Maciej Lukaszewicz

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-, β- 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 m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2, respectively, than for in vitro transcribed mRNA capped with m27,3′-OGpppG. Higher expression of cancer antigens would make mRNAs containing m27,2′-OGppBH3pG D1 and m27,2′-OGppBH3pG D2 favorable for anticancer immunization.

Phosphoroselenoate Dinucleotides for Modification of mRNA 5′ End

er, this structure has a disproportionately large effect on mRNA function since it represents an anchoring point for a variety of proteins involved in mRNA-related physiological processes, as well as protecting mRNA from 5’!3’ exonucleolytic degradation. 2] Hence, synthetic cap analogues are invaluable tools for studying mRNA translation and turnover. Moreover, dinucleotide cap analogues can be easily incorporated into the mRNA 5’ end during in vitro transcription; this makes them useful for biotechnological purposes (protein production) and, potentially, for medical applications related to mRNA-mediated gene therapy. 5] Here, we describe the synthesis and properties of two P diastereomers of a cap analogue modified with phosphoroselenoate moiety at the b position of the triphosphate bridge (m2 GppSepG, D1 and D2; Figure 1 B). The compounds additionally contain a 2’-O-methyl group in the mGuo moiety to ensure their incorporation into mRNA during in vitro transcription exclusively in the correct orientation (antireverse cap analogues, ARCAs), and hence they can be referred to as b-SeARCAs. The idea of synthesising b-Se-ARCAs arose from our previous finding that mRNAs with a cap structure modified with a phosphorothioate moiety at the b position of the triphosphate bridge, m2 GppSpG (D2), were resistant to Dcp2 pyrophosphatase, which is a decapping enzyme essential for triggering 5’!3’ mRNA decay. Hence, such mRNAs were more stable as well as fiveand twofold more efficiently translated in vivo (mammalian cells) than mRNAs capped with mGpppG and m2 GpppG, respectively. Phosphoroselenoates (PSe) are closely related to phosphorothioates (PS) in terms of chemical and biochemical properties, thus, we expected that also PSe cap analogues would share these interesting characteristics. One important superiority of nucleotide phosphoroselenoate analogues over phosphorothioate analogues is their usefulness in nucleic acid X-ray crystallography for multiwavelength anomalous diffraction (MAD) phasing technique. This fact combined with the expected resistance of m2 GppSepG to Dcp2, led us to the conclusion that such compounds (or mRNA fragments capped with such compounds) could serve as perfect ligands for co-crystallisation with Dcp2, as well as other proteins involved in mRNA 5’-end recognition. To the best of our knowledge, b-Se-ARCAs also represent the first examples of nucleotide analogues that are modified with a PSe moiety not at the a position with respect to nucleoside. The synthesis of m2 GppSepG is depicted in Scheme 1. The selenophosphate (1) was prepared by using a modification of previously reported procedures. 12] Tris(trimethylsilyl)phosphite was treated with a suspension of selenium in pyridine to give tris(trimethylsilyl)phosphoroselenoate, which was then desilylated by methanol in the presence of triethylamine to give selenophosphate triethylammonium (TEA) salt. In agreement with previous reports, 13] we found selenophosphate to be extremely labile and prone to oxidation. However, despite its instability, a freshly prepared selenophosphate TEA salt was efficiently coupled with nucleoside 5’-monophosphate P-imidazolide derivatives to produce the corresponding nucleoside 5’O-(2-selenodiphosphates) under similar conditions to those we developed previously for the synthesis of the analogous PS nucleotides. Figure 1. A) Structure of eukaryotic mRNA 5’ end (cap). B) Structure of the phosphoroselenoate mRNA cap analogue synthesised in this work.

Analysis of decapping scavenger cap complex using modified cap analogs reveals molecular determinants for efficient cap binding

Decapping scavenger (DcpS) assists in precluding inhibition of cap‐binding proteins by hydrolyzing cap species remaining after mRNA 3′→5′ degradation. Its significance was reported in splicing, translation initiation and microRNA turnover. Here we examine the structure and binding mode of DcpS from Caenorhabditis elegans (CeDcpS) using a large collection of chemically modified methylenebis(phosphonate), imidodiphosphate and phosphorothioate cap analogs. We determine that CeDcpS is a homodimer and propose high accuracy structural models of apo‐ and m7GpppG‐bound forms. The analysis of CeDcpS regioselectivity uncovers that the only site of hydrolysis is located between the β and γ phosphates. Structure–affinity relationship studies of cap analogs for CeDcpS reveal molecular determinants for efficient cap binding: a strong dependence on the type of substituents in the phosphate chain, and reduced binding affinity for either methylated hydroxyl groups of m7Guo or an extended triphosphate chain. Docking analysis of cap analogs in the CeDcpS active site explains how both phosphate chain mobility and the orientation in the cap‐binding pocket depend on the number of phosphate groups, the substituent type and the presence of the second nucleoside. Finally, the comparison of CeDcpS with its well known human homolog provides general insights into DcpS–cap interactions.

Clickable trimethylguanosine cap analogs modified within the triphosphate bridge: synthesis, conjugation to RNA and susceptibility to degradation

The trimethylguanosine (m3G) cap present at the 5′ end of small nuclear RNAs (snRNAs) has been proposed as an effective nuclear localization signal (NLS) for nucleus-targeting therapeutics such as antisense oligonucleotides. To provide novel tools for studies on m3G-mediated transport and m3G degradation, we synthesized a series of novel m3G cap analogs that combine modifications potentially affecting its activity as an NLS and stability in vivo with a modification enabling simple conjugation to biomolecules. The synthesized dinucleotide m3G analogs carry a single phosphate-modification (phosphorothioate, methylenebisphosphonate or imidodiphosphate) at the selected position of the triphosphate bridge in order to increase their resistance to enzymatic cleavage and a (2-azidoethyl)-carbamoylmethyl group at the 2′-position of adenosine as a second nucleotide to enable conjugation to alkyne-containing biomolecules by copper catalyzed azide–alkyne cycloaddition (CuAAC). The susceptibility of m3G cap analogs to non-specific and specific degradation was studied in fetal bovine serum and in an in vitro decapping assay with hNUDT16 enzyme, respectively. The susceptibility of m3G cap analogs to hNUDT16 mediated decapping was also determined after their CuAAC-mediated conjugation to a model oligonucleotide bearing a 5′-alkyne group. Depending on the type and the position of introduced modifications, they modulate the susceptibility to specific and non-specific degradation of conjugated molecules to various extent, with O to NH substitution at the α/β position providing the greatest m3G stability against hNUDT16.

Synthesis and evaluation of fluorescent cap analogues for mRNA labelling

We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-β methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins.

How to find the optimal partner--studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine.

Snurportin 1 is an adaptor protein that mediates the active nuclear import of uridine-rich small nuclear RNAs (U snRNA) by the importin-β receptor pathway. Its cellular activity influences the overall transport yield of small ribonucleoprotein complexes containing N(2),N(2),7-trimethylguanosine (TMG) capped U snRNA. So far little is still known about structural requirements related to molecular recognition of the trimethylguanosine moiety by snurportin in solution. Since these interactions are of a great biomedical importance, we synthesized a series of new 7-methylguanosine cap analogues with extended substituents at the exocyclic 2-amino group to gain a deeper insight into how the TMG-cap is adapted into the snurportin cap-binding pocket. Prepared chemical tools were applied in binding assays using emission spectroscopy. Surprisingly, our results revealed strict selectivity of snurportin towards the TMG-cap structure that relied mainly on its structural stiffness and compactness.

Hydrolytic activity of human Nudt16 enzyme on dinucleotide cap analogs and short capped oligonucleotides

Human Nudt16 (hNudt16) is a member of the Nudix family of hydrolases, comprising enzymes catabolizing various substrates including canonical (d)NTPs, oxidized (d)NTPs, nonnucleoside polyphosphates, and capped mRNAs. Decapping activity of the Xenopus laevis (X29) Nudt16 homolog was observed in the nucleolus, with a high specificity toward U8 snoRNA. Subsequent studies have reported cytoplasmic localization of mammalian Nudt16 with cap hydrolysis activity initiating RNA turnover, similar to Dcp2. The present study focuses on hNudt16 and its hydrolytic activity toward dinucleotide cap analogs and short capped oligonucleotides. We performed a screening assay for potential dinucleotide and oligonucleotide substrates for hNudt16. Our data indicate that dinucleotide cap analogs and capped oligonucleotides containing guanine base in the first transcribed nucleotide are more susceptible to enzymatic digestion by hNudt16 than their counterparts containing adenine. Furthermore, unmethylated dinucleotides (GpppG and ApppG) and respective oligonucleotides (GpppG-16nt and GpppA-16nt) were hydrolyzed by hNudt16 with greater efficiency than were m7GpppG and m7GpppG-16nt. In conclusion, we found that hNudt16 hydrolysis of dinucleotide cap analogs and short capped oligonucleotides displayed a broader spectrum specificity than is currently known.

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

Abstract 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.

Synthesis and biochemical studies of tetraphosphate 5′ mRNA cap analogs bearing bisphosphonate modification

Dinucleotide cap analogs containing tetraphosphate bridge modified by replacing one of the bridging oxygen atoms with methylene group were synthesized. The analogs have been examined for their stability towards enzymatic hydrolysis by human DcpS and binding affinity for eIF4E. Their inhibitory properties in rabbit reticulocyte lysate (RRL) translational system were studied.