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.

An intramolecular disulphide bond in human 4E-T affects its binding to eIF4E1a protein.

The cap at the 5'terminus of mRNA is a key determinant of gene expression in eukaryotic cells, which among others is required for cap dependent translation and protects mRNA from degradation. These properties of cap are mediated by several proteins. One of them is 4E-Transporter (4E-T), which plays an important role in translational repression, mRNA decay and P-bodies formation. 4E-T is also one of several proteins that interact with eukaryotic initiation factor 4E (eIF4E), a cap binding protein which is a key component of the translation initiation machinery. The molecular mechanisms underlying the interactions of these two proteins are crucial for mRNA processing. Studying the interactions between human eIF4E1a and the N-terminal fragment of 4E-T that possesses unstructured 4E-binding motifs under non-reducing conditions, we observed that 4E-T preferentially forms an intramolecular disulphide bond. This "disulphide loop" reduces affinity of 4E-T for eIF4E1a by about 300-fold. Considering that only human 4E-T possesses two cysteines located between the 4E binding motifs, we proposed that the disulphide bond may act as a switch to regulate interactions between the two proteins.

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.

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.

Amino-Functionalized 5' Cap Analogs as Tools for Site-Specific Sequence-Independent Labeling of mRNA.

mRNA is a template for protein biosynthesis, and consequently mRNA transport, translation, and turnover are key elements in the overall regulation of gene expression. Along with growing interest in the mechanisms regulating mRNA decay and localization, there is an increasing need for tools enabling convenient fluorescent labeling or affinity tagging of mRNA. We report new mRNA 5' cap analog-based tools that enable site-specific labeling of RNA within the cap using N-hydroxysuccinimide (NHS) chemistry. We explored two complementary methods: a co-transcriptional labeling method, in which the label is first attached to a cap analog and then incorporated into RNA by in vitro transcription, and a post-transcriptional labeling method, in which an amino-functionalized cap analog is incorporated into RNA followed by chemical labeling of the resulting transcript. After testing the biochemical properties of RNAs carrying the novel modified cap structures, we demonstrated the utility of fluorescently labeled RNAs in decapping assays, RNA decay assays, and RNA visualization in cells. Finally, we also demonstrated that mRNAs labeled by the reported method are translationally active. We envisage that the novel analogs will provide an alternative to radiolabeling of mRNA caps for in vitro studies and open possibilities for new applications related to the study of mRNA fates in vivo.

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.

Diverse cap-binding properties of Drosophila eIF4E isoforms.

The majority of eukaryotic mRNAs are translated in a cap-dependent manner, which requires recognition of the mRNA 5' cap by eIF4E protein. Multiple eIF4E family members have been identified in most eukaryotic organisms. Drosophila melanogaster (Dm) has eight eIF4E related proteins; seven of them belong to Class I and one to Class II. Their biological roles with the exception of Dm eIF4E-1, Dm eIF4E-3 and Dm 4EHP, remain unknown. Here, we compare the molecular basis of Dm eIF4E's interactions with cap and eIF4G peptide by using homology modelling and fluorescence binding assays with various cap analogues. We found that despite the presence of conserved key residues responsible for cap recognition, the differences in binding different cap analogues among Class I Dm eIF4E isoforms are up to 14-fold. The highest affinity for cap analogues was observed for Dm eIF4E-3. We suggest that Dm eIF4E-3 and Dm eIF4E-5 bind the second nucleoside of the cap in an unusual manner via stacking interactions with a histidine or a phenylalanine residue, respectively. Moreover, the analysis of ternary complexes of eIF4G peptide-eIF4E-cap analogue showed cooperativity between eIF4G and cap binding only for Dm eIF4E-4, which exhibits the lowest affinity for cap analogues among all Dm eIF4Es.

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.

Five eIF4E isoforms from Arabidopsis thaliana are characterized by distinct features of cap analogs binding.

The assembly of the ribosome on majority of eukaryotic mRNAs is initiated by the recruitment of eIF4E protein to the mRNA 5' end cap structure. Flowering plants use two eIF4E isoforms, named eIF4E and eIF(iso)4E, as canonical translation initiation factors and possess a homolog of mammalian 4EHP (or eIF4E-2) termed nCBP. Plants from Brassicaceae family additionally conserve a close paralog of eIF4E which in Arabidopsis thaliana has two copies named eIF4E1b and eIF4E1c. In order to assess the efficiency of plant non-canonical (eIF4E1b/1c and nCBP) and canonical (eIF4E and eIF(iso)4E) eIF4E proteins to bind mRNAs we utilized fluorescence titrations to determine accurate binding affinities of five A.thaliana eIF4E isoforms for a series of cap analogs. We found that eIF4E binds cap analogs from 4-fold to 10-fold stronger than eIF(iso)4E, while binding affinities of nCBP and eIF(iso)4E are comparable. Furthermore, eIF4E1c interacts similarly strongly with the cap as eIF4E, but eIF4E1b binds cap analogs ca. 2-fold weaker than eIF4E1c, regardless of the 95% sequence identity between these two proteins. The use of differentially chemically modified cap analogs in binding studies and a detailed analysis of the obtained homology models gave us insight into the molecular characteristic of varying cap-binding abilities of Arabidopsis eIF4E isoforms.

