Trimethylguanosine cap-fluorescent molecular rotor (TMG-FMR) conjugates are potent, specific snurportin1 ligands enabling visualization in living cells.

The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.

Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap® Analog by In Vitro Transcription.

Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.

Isoxazole-containing 5' mRNA cap analogues as inhibitors of the translation initiation process.

Herein we describe a synthesis of new isoxazole-containing 5' mRNA cap analogues via a cycloaddition reaction. The obtained analogues show a capability to inhibit cap-dependent translation in vitro and are characterized by a new binding mode in which an isoxazolic ring, instead of guanine, is involved in the stacking effect. Our study provides valuable information toward designing new compounds that can be potentially used as anticancer therapeutics.

Highly Regioselective Methylation of Guanosine Nucleotides: An Efficient Synthesis of 7-Methylguanosine Nucleotides.

This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5'-O-monophosphate (m7 GMP), 7-methylguanosine 5'-O-diphosphate (m7 GDP), 7-methyl-2'-deoxyguanosine 5'-O-triphosphate (m7 2'dGTP), and 7-methylguanosine 5'-O-triphosphate (m7 GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7-methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 7-methylguanosine nucleotides.

N1-Propargylguanosine Modified mRNA Cap Analogs: Synthesis, Reactivity, and Applications to the Study of Cap-Binding Proteins.

The mRNA 5' cap consists of N7-methylguanosine bound by a 5',5'-triphosphate bridge to the first nucleotide of the transcript. The cap interacts with various specific proteins and participates in all key mRNA-related processes, which may be of therapeutic relevance. There is a growing demand for new biophysical and biochemical methods to study cap-protein interactions and identify the factors which inhibit them. The development of such methods can be aided by the use of properly designed fluorescent molecular probes. Herein, we synthesized a new class of m7Gp3G cap derivatives modified with an alkyne handle at the N1-position of guanosine and, using alkyne-azide cycloaddition, we functionalized them with fluorescent tags to obtain potential probes. The cap derivatives and probes were evaluated in the context of two cap-binding proteins, eukaryotic translation initiation factor (eIF4E) and decapping scavenger (DcpS). Biochemical and biophysical studies revealed that N1-propargyl moiety did not significantly disturb cap-protein interaction. The fluorescent properties of the probes turned out to be in line with microscale thermophoresis (MST)-based binding assays.

Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5' Caps.

Eukaryotic RNAs are heavily processed, including co- and post-transcriptional formation of various 5' caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7m G cap is hypermethylated at the N2 -position, whereas in higher eukaryotes and viruses 2'-O-methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid-phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site-specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di-and trimethylated caps, as well as RNAs with caps methylated at the 2'-O-position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.

7-Methylguanosine monophosphate analogues with 5'-(1,2,3-triazoyl) moiety: Synthesis and evaluation as the inhibitors of cNIIIB nucleotidase.

The hydrolysis of nucleoside 5'-monophosphates to the corresponding nucleosides and inorganic phosphate is catalysed by 5'-nucleotidases, thereby contributing to the control of endogenous nucleotide turnover and affecting the fate of exogenously delivered nucleotide- and nucleoside-derived therapeutics in cells. A recently identified nucleotidase cNIIIB shows preference towards 7-methylguanosine monophosphate (m7GMP) as a substrate, which suggests its potential involvement in mRNA degradation. However, the extent of biological functions and the significance of cNIIIB remains to be elucidated. Here, we synthesised a series of m7GMP analogues carrying a 1,2,3-triazole moiety at the 5' position as the potential inhibitors of human cNIIIB. The compounds were synthesised by using the copper-catalysed azide-alkyne cycloaddition (CuAAC) between 5'-azido-5'-deoxy-7-methylguanosine and different phosphate or phosphonate derivatives carrying terminal alkyne. The analogues were evaluated as cNIIIB inhibitors using HPLC and malachite green assays, demonstrating that compound 1a, carrying a 1,2,3-triazoylphosphonate moiety, inhibits cNIIIB activity at micromolar concentrations (IC50 87.8 ±â€¯7.5 µM), while other analogues showed no activity. In addition, compound 1d was identified as an artifical substrate for HscNIIIB. Further characterization of inhibitor 1a revealed that it is poorly recognised by other m7G-binding proteins, eIF4E and DcpS, indicating its selectivity towards cNIIIB. The first inhibitor (1a) and unnatural substrate (1d) of cNIIIB, identified here, can be used as molecular probes for the elucidation of biological roles of cNIIIB, including the verification of its proposed function in mRNA metabolism.

Cap analogs modified with 1,2-dithiodiphosphate moiety protect mRNA from decapping and enhance its translational potential.

Along with a growing interest in mRNA-based gene therapies, efforts are increasingly focused on reaching the full translational potential of mRNA, as a major obstacle for in vivo applications is sufficient expression of exogenously delivered mRNA. One method to overcome this limitation is chemically modifying the 7-methylguanosine cap at the 5' end of mRNA (m7Gppp-RNA). We report a novel class of cap analogs designed as reagents for mRNA modification. The analogs carry a 1,2-dithiodiphosphate moiety at various positions along a tri- or tetraphosphate bridge, and thus are termed 2S analogs. These 2S analogs have high affinities for translation initiation factor 4E, and some exhibit remarkable resistance against the SpDcp1/2 decapping complex when introduced into RNA. mRNAs capped with 2S analogs combining these two features exhibit high translation efficiency in cultured human immature dendritic cells. These properties demonstrate that 2S analogs are potentially beneficial for mRNA-based therapies such as anti-cancer immunization.

Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m(7)GpppG.

Fluorescent mRNA molecules offer a wide range of applications for studying capping/decapping reactions, translation, and other biophysical studies. Furthermore, fluorescent tags prove invaluable for tracking RNA molecules in cells. Here, we describe an efficient synthesis of a fluorescent cap analog, anthranioyl-GTP, its purification, and in vitro cap labeling of transcribed mRNA catalyzed by the recombinant vaccinia capping enzyme to produce anthranioyl-m(7)GpppG-capped RNA.

Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety.

The first example of the synthesis of new dinucleotide cap analog containing propargyl group such as m(7,3'-O-propargyl)G[5']ppp[5']G is reported. The effect of propargyl cap analog with standard cap was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. It is noteworthy that propargyl cap analog outperforms standard cap by 3.1 fold in terms of translational properties. The propargyl cap analog forms a more stable complex with translation initiation factor eIF4E based on the molecular modeling studies.

Phosphate-modified analogues of m(7)GTP and m(7)Gppppm(7)G-Synthesis and biochemical properties.

The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between ß and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m(7)GMP analogue with a given P(1),P(2)-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m(7)GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.

Design and Facile Synthesis of New Dinucleotide Cap Analog Containing Both 2' and 3'-OH Modification on M7Guanosine Moiety.

The first example of the synthesis of new dinucleotide cap analog containing 2('),3(')-diacetyl group on m(7)guanosine moiety is described. The desired modified cap analog, m(7,2)(')(,3)(')(-diacetyl)G[5(')]ppp[5(')]G has been obtained by the coupling reaction of triethylamine salt of m(7,2)(')(,3)(')(-diacetyl)GDP with ImGMP in presence of ZnCl2 as a catalyst in 62% yield with high purity. The structure of new cap analog has been confirmed by (1)H and (31)P NMR and mass data.

Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5',5'-triphosphate bridge.

A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,ß-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,ß-bridging (m3(2,2,7)GppNHpG) or ß-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.

Synthesis, properties, and biological activity of boranophosphate analogs of the mRNA cap: versatile tools for manipulation of therapeutically relevant cap-dependent processes.

Modified mRNA cap analogs aid in the study of mRNA-related processes and may enable creation of novel therapeutic interventions. We report the synthesis and properties of 11 dinucleotide cap analogs bearing a single boranophosphate modification at either the α-, ß- or γ-position of the 5',5'-triphosphate chain. The compounds can potentially serve either as inhibitors of translation in cancer cells or reagents for increasing expression of therapeutic proteins in vivo from exogenous mRNAs. The BH3-analogs were tested as substrates and binding partners for two major cytoplasmic cap-binding proteins, DcpS, a decapping pyrophosphatase, and eIF4E, a translation initiation factor. The susceptibility to DcpS was different between BH3-analogs and the corresponding analogs containing S instead of BH3 (S-analogs). Depending on its placement, the boranophosphate group weakened the interaction with DcpS but stabilized the interaction with eIF4E. The first of the properties makes the BH3-analogs more stable and the second, more potent as inhibitors of protein biosynthesis. Protein expression in dendritic cells was 2.2- and 1.7-fold higher for mRNAs capped with m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m2 (7,3'-O)GpppG. Higher expression of cancer antigens would make mRNAs containing m2 (7,2'-O)GppBH3pG D1 and m2 (7,2'-O)GppBH3pG D2 favorable for anticancer immunization.

Triazole-containing monophosphate mRNA cap analogs as effective translation inhibitors.

Synthetic analogs of the 5' end of mRNA (cap structure) are widely used in molecular studies on mechanisms of cellular processes such as translation, intracellular transport, splicing, and turnover. The best-characterized cap binding protein is translation initiation factor 4E (eIF4E). Recognition of the mRNA cap by eIF4E is a critical, rate-limiting step for efficient translation initiation and is considered a major target for anticancer therapy. Here, we report a facile methodology for the preparation of N2-triazole-containing monophosphate cap analogs and present their biological evaluation as inhibitors of protein synthesis. Five analogs possessing this unique hetero-cyclic ring spaced from the m7-guanine of the cap structure at a distance of one or three carbon atoms and/or additionally substituted by various groups containing the benzene ring were synthesized. All obtained compounds turned out to be effective translation inhibitors with IC50 similar to dinucleotide triphosphate m(7)GpppG. As these compounds possess a reduced number of phosphate groups and, thereby, a negative charge, which may support their cell penetration, this type of cap analog might be promising in terms of designing new potential therapeutic molecules. In addition, an exemplary dinucleotide from a corresponding mononucleotide containing benzyl substituted 1,2,3-triazole was prepared and examined. The superior inhibitory properties of this analog (10-fold vs. m(7)GpppG) suggest the usefulness of such compounds for the preparation of mRNA transcripts with high translational activity.

Cap analogs containing 6-thioguanosine--reagents for the synthesis of mRNAs selectively photo-crosslinkable with cap-binding biomolecules.

Numerous biomolecules recognize the 7-methylguanosine cap structure present at the 5' ends of eukaryotic mRNAs. Photo-crosslinking is a valuable technique to study these interactions. We report three anti-reverse cap analogs containing a photo-activable nucleoside, 6-thioguanosine ((6S)G), that enable the synthesis of capped RNAs with (6S)G positioned exclusively as the first transcribed nucleotide. The effect of the 6-thioguanosine moiety on binding to the translation factor eIF4E and the efficiency of mRNA translation was determined. The utility of mRNAs with a (6S)G-modified cap in crosslinking experiments is shown by mapping the histone H4 cap-binding pocket.

Chemical synthesis of dinucleotide cap analogs.

This unit describes a reliable, efficient and general method for the synthesis of standard cap analog (mCAP), m(7)G[5']ppp[5']G, and anti-reverse cap analog (ARCA), m(7,3')(O)G[5']ppp[5']G. The synthesis of required intermediate m(7)GDP or m(2)(7,3'O)GDP has been achieved through regioselective methylation of the corresponding diphosphate using dimethyl sulfate under aqueous conditions. Then, the coupling reaction of m(7)GDP or m(2)(7,3'O)GDP with ImGMP using ZnCl(2)/DMF system affords the corresponding cap analog in good yields.

Synthesis and biological validation of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs for mRNA translation.

Design, synthesis and biological validation of dinucleotide cap analogs, N(7)-(4-chlorophenoxyethyl)-G(5')ppp(5')G (5a) and N(7)-(4-chlorophenoxyethyl)-m(3'-O)G(5')ppp(5')G (5b) are reported. The effect of N(7)-(4-chlorophenoxyethyl) substitution on cap analogs has been evaluated with respect to its in vitro transcription by using T7 RNA polymerase capping efficiency, and translational activity. The gel shift assay indicates that the new cap analogs (5a, 5b) showed 77% and 76% capping efficiency respectively, whereas the standard cap analog, m(7)G(5')ppp(5')G has a capping efficiency of 63%. The capping efficiency experiment clearly demonstrates that the N(7)-modified analogs are good substrate for T7 RNA polymerase. It is noteworthy that the mRNA poly(A) capped with N(7)-(4-chlorophenoxyethyl)-m(3'-O)G(5')ppp(5')G (5b) was translated ∼1.64-fold more efficiently, while compound (5a) was translated ∼0.72-fold less efficiently than mRNA capped with standard cap analog. The observed low translation activity for (5a) could be due to stability in the form of dinucleotide cap analogs. Based on the substrate compatability of the N(7) modification in dinucleotide form, these new analogs may be used for structure function studies as well as protein production.

Synthesis of N²-modified 7-methylguanosine 5'-monophosphates as nematode translation inhibitors.

Preparative scale synthesis of 14 new N(2)-modified mononucleotide 5' mRNA cap analogues was achieved. The key step involved use of an S(N)Ar reaction with protected 2-fluoro inosine and various primary and secondary amines. The derivatives were tested in a parasitic nematode, Ascaris suum, cell-free system as translation inhibitors. The most effective compound with IC(50) ∼0.9µM was a N(2)-p-metoxybenzyl-7-methylguanosine-5'-monophosphate 35.

Synthesis of ¹³C- and ¹4C-labeled dinucleotide mRNA cap analogues for structural and biochemical studies.

Herein we describe the first simple and short method for specific labeling of mono- and trimethylated dinucleotide mRNA cap analogues with (13)C and (14)C isotopes. The labels were introduced within the cap structures either at the N7 for monomethylguanosine cap or N7 and N2 position for trimethylguanosine cap. The compounds designed for structural and biochemical studies will be useful tools for better understanding the role of the mRNA cap structures in pre-mRNA splicing, nucleocytoplasmic transport, translation initiation and mRNA degradation.