5-Substituted uridines, 2-thiouridines and 2-selenouridines represent the most common wobble-positioned bacterial tRNA modifications, with the 5-methylaminomethyl (mnm5 substituent being particularly widespread. Their biological role in the precise recognition of synonymous purine-ending codons is still under investigation. Modified uridines are also known to enhance the stability and base pairing specificity of therapeutic nucleic acids. However, a full understanding of the O2/S2/Se2 chalcogen effect, particularly in the presence of the mnm⁵ substituent, remains limited. To address this, a systematic comparative study was conducted on the thermodynamic and structural contributions of mnm⁵ and 2-chalcogen modifications to RNA duplex properties. The duplexes were designed to contain seventeen base pairs with the sequence 5’- GUUGACUU*UUAAUCAAC-3’/3’-CAACUGA(A/G)AAUUAGUUG-5’, where U* denotes U, S2U, Se2U or their mnm5-substituted analogs. We found that chalcogens modulate the stability of duplexes with opposing adenosine in the following order: uridines < Se2-uridines < S2-uridines, with the mnm5 substituent exerting a significantly destabilizing effect. In duplexes with opposing guanosine, the influence of chalcogens is less pronounced, whether alone or in combination with mnm5, however, Se2-uridines promote duplex formation more effectively than their 2-thio and 2-oxo counterparts. This effect is likely associated with their high ionization propensity. The base pairing specificity for A over G was found to follow the order: uridines < Se2-uridines < S2-uridines. The degenerate behaviour of mnm5-uridines toward A and G was observed manifested by their tendency to reduce the base pairing discrimination between purine nucleosides. All studied RNA duplexes exhibited circular dichroism (CD) spectra characteristic of A-RNA double stranded helices. In addition, the first chemical synthesis of an mnm⁵Se²U-modified RNA oligomer is reported.

A novel and efficient way for the synthesis of N6 -hydantoin-modified adenosines, which utilizes readily available N6 -(N-Boc-α-aminoacyl)-adenosine derivatives, was developed. The procedure is based on the epimerization-free, Tf2 O-mediated conversion of the Boc group into an isocyanate moiety, followed by intramolecular cyclization. Using this method two recently discovered hydantoin modified tRNA adenosines, that is, cyclic N6 -threonylcarbamoyl-adenosine (ct6 A) and 2-methylthio-N6 -threonylcarbamoyladenosine (ms2 ct6 A) were prepared in good yields.

The 5-substituted 2-selenouridines are natural components of the bacterial tRNA epitranscriptome. Because selenium-containing biomolecules are redox-active entities, the oxidation susceptibility of 2-selenouridine (Se2U) was studied in the presence of hydrogen peroxide under various conditions and compared with previously reported data for 2-thiouridine (S2U). It was found that Se2U is more susceptible to oxidation and converted in the first step to the corresponding diselenide (Se2U)2, an unstable intermediate that decomposes to uridine and selenium. The reversibility of the oxidized state of Se2U was demonstrated by the efficient reduction of (Se2U)2 to Se2U in the presence of common reducing agents. Thus, the 2-selenouridine component of tRNA may have antioxidant potential in cells because of its ability to react with both cellular ROS components and reducing agents. Interestingly, in the course of the reactions studied, we found that (Se2U)2 reacts with Se2U to form new ‘oligomeric nucleosides′ as linear and cyclic byproducts.

An efficient method of ureido linkage formation during epimerization-free one-pot synthesis of protected hypermodified N-6-threonylcarbamoyladenosine (t(6)A) and its 2-SMe analog (ms(2)t(6)A) was developed. The method is based on a Tf2O-mediated direct conversion of the N-Boc-protecting group of N-Boc-threonine into the isocyanate derivative, followed by reaction with the N(6)exo-amine function of the sugar protected nucleoside (yield 86-94%). Starting from 2 ',3 ',5 '-tri-O-acetyl protected adenosine or 2-methylthioadenosine, the corresponding 3 '-O-phosphoramidite monomers were obtained in 48% and 42% overall yield (5 step synthesis). In an analogous synthesis, using the 2 '-O-(tert-butyldimethylsilyl)-3 ',5 '-O-(di-tert-butylsilylene) protection system at the adenosine ribose moiety, the t(6)A-phosphoramidite monomer was obtained in a less laborious manner and in a remarkably better yield of 74%.