Distinct features of cap binding by eIF4E1b proteins.

eIF4E1b, closely related to the canonical translation initiation factor 4E (eIF4E1a), cap-binding protein is highly expressed in mouse, Xenopus and zebrafish oocytes. We have previously characterized eIF4E1b as a component of the CPEB mRNP translation repressor complex along with the eIF4E-binding protein 4E-Transporter, the Xp54/DDX6 RNA helicase and additional RNA-binding proteins. eIF4E1b exhibited only very weak interactions with m(7)GTP-Sepharose and, rather than binding eIF4G, interacted with 4E-T. Here we undertook a detailed examination of both Xenopus and human eIF4E1b interactions with cap analogues using fluorescence titration and homology modeling. The predicted structure of eIF4E1b maintains the α+ß fold characteristic of eIF4E proteins and its cap-binding pocket is similarly arranged by critical amino acids: Trp56, Trp102, Glu103, Trp166, Arg112, Arg157 and Lys162 and residues of the C-terminal loop. However, we demonstrate that eIF4E1b is 3-fold less well able to bind the cap than eIF4E1a, both proteins being highly stimulated by methylation at N(7) of guanine. Moreover, eIF4E1b proteins are distinguishable from eIF4E1a by a set of conserved amino acid substitutions, several of which are located near to cap-binding residues. Indeed, eIF4E1b possesses several distinct features, namely, enhancement of cap binding by a benzyl group at N(7) position of guanine, a reduced response to increasing length of the phosphate chain and increased binding to a cap separated by a linker from Sepharose, suggesting differences in the arrangement of the protein's core. In agreement, mutagenesis of the amino acids differentiating eIF4E1b from eIF4E1a reduces cap binding by eIF4E1a 2-fold, demonstrating their role in modulating cap binding.

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.

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei.

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

Trypanosoma brucei translation initiation factor homolog EIF4E6 forms a tripartite cytosolic complex with EIF4G5 and a capping enzyme homolog.

Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene-one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5' cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.

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.

Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5' cap.

Initiation is the rate-limiting step during mRNA 5' cap-dependent translation, and thus a target of a strict control in the eukaryotic cell. It is shown here by analytical ultracentrifugation and fluorescence spectroscopy that the affinity of the human translation inhibitor, eIF4E-binding protein (4E-BP1), to the translation initiation factor 4E is significantly higher when eIF4E is bound to the cap. The 4E-BP1 binding stabilizes the active eIF4E conformation and, on the other hand, can facilitate dissociation of eIF4E from the cap. These findings reveal the particular allosteric effects forming a thermodynamic cycle for the cooperative regulation of the translation initiation inhibition.

The synthesis of isopropylidene mRNA cap analogs modified with phosphorothioate moiety and their evaluation as promoters of mRNA translation.

Synthetic mRNA cap analogs are valuable tools in the preparation of modified mRNA transcripts with improved translational activity and increased cellular stability, and have recently attracted more attention because of their great potential in therapeutic applications. We have synthesized and tested isopropylidene dinucleotide cap analogs bearing a phosphorothioate group at the ß position of the 5',5'-triphosphate bridge (two diastereomers of 2',3'-iPr-m(7)GppSpG), as synthetically simpler alternatives to previously obtained phosphorothioate cap analogs. To evaluate the utility of the new compounds in biological systems we determined their affinity to translation initiation factor 4E (eIF4E), and tested their translational properties in rabbit reticulocyte lysates (RRL) and in human immature dendritic cells (hiDCs). In order to explain the properties of isopropylidene analogs we performed (1)H NMR conformational analysis and correlated the absolute configuration at the ß-phosphorous atom with previously synthesized m(7)GppSpG.

Synthesis of biotin labelled cap analogue--incorporable into mRNA transcripts and promoting cap-dependent translation.

Analogues of the eukaryotic messenger RNA 5' end (m(7)G cap) are useful tools for studying mRNA fate and serve as reagents for in vitro preparation of 5' capped mRNAs. We designed a biotin-labeled dinucleotide cap analogue that can be incorporated into transcripts to produce 5'-capped and biotinylated mRNAs which retain their biological functionality and may be employed for biotin-(strept)avidin technologies.

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila.

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5' cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins.

Cap-binding proteins have been routinely isolated using m7GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m7GTP. Here, we report the synthesis of new affinity resins, m7GpCH2pp- and m7GpCH2ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m7GpppA-Sepharose, bind recombinant mouse eIF4E²8⁻²¹7 specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m7GpCH2pp- and m7GpCH2ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m7GpCH2ppA-Sepharose. Our data prove the applicability of these novel resins, especially m7GpCH2ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.