Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates.

Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry; however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered by their rapid in vivo metabolism. Although a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. This operationally simple approach provides access to pure stereoisomers of nucleoside α-thiodiphosphates and α-thiotriphosphates, as well as symmetrical or unsymmetrical dinucleoside thiodiphosphates and thiotriphosphates (including RNA cap reagents). We demonstrate that ligand-receptor interactions can be dramatically influenced by P-stereochemistry, showing that such thioisosteric replacements can have profound effects on the potency and stability of lead candidates.

Chemical Modifications of mRNA Ends for Therapeutic Applications.

Messenger ribonucleic acid (mRNA) is the universal cellular instruction for ribosomes to produce proteins. Proteins are responsible for most of the functions of living organisms, and their abnormal structure or activity is the cause of many diseases. mRNA, which is expressed in the cytoplasm and, unlike DNA, does not need to be delivered into the nucleus, appears to be an ideal vehicle for pursuing the idea of gene therapy in which genetic information about proteins is introduced into an organism to exert a therapeutic effect. mRNA molecules of any sequence can be synthesized using the same set of reagents in a cell-free system via a process called in vitro transcription (IVT), which is very convenient for therapeutic applications. However, this does not mean that the path from the idea to the first mRNA-based therapeutic was short and easy. It took 30 years of trial and error in the search for solutions that eventually led to the first mRNA vaccines created in record time during the SARS-CoV-2 pandemic. One of the fundamental problems in the development of RNA-based therapeutics is the legendary instability of mRNA, due to the transient nature of this macromolecule. From the chemical point of view, mRNA is a linear biopolymer composed of four types of ribonucleic subunits ranging in length from a few hundred to hundreds of thousands of nucleotides, with unique structures at its ends: a 5'-cap at the 5'-end and a poly(A) tail at the 3'-end. Both are extremely important for the regulation of translation and mRNA durability. These elements are also convenient sites for sequence-independent labeling of mRNA to create probes for enzymatic assays and tracking of the fate of mRNA in cells and living organisms. Synthetic 5'-cap analogs have played an important role in the studies of mRNA metabolism, and some of them have also been shown to significantly improve the translational properties of mRNA or affect mRNA stability and reactogenicity. The most effective of these is used in clinical trials of mRNA-based anticancer vaccines. Interestingly, thanks to the knowledge gained from the biophysical studies of cap-related processes, even relatively large modifications such as fluorescent tags can be attached to the cap structure without significant effects on the biological properties of the mRNA, if properly designed cap analogs are used. This has been exploited in the development of molecular tools (fluorescently labeled mRNAs) to track these macromolecules in complex biological systems, including organisms. These tools are extremely valuable for better understanding of the cellular mechanisms involved in mRNA metabolism but also for designing therapeutic mRNAs with superior properties. Much less is known about the usefulness/utility of poly(A) tail modifications in the therapeutic context, but it is clear that chemical modifications of poly(A) can also affect biochemical properties of mRNA. This Account is devoted to chemical modifications of both the 5'- and 3'-ends of mRNA aimed at improving the biological properties of mRNA, without interfering with its translational function, and is based on the authors' more than 20 years of experience in this field.

Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PLpro and Mpro proteases, and nsp14 guanine N7-methyltransferase.

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage-a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues-bis(2-carbamoylaryl)phenyl diselenides-in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.

Synthesis and Evaluation of Diguanosine Cap Analogs Modified at the C8-Position by Suzuki-Miyaura Cross-Coupling: Discovery of 7-Methylguanosine-Based Molecular Rotors.

Chemical modifications of the mRNA cap structure can enhance the stability, translational properties, and half-life of mRNAs, thereby altering the therapeutic properties of synthetic mRNA. However, cap structure modification is challenging because of the instability of the 5'-5'-triphosphate bridge and N7-methylguanosine. The Suzuki-Miyaura cross-coupling reaction between boronic acid and halogen compound is a mild, convenient, and potentially applicable approach for modifying biomolecules. Herein, we describe two methods to synthesize C8-modified cap structures using the Suzuki-Miyaura cross-coupling reaction. Both methods employed phosphorimidazolide chemistry to form the 5',5'-triphosphate bridge. However, in the first method, the introduction of the modification via the Suzuki-Miyaura cross-coupling reaction at the C8 position occurs postsynthetically, at the dinucleotide level, whereas in the second method, the modification was introduced at the level of the nucleoside 5'-monophosphate, and later, the triphosphate bridge was formed. Both methods were successfully applied to incorporate six different groups (methyl, cyclopropyl, phenyl, 4-dimethylaminophenyl, 4-cyanophenyl, and 1-pyrene) into either the m7G or G moieties of the cap structure. Aromatic substituents at the C8-position of guanosine form a push-pull system that exhibits environment-sensitive fluorescence. We demonstrated that this phenomenon can be harnessed to study the interaction with cap-binding proteins, e.g., eIF4E, DcpS, Nudt16, and snurportin.

2'-O-Methylation of the second transcribed nucleotide within the mRNA 5' cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion.

In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2'-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2'-O-methylated adenosine, it can be additionally modified to N6,2'-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between 'self' and 'non-self' in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2'-O-methylation of the second transcribed nucleotide within the mRNA 5' cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2'-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as 'self' and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.

Introducing SuFNucs: Sulfamoyl-Fluoride-Functionalized Nucleosides That Undergo Sulfur Fluoride Exchange Reaction.

The reaction between ribonucleosides and ex situ generated sulfonyl fluoride has been developed. The reaction takes place at the -NH2 groups of nucleobases, and the resulting nucleosides are equipped with a sulfamoyl fluoride moiety, dubbed SuFNucs. These species undergo a selective sulfur fluoride exchange (SuFEx) reaction with various amines, leading to sulfamide-functionalized derivatives of adenosine, guanosine, and cytidine (SulfamNucs). The scope and examples of further SuFNucs fuctionalization leading to nucleotides, oligonucleotides, and peptide-nucleoside conjugates are presented.

Fluorinated Phosphoadenosine 5'-Phosphosulfate Analogues for Continuous Sulfotransferase Activity Monitoring and Inhibitor Screening by 19F NMR Spectroscopy.

Sulfotransferases (STs) are ubiquitous enzymes that participate in a vast number of biological processes involving sulfuryl group (SO3) transfer. 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the universal ST cofactor, serving as the "active sulfate" source in cells. Herein, we report the synthesis of three fluorinated PAPS analogues that bear fluorine or trifluoromethyl substituents at the C2 or C8 positions of adenine and their evaluation as substitute cofactors that enable ST activity to be quantified and real-time-monitored by fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy. Using plant AtSOT18 and human SULT1A3 as two model enzymes, we reveal that the fluorinated PAPS analogues show complementary properties with regard to recognition by enzymes and the working 19F NMR pH range and are attractive versatile tools for studying STs. Finally, we developed an 19F NMR assay for screening potential inhibitors against SULT1A3, thereby highlighting the possible use of fluorinated PAPS analogues for the discovery of drugs for ST-related diseases.

Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications.

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5',5'-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging - they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands - folic acid, biotin, glucose, and cholesterol - to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.

Upregulation of RNA cap methyltransferase RNMT drives ribosome biogenesis during T cell activation.

The m7G cap is ubiquitous on RNAPII-transcribed RNA and has fundamental roles in eukaryotic gene expression, however its in vivo role in mammals has remained unknown. Here, we identified the m7G cap methyltransferase, RNMT, as a key mediator of T cell activation, which specifically regulates ribosome production. During T cell activation, induction of mRNA expression and ribosome biogenesis drives metabolic reprogramming, rapid proliferation and differentiation generating effector populations. We report that RNMT is induced by T cell receptor (TCR) stimulation and co-ordinates the mRNA, snoRNA and rRNA production required for ribosome biogenesis. Using transcriptomic and proteomic analyses, we demonstrate that RNMT selectively regulates the expression of terminal polypyrimidine tract (TOP) mRNAs, targets of the m7G-cap binding protein LARP1. The expression of LARP1 targets and snoRNAs involved in ribosome biogenesis is selectively compromised in Rnmt cKO CD4 T cells resulting in decreased ribosome synthesis, reduced translation rates and proliferation failure. By enhancing ribosome abundance, upregulation of RNMT co-ordinates mRNA capping and processing with increased translational capacity during T cell activation.

RNA Ligation for Mono and Dually Labeled RNAs.

Labeled RNAs are invaluable probes for investigation of RNA function and localization. However, mRNA labeling remains challenging. Here, we developed an improved method for 3'-end labeling of in vitro transcribed RNAs. We synthesized novel adenosine 3',5'-bisphosphate analogues modified at the N6 or C2 position of adenosine with an azide-containing linker, fluorescent label, or biotin and assessed these constructs as substrates for RNA labeling directly by T4 ligase or via postenzymatic strain-promoted alkyne-azide cycloaddition (SPAAC). All analogues were substrates for T4 RNA ligase. Analogues containing bulky fluorescent labels or biotin showed better overall labeling yields than postenzymatic SPAAC. We successfully labeled uncapped RNAs, NAD-capped RNAs, and 5'-fluorescently labeled m7 Gp3 Am -capped mRNAs. The obtained highly homogenous dually labeled mRNA was translationally active and enabled fluorescence-based monitoring of decapping. This method will facilitate the use of various functionalized mRNA-based probes.