Sulfur- and selenium-modified uridines present in the wobble position of transfer RNAs (tRNAs) play an important role in the precise reading of genetic information and tuning of protein biosynthesis in all three domains of life. Both sulfur and selenium chalcogens functionally operate as key elements of biological molecules involved in the protection of cells against oxidative damage. In this work, 2-thiouracil (S2Ura) and 2-selenouracil (Se2Ura) were treated with hydrogen peroxide at 1:0.5, 1:1, and 1:10 molar ratios and at selected pH values ranging from 5 to 8. It was found that Se2Ura was more prone to oxidation than its sulfur analog, and if reacted with H2O2 at a 1:1 or lower molar ratio, it predominantly produced diselenide Ura-Se-Se-Ura, which spontaneously transformed to a previously unknown Se-containing two-ring compound. Its deselenation furnished the major reaction product, a structure not related to any known biological species. Under the same conditions, only a small amount of S2Ura was oxidized to form Ura-SO2H and uracil (Ura). In contrast, 10-fold excess hydrogen peroxide converted Se2Ura and S2Ura into corresponding Ura-SeOnH and Ura-SOnH intermediates, which decomposed with the release of selenium and sulfur oxide(s) to yield Ura as either a predominant or exclusive product, respectively. Our results confirmed significantly different oxidation pathways of 2-selenouracil and 2-thiouracil.

The chemical synthesis of modified oligoribonucleotides represents a powerful approach to study the structure, stability, and biological activity of RNAs. Selected RNA modifications have been proven to enhance the drug-like properties of RNA oligomers providing the oligonucleotide-based therapeutic agents in the antisense and siRNA technologies. The important sites of RNA modification/functionalization are the nucleobase residues. Standard phosphoramidite RNA chemistry allows the site-specific incorporation of a large number of functional groups to the nucleobase structure if the building blocks are synthetically obtainable and stable under the conditions of oligonucleotide chemistry and work-up. Otherwise, the chemically modified RNAs are produced by post-synthetic oligoribonucleotide functionalization. This review highlights the post-synthetic RNA modification approach as a convenient and valuable method to introduce a wide variety of nucleobase modifications, including recently discovered native hypermodified functional groups, fluorescent dyes, photoreactive groups, disulfide crosslinks, and nitroxide spin labels.

5-Substituted 2-selenouridines (R5Se2U) are post-transcriptional modifications present in the first anticodon position of transfer RNA. Their functional role in the regulation of gene expression is elusive. Here, we present efficient syntheses of 5-methylaminomethyl-2-selenouridine (1, mnm5Se2U), 5-carboxymethylaminomethyl-2-selenouridine (2, cmnm5Se2U), and Se2U (3) alongside the crystal structure of the latter nucleoside. By using pH-dependent potentiometric titration, pKa values for the N3H groups of 1–3 were assessed to be significantly lower compared to their 2-thio- and 2-oxo-congeners. At physiological conditions (pH 7.4), Se2-uridines 1 and 2 preferentially adopted the zwitterionic form (ZI, ca. 90%), with the positive charge located at the amino alkyl side chain and the negative charge at the Se2-N3-O4 edge. As shown by density functional theory (DFT) calculations, this ZI form efficiently bound to guanine, forming the so-called “new wobble base pair”, which was accepted by the ribosome architecture. These data suggest that the tRNA anticodons with wobble R5Se2Us may preferentially read the 5′-NNG-3′ synonymous codons, unlike their 2-thio- and 2-oxo-precursors, which preferentially read the 5′-NNA-3′ codons. Thus, the interplay between the levels of U-, S2U- and Se2U-tRNA may have a dominant role in the epitranscriptomic regulation of gene expression via reading of the synonymous 3′-A- and 3′-G-ending codons.

The synthesis of the protected form of 2-methylthio-N6 -threonylcarbamoyl adenosine (ms2 t6 A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms2 t6 A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms2 t6 A-oligonucleotide (the anticodon stem and loop fragment of tRNALys from T. brucei). Finally, stereochemically secure ms2 t6 A→ms2 ct6 A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms2 ct6 A unit. The applied post-synthetic approach provides two sequentially homologous ms2 t6 A- and ms2 ct6 A-oligonucleotides that are suitable for further comparative structure-activity relationship studies.

Two novel methods for the preparation of the virtually equimolar mixtures of (S)- and (R)-diastereomers of 5-methoxycarbonylhydroxymethyluridine (mchm5U) have been developed.

Hydantoin modified ms2ct6A nucleoside was introduced into the RNA chain by post-synthetic ms2t6A→ms2ct6A cyclization at the oligonucleotide level. The precursor 17 nt ms2t6A-oligonucleotide (ASL of tRNALys from T. brucei) was prepared using phosphoramidite chemistry. The hypermodified ms2t6A-phosphoramidite was synthesized efficiently from adenosine or guanosine by the optimized carbamate and, for the first time, the isocyanate chemistry. Obtained ms2t6A- and ms2ct6A-RNA can be used for comparative studies of structure–function relationships. More information can be found in the Full Paper by E. Sochacka et al. on page 13309.

Post-transcriptional chemical modifications of (t) RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.

The 5-substituted 2-thiouridines (R5S2Us) present in the first (wobble) position of the anticodon of transfer RNAs (tRNAs) contribute to accuracy in reading mRNA codons and tuning protein synthesis. Previously, we showed that, under oxidative stress conditions in vitro, R5S2Us were sensitive to hydrogen peroxide (H2O2) and that their oxidative desulfuration produced 5-substituted uridines (R5Us) and 4-pyrimidinone nucleosides (R5H2Us) at a ratio that depended on the pH and an R5 substituent. Here, we demonstrate that the desulfuration of 2-thiouridines, either alone or within an RNA/tRNA chain, is catalyzed by cytochromec (cytc). Its kinetics are similar to those of Fenton-type catalytic 2-thiouridine (S2U) desulfuration. Cytc/H2O2- and Fe-II-mediated reactions deliver predominantly 4-pyrimidinone nucleoside (H2U)-type products. The pathway of the cytc/H2O2-peroxidase-mediated S2UH2U transformation through uridine sulfenic (U-SOH), sulfinic (U-SO2H), and sulfonic (U-SO3H) intermediates is confirmed by LC-MS. The cytc/H2O2-mediated oxidative damage of S2U-tRNA may have biological relevance through alteration of the cellular functions of transfer RNA.

To date the only tRNAs containing nucleosides modified with a selenium (5‐carboxymethylaminomethyl‐2‐selenouridine and 5‐methylaminomethyl‐2‐selenouridine) have been found in bacteria. By using tRNA anticodon‐stem‐loop fragments containing S2U, Se2U, or geS2U, we found that in vitro tRNA 2‐selenouridine synthase (SelU) converts S2U‐RNA to Se2U‐RNA in a two‐step process involving S2U‐RNA geranylation (with ppGe) and subsequent selenation of the resulting geS2U‐RNA (with SePO33−). No ‘direct’ S2U‐RNA→Se2U‐RNA replacement is observed in the presence of SelU/SePO33− only (without ppGe). These results suggest that the in vivo S2U→Se2U and S2U→geS2U transformations in tRNA, so far claimed to be the elementary reactions occurring independently in the same domain of the SelU enzyme, should be considered a combination of two consecutive events – geranylation (S2U→geS2U) and selenation (geS2U→Se2U).

A post-synthetic reaction of 5-pivaloyloxymethyluridine (present in a support-bound RNA oligomer) with various nucleophilic reagents furnished efficiently the corresponding products bearing one of the tRNA wobble 5-methyluridines (mnm5U, cmnm5U, τm5U, nm5U, inm5U or cnm5U). The syntheses of oligoribonucleotides modified with inm5U and cnm5U are reported for the first time.

Modified nucleosides present in the wobble position of the tRNA anticodons regulate protein translation through tuning the reading of mRNA codons. Among 40 of such nucleosides, there are modified uridines containing either a sulfur atom at the C2 position and/or a substituent at the C5 position of the nucleobase ring. It is already evidenced that tRNAs with 2-thiouridines at the wobble position preferentially read NNA codons, while the reading mode of the NNG codons by R5U/R5S2U-containing anticodons is still elusive. For a series of 18 modified uridines and 2-thiouridines, we determined the pKa values and demonstrated that both modifying elements alter the electron density of the uracil ring and modulate the acidity of their N3H proton. In aqueous solutions at physiological pH the 2-thiouridines containing aminoalkyl C5-substituents are ionized in ca. 50%. The results, confirmed also by theoretical calculations, indicate that the preferential binding of the modified units bearing non-ionizable 5-substituents to guanosine in the NNG codons may obey the alternative C-G-like (Watson-Crick) mode, while binding of those bearing aminoalkyl C5-substituents (protonated under physiological conditions) and especially those with a sulfur atom at the C2 position, adopt a zwitterionic form and interact with guanosine via a 'new wobble' pattern.

A t6A nucleoside was efficiently and stereospecifically transformed into a hydantoin cyclic form of N6-l-threonylcarbamoyladenosine (ct6A) by the use of polymer bounded carbodiimide (EDC-P) and HOBt. The procedure was successfully applied for a post-synthetic conversion of t6A-containing RNA 17-mers (of the sequences of anticodon stem and loop (ASL) fragments of S. pombe tRNAi and E. coli tRNALys) into the products bearing the ct6A unit.