Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo.

Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-capped RNA species is superior for protein expression and for efficient priming and expansion of naïve antigen-specific T cells in mice. Our findings establish 5' mRNA cap analogs as yet another module for tuning immunopharmacological properties of recombinant antigen-encoding RNA for vaccination purposes.

A cap binding protein that may mediate nuclear export of RNA polymerase II-transcribed RNAs.

It has previously been shown that efficient export of U1 snRNA or of microinjected, in vitro synthesized, RNA transcripts from the nucleus of Xenopus oocytes is facilitated by their monomethyl guanosine cap structures. Nuclear exit of these transcripts could be competitively inhibited by microinjection of an excess of a cap analog, the dinucleotide m7GpppG (Hamm, J., and I. W. Mattaj. 1990. Cell. 63:109-118). We have now analyzed the ability of several other related cap analogs to inhibit the export of U1 snRNA from the nucleus. The results define the recognition specificity of a factor(s) involved in RNA transport, and indicate that the cap binding activity (CBA) involved in RNA export is different from cap binding proteins (CBPs) involved in the initiation of translation. A CBP, whose specificity for different analogs correlates with the ability of the analogs to inhibit U1 snRNA export, is identified in nuclear extracts prepared from HeLa cells. We propose that this protein may have a role in the export of capped RNAs from the nucleus.

Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport.

The requirements for nuclear targeting of a number of U snRNAs have been studied by analyzing the behavior of in vitro-generated transcripts after microinjection into the cytoplasm of Xenopus oocytes. Like the previously studied U1 snRNA, U2 snRNA is excluded from the nucleus when it does not have the 2,2,7mGpppN cap structure typical of the RNA polymerase II (pol II)-transcribed U snRNAs. Surprisingly, two other pol II-transcribed U snRNAs, U4 and U5, have a much less stringent requirement for the trimethyl cap structure. The gamma-monomethyl triphosphate cap structure of the RNA polymerase III-transcribed U6 snRNA, on the other hand, is shown not to play a role in nuclear targeting. Wheat germ agglutinin, which is known to prevent the import of many proteins into the nucleus, inhibits nuclear uptake of U6, but not of U1 or U5 snRNAs. Conversely, a 2,2,7mGpppG dinucleotide analogue of the trimethyl cap structure inhibits transport of the pol II U snRNAs, but does not detectably affect the transport of either U6 snRNA or a karyophilic protein. From these results it can be deduced that U6 enters the nucleus by a pathway similar or identical to that used by karyophilic proteins. The composite nuclear localization signals of the trimethyl cap-containing U snRNPs, however, do not function in the same way as previously defined nuclear targeting signals.

Kinetics of C. elegans DcpS cap hydrolysis studied by fluorescence spectroscopy.

DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine K(m) and V(max)values. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m(7)Gp(CH(2))ppG and m(7)Gpp(CH(2))pG) and less potently by ARCA (m(7,3' O)GpppG).

Correlations of conformational parameters and equilibrium conformational states in a variety of beta-D-arabinonucleosides and their analogues.

H nuclear magnetic resonance spectroscopy has been applied to a study of the conformations of a variety of purine and pyrimidine beta-D-arabinofuranosyl nucleosides. The experimental results, together with data collected from the literature, demonstrated the existence of reasonably good correlations between the coupling constants made it possible to define more accurately, than hitherto possible, the conformational states between which equilibria exist in solution. The equilibrium for the arabinonucleosides differs from that previously established for ribonucleosides; in particular, structural modifications and solvent effects may appreciably modify the conformational states between which equilibria exist. Preliminary measurements on some arabinosides in the syn conformation about the glycosidic bond indicated that these do not conform to the foregoing correlations, and will require separate study. A correlation has also been established between the conformation of the arabinose ring and that of the exocyclic 5'-CH2OH group. For both purine and pyrimidine arabinonucleosides, the conformational state 3E of the arabinose ring coexists to some extent with a gauche-gauche conformation of the exocyclic 5'-CH2OH, as in the case of pyrimidine (but not purine) ribonucleosides. Application of the foregoing to some biological problems is described.

Conformation in aqueous medium of the neutral, protonated and anionic forms of 9-beta-D-arabinofuranosyladenine.

Proton magnetic resonance spectroscopy was employed to study the solution conformations of the neutral, protonated and dissociated forms of the therapeutically active 9-beta-D-arabinofuranosyladenine (araA). In particular, in strongly basic medium, increasing alkalinity led to pronounced changes in chemical shifts and coupling constants of some pentose protons, due to ionization of the pentose hydroxyls, especially the 2'-OH. The neutral form of araA may be characterized as approx. 25% C(2')endo and approx. 60% gauche-gauche, hence somewhat different from that of the therapeutically active 1-beta-D-arabinofuranosylcytosine (araC). By contrast, the conformations of the anionic forms of both of these are identical, predominantly (greater than 80%) C(2')endo and gauche-gauche. With the aid of the 3'-O-methyl derivatives of araA and araC, where only the 2'-OH ionizes, and the accompanying conformational changes are similar, it follows that the conformation C(2')endo and gauche-gauche for all the foregoing is constrained to this form via a strong intramolecular hydrogen bond, viz. O(5')H...O(2')(-). The influence of the foregoing hydrogen bond on the chemical shifts of the adenine H(8) in the araA anion points to the existence of the latter in the form anti. A similar effect of the doubly ionized phosphate group on H(8) in 5'-araAMP shows the nucleotide to also prefer the form anti, as previously demonstrated for 5'-AMP. The conformations of the sugar rings of the neutral forms of araA and adenosine in aqueous medium differ appreciably, whereas in the solid state they are very similar. PMR spectroscopy is shown to be an effective method for following sugar hydroxyl dissociation. The extent of ionization of a given hydroxyl is provided by the resulting chemical shifts of neighbouring (geminal and vicinal) protons. When ionization is accompanied by a change in conformation, the process may be followed also by changes in proton-proton vicinal coupling constants.

1H-NMR studies on association of mRNA cap-analogues with tryptophan-containing peptides.

1H-NMR spectroscopy was applied to a study of the mode of interaction, in aqueous medium in the pH range 5.2-8.5 and at low and high temperatures, between several mono- and dinucleotide analogues of the mRNA cap m7GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the suspected binding sites in the mRNA cap-binding protein (CBP). A program, GEOSHIFT, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompