorthogonal functionalities to N⁷ of the cap guanine (Holstein et al., 2016; Muttach et al., 2017). Functional groups as bulky as a norbornene connected to a benzylic linker have been transferred using this enzyme (Figure 1-14). The modified cap-structure was then labeled using rapid and fluorogenic iEDDA reaction in cell lysate (Muttach et al., 2017).

Perhaps the most versatile example of methyltransferase enzymes reported so far is the C/D-box dependent archaeal methyltransferase. This RNA guided enzyme alkylates internal 2'-OH positions of RNA based on complementarity to the guide C/D box guide RNA. The approach shows great promise for versatile site-specific labeling of RNA. However the reactivity of the enzyme is extremely low towards the tested SAM analogs (Figure 1-14) (Tomkuvienė et al., 2012). This drawback may be circumvented using protein engineering methods, however, no further development has so far been reported on this system.

Figure 1-14 RNA methyltransferases in RNA labeling. (A) Common bioorthogonal reactions employed for indirect labeling of RNA, derivatized using RNA modifying enzymes. (B) General architecture of SAM analogs and the side chains transferred using various methyltransferases. (C) Schematic depiction of RNA methyltransferase-catalyzed RNA labeling. Gla-Tgs2-Var1 and Ecm1 are specific for mRNA cap structure. C/D RNP can be targeted towards different RNA sequences based on guide RNA complementarity. Trm1 and Trm11 methyltransferases modify specific G-nucleotides within their cognate tRNA.

1.4.2.2.2 Other tRNA-modifying enzymes

Except for RNA methyltransferases other classes of tRNA modifying enzymes also exist which have shown great potential for RNA labeling. The archaeal tRNA^Ile2- agmatidine synthetase (Tias) is one example of such enzymes. The enzyme naturally catalyzes ATP dependent substitution, with agmatine, of the O² at C34 of its cognate tRNA (Figure 1-15 A) (Osawa et al., 2011). Li et al demonstrated that the enzyme can readily accept a variety of alkynyl or azido functionalized agmatine analogs, including propargylamine (Figure 1-15 A). They then co-expressed Tias with a 5S rRNA-tRNA^Ile2 fusion transcript in a mammalian cell-line. The cells were then treated with propargylamine. Following this treatment, the cells were fixed. CuAAC reaction was then used to label propargylamine modified RNA in situ with SulfoCy5-Azide (Figure 1-15 B) (Li et al., 2015).

Another tRNA modifying enzyme-based system, developed by Alexander et al, is called RNA-transglycosylation at guanine (RNA-TAG) (Alexander et al., 2015). In this method, bacterial tRNA guanine transglycosylase (TGT) is taken advantage of for RNA labeling.

This enzyme is responsible for the substitution of specific guanine nucleotides with the noncanonical guanine analog PreQ1 (Figure 1-15 C). Various PreQ1 analogs with modifications at their exocyclic primary amine have been shown to be efficiently accepted as TGT substrate. Direct attachment of bulky moieties such as Cy7, bodipy, thiazole-orange, or biotin has also been possible using this enzyme. In case of the thiazole-orange conjugated PreQ1, a 40-fold increase in fluorescent intensity was observed upon RNA labeling. The enzyme recognizes a 17-nucleotide stem-loop called ECY-A1 as the core target sequence.

Insertion of ECY-A1 into the 3'-UTR of the mCherry mRNA led to its TGT mediated labeling in vitro (Figure 1-15 D) (Alexander et al., 2015). Photocleavable groups have also been attached to 5'-UTR of an in vitro transcribed EGFP mRNA. The mRNA was transfected into a mammalian cell-line and its translation was induced upon photocleavage (Zhang et al., 2018).

Figure 1-15 tRNA-modifying enzyme-mediated RNA labeling. (A) Agmatidine synthetase activity of Tias and the substrate scope of the enzyme. (B) Insertion of the tRNA^Ile2 into the RNA of interest (ROI), allows Tias-mediated two-step labeling of target RNA. (C) TGT mediated RNA labeling. The exocyclic amine of PreQ1 substrate can be derivatized directly with a variety of bulky moieties. (D) Insertion of the ECY-A1 stem-loop into ROI allows TGT mediated direct labeling of RNA.

1.4.2.3 Catalytic nucleic acid-based RNA labeling