Efficient Synthesis of Trifluoromethylated Purine Ribonucleosides and Ribonucleotides.

The protocols presented in this article describe highly detailed synthesis of trifluoromethylated purine nucleotides and nucleosides (G and A). The procedure involves trifluoromethylation of properly protected (acetylated) nucleosides, followed by deprotection leading to key CF3 -containing nucleosides. This gives synthetic access to 8-CF3 -substituted guanosine derivatives and three adenosine derivatives (8-CF3 , 2-CF3 , and 2,8-diCF3 ). In further steps, phosphorylation and phosphate elongation (for selected examples) result in respective trifluoromethylated nucleoside mono-, di-, and triphosphates. Support protocols are included for compound handling, purification procedures, analytical sample preparation, and analytical techniques used throughout the performance of the basic protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of trifluoromethylated guanosine and adenosine derivatives Basic Protocol 2: Synthesis of trifluoromethylated guanosine and adenosine monophosphates Basic Protocol 3: Synthesis of phosphorimidazolides of 8-CF3 GMP and 8-CF3 AMP Basic Protocol 4: Synthesis of trifluoromethylated guanosine and adenosine oligophosphates Support Protocol 1: TLC sample preparation and analysis Support Protocol 2: Purification protocol for Basic Protocol 1 Support Protocol 3: HPLC analysis and preparative HPLC Support Protocol 4: Ion-exchange chromatography.

Phosphodiester modifications in mRNA poly(A) tail prevent deadenylation without compromising protein expression.

Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases-SP6, T7, and poly(A) polymerase. To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.

Exploring tryptamine conjugates as pronucleotides of phosphate-modified 7-methylguanine nucleotides targeting cap-dependent translation.

Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in many cancers deregulating translational control of the cell cycle. mRNA 5' cap analogs targeting eIF4E are small molecules with the potential to counteract elevated levels of eIF4E in cancer cells. However, the practical utility of typical cap analogs is limited because of their reduced cell membrane permeability. Transforming the active analogs into their pronucleotide derivatives is a promising approach to overcome this obstacle. 7-Benzylguanosine monophosphate (bn7GMP) is a cap analog that has been successfully transformed into a cell-penetrating pronucleotide by conjugation of the phosphate moiety with tryptamine. In this work, we explored whether a similar strategy is applicable to other cap analogs, particularly phosphate-modified 7-methylguanine nucleotides. We report the synthesis of six new tryptamine conjugates containing N7-methylguanosine mono- and diphosphate and their analogs modified with thiophosphate moiety. These new potential pronucleotides and the expected products of their activation were characterized by biophysical and biochemical methods to determine their affinity towards eIF4E, their ability to inhibit translation in vitro, their susceptibility to enzymatic degradation and their turnover in cell extract. The results suggest that compounds containing the thiophosphate moiety may act as pronucleotides that release low but sustainable concentrations of 7-methylguanosine 5'-phosphorothioate (m7GMPS), which is a translation inhibitor with in vitro potency higher than bn7GMP.

Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity.

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5' cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3'-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

Synthesis of Trifluoromethylated Purine Ribonucleotides and Their Evaluation as 19F NMR Probes.

Protected guanosine and adenosine ribonucleosides and guanine nucleotides are readily functionalized with CF3 substituents within the nucleobase. Protected guanosine is trifluoromethylated at the C8 position under radical-generating conditions in up to 95% yield and guanosine 5'-oligophosphates in up to 35% yield. In the case of adenosine, the selectivity of trifluoromethylation depends heavily on the functional group protection strategy and leads to a set of CF3-modified nucleosides with different substitution patterns (C8, C2, or both) in up to 37% yield. Further transformations based on phosphorimidazolide chemistry afford various CF3-substituted mono- and dinucleoside oligophosphates in good yields. The utility of the trifluoromethylated nucleotides as probes for 19F NMR-based real-time enzymatic reaction monitoring is demonstrated with three different human nucleotide hydrolases (Fhit, DcpS, and cNIIIB). Substrate and product(s) resonances were sufficiently separated to enable effective tracking of each enzymatic activity of interest.

Kinetic analysis of IFIT1 and IFIT5 interactions with different native and engineered RNAs and its consequences for designing mRNA-based therapeutics.

In response to foreign RNA, cellular antiviral mechanisms stimulate high expression of interferon-induced proteins with tetratricopeptide repeats (IFITs). Two members of the IFIT protein family, IFIT1 and IFIT5, are capable of binding the very terminal 5' end of mRNA. In eukaryotes, these mRNA termini contain a cap structure (m7GpppN, cap 0) that is often subjected to further modifications. Here, we performed a thorough examination of IFIT1 and IFIT5 binding to a wide spectrum of differently capped as well as fully uncapped mRNAs. The kinetic analysis of IFIT1 and IFIT5 interactions with mRNA ligands indicates that the cap structure modifications considerably influence the stability of IFIT1/RNA complexes. The most stable complexes were formed between IFIT1 and GpppG/A- and m7GpppG/A-RNAs. Unexpectedly, we found that NAD+- and NADH-capped RNAs associate with IFIT5 with kinetic parameters comparable to pppG-RNA. Finally, we measured interactions of IFIT1 with mRNAs bearing modified synthetic cap analogs that start to become the important tools in biotechnological and medicinal research. We found that incorporation of modified cap analogs to the RNA protects the latter, to a certain degree, from the translational inhibition caused by IFIT1 protein.

Purine and pyrimidine dinucleoside polyphosphates differentially affect the phenylpropanoid pathway in Vitis vinifera L. cv. Monastrell suspension cultured cells.

It is known that the concentration of dinucleoside polyphosphates (NpnN's) in cells increases under stress and that adverse environmental factors induce biosynthesis of phenylpropanoids, which protect the plant against stress. Previously, we showed that purine NpnN's such as Ap3A and Ap4A induce both the activity of enzymes of the phenylpropanoid pathway and the expression of relevant genes in Arabidopsis seedlings. Moreover, we showed that Ap3A induced stilbene biosynthesis in Vitis vinifera cv. Monastrell suspension cultured cells. Data presented in this paper show that pyrimidine-containing NpnN's also modify the biosynthesis of stilbenes, affecting the transcript level of genes encoding key enzymes of the phenylpropanoid pathway and of these, Up4U caused the most effective accumulation of trans-resveratrol in the culture media. Similar effect was caused by Ap3A and Gp3G. Other pyrimidine NpnN's, such as Cp3C, Cp4C, and Ap4C, strongly inhibited the biosynthesis of stilbenes, but markedly (6- to 8-fold) induced the expression of the cinnamoyl-CoA reductase gene that controls lignin biosynthesis. Purine counterparts also clearly induced biosynthesis of trans-resveratrol and trans-piceid, but only slightly induced the expression of genes involved in lignin biosynthesis. In cells, Up3U caused a greater accumulation of trans-resveratrol and trans-piceid than did Up4U. Each of the NpnN's studied induced expression of the gene encoding the resveratrol transporter VvABCG44, which operates within the Vitis vinifera cell membrane. AMP, GMP, UMP, and CMP, potential products of NpnN degradation, did not affect the accumulation of stilbenes. The results obtained strongly support that NpnN's play a role as signaling molecules in plants.

5'-Phosphorothiolate Dinucleotide Cap Analogues: Reagents for Messenger RNA Modification and Potent Small-Molecular Inhibitors of Decapping Enzymes.

The 5' cap consists of 7-methylguanosine (m7G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented ß-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and ß-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting ß-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.

mRNA cap analogues substituted in the tetraphosphate chain with CX2: identification of O-to-CCl2 as the first bridging modification that confers resistance to decapping without impairing translation.

Analogues of the mRNA 5'-cap are useful tools for studying mRNA translation and degradation, with emerging potential applications in novel therapeutic interventions including gene therapy. We report the synthesis of novel mono- and dinucleotide cap analogues containing dihalogenmethylenebisphosphonate moiety (i.e. one of the bridging O atom substituted with CCl2 or CF2) and their properties in the context of cellular translational and decapping machineries, compared to phosphate-unmodified and previously reported CH2-substituted caps. The analogues were bound tightly to eukaryotic translation initiation factor 4E (eIF4E), with CCl2-substituted analogues having the highest affinity. When incorporated into mRNA, the CCl2-substituted dinucleotide most efficiently promoted cap-dependent translation. Moreover, the CCl2-analogues were potent inhibitors of translation in rabbit reticulocyte lysate. The crystal structure of eIF4E in complex with the CCl2-analogue revealed a significantly different ligand conformation compared to that of the unmodified cap analogue, which likely contributes to the improved binding. Both CCl2- and CF2- analogues showed lower susceptibility to hydrolysis by the decapping scavenger enzyme (DcpS) and, when incorporated into RNA, conferred stability against major cellular decapping enzyme (Dcp2) to transcripts. Furthermore, the use of difluoromethylene cap analogues was exemplified by the development of 19F NMR assays for DcpS activity and eIF4E binding.