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.

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.

Towards superior mRNA caps accessible by click chemistry: synthesis and translational properties of triazole-bearing oligonucleotide cap analogs.

Messenger RNA (mRNA)-based gene delivery is a powerful strategy for the development of vaccines and therapeutics. Consequently, approaches that enable efficient synthesis of mRNAs with high purity and biological activity are in demand. Chemically modified 7-methylguanosine (m7G) 5' caps can augment the translational properties of mRNA; however, efficient synthesis of structurally complex caps, especially on a large scale, is challenging. Previously, we proposed a new strategy to assemble dinucleotide mRNA caps by replacing the traditional pyrophosphate bond formation by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Here, we used CuAAC to synthesize 12 novel triazole-containing tri- and tetranucleotide cap analogs with the aim of exploring the chemical space around the first transcribed nucleotide in mRNA and overcoming some of the limitations previously reported for the triazole-containing dinucleotide analogs. We evaluated the efficiency of incorporation into RNA for these analogs and their influence on the translational properties of in vitro transcribed (IVT) mRNAs in rabbit reticulocyte lysate and JAWS II cultured cells. The incorporation of the triazole moiety within the 5',5'-oligophosphate of trinucleotide cap produced compounds that were well incorporated into RNA by T7 polymerase while replacing the 5',3'-phosphodiester bond with triazole impaired incorporation and translation efficiency, despite a neutral effect on the interaction with the translation initiation factor eIF4E. One of the compounds (m7Gppp-tr-C2H4pAmpG), had translational activity and other biochemical properties comparable to natural cap 1 structure, thus being a promising mRNA capping reagent for potential in cellulo and in vivo applications in the field of mRNA-based therapeutics.

Fluorescence Anisotropy Assay with Guanine Nucleotides Provides Access to Functional Analysis of Gαi1 Proteins.

Gα proteins as part of heterotrimeric G proteins are molecular switches essential for G protein-coupled receptor- mediated intracellular signaling. The role of the Gα subunits has been examined for decades with various guanine nucleotides to elucidate the activation mechanism and Gα protein-dependent signal transduction. Several approaches describe fluorescent ligands mimicking the GTP function, yet lack the efficient estimation of the proteins' GTP binding activity and the fraction of active protein. Herein, we report the development of a reliable fluorescence anisotropy-based method to determine the affinity of ligands at the GTP-binding site and to quantify the fraction of active Gαi1 protein. An advanced bacterial expression protocol was applied to produce active human Gαi1 protein, whose GTP binding capability was determined with novel fluorescently labeled guanine nucleotides acting as high-affinity Gαi1 binders compared to the commonly used BODIPY FL GTPγS. This study thus contributes a new method for future investigations of the characterization of Gαi and other Gα protein subunits, exploring their corresponding signal transduction systems and potential for biomedical applications.

Fluorescence-Based Activity Screening Assay Reveals Small Molecule Inhibitors of Vaccinia Virus mRNA Decapping Enzyme D9.

Vaccinia virus (VACV) represents a family of poxviruses, which possess their own decapping machinery as a part of their strategy to eliminate host mRNAs and evade the innate immune response. D9 is one of the two encoded VACV decapping enzymes that is responsible for cap removal from the 5' end of both host mRNA transcripts and viral double-stranded RNAs. Little is known about the structural requirements for D9 inhibition by small molecules. Here, we identified a minimal D9 substrate and used it to develop a real-time fluorescence assay for inhibitor discovery and characterization. We screened a panel of nucleotide-derived substrate analogues and pharmacologically active candidates to identify several compounds with nano- and low micromolar IC50 values. m7GpppCH2p was the most potent nucleotide inhibitor (IC50 ∼ 0.08 µM), and seliciclib and CP-100356 were the most potent drug-like compounds (IC50 0.57 and 2.7 µM, respectively). The hits identified through screening inhibited D9-catalyzed decapping of 26 nt RNA substrates but were not active toward VACV D10 or human decapping enzyme, Dcp1/2. The inhibition mode for one of the compounds (CP-100356) was elucidated based on the X-ray cocrystal structure, opening the possibility for structure-based design of novel D9 inhibitors and binding probes.

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.

Novel N7-Arylmethyl Substituted Dinucleotide mRNA 5' cap Analogs: Synthesis and Evaluation as Modulators of Translation.

Dinucleotide analogs of the messenger RNA cap (m7GpppN) are useful research tools and have potential applications as translational inhibitors or reagents for modification of in vitro transcribed mRNAs. It has been previously reported that replacing the methyl group at the N7-position with benzyl (Bn) produces a dinucleotide cap with superior properties. Here, we followed up on this finding by synthesizing 17 novel Bn7GpppG analogs and determining their structure-activity relationship regarding translation and translational inhibition. The compounds were prepared in two steps, including selective N7-alkylation of guanosine 5'-monophosphate by arylmethyl bromide followed by coupling with imidazole-activated GDP, with total yields varying from 22% to 62%. The compounds were then evaluated by determining their affinity for eukaryotic translation initiation factor 4E (eIF4E), testing their susceptibility to decapping pyrophosphatase, DcpS-which is most likely the major cellular enzyme targeting this type of compound-and determining their translation inhibitory properties in vitro. We also synthesized mRNAs capped with the evaluated compounds and tested their translational properties in A549 cells. Our studies identified N7-(4-halogenbenzyl) substituents as promising modifications in the contexts of either mRNA translation or translational inhibition. Finally, to gain more insight into the consequences at the molecular level of N7-benzylation of the mRNA cap, we determined the crystal structures of three compounds with eIF4E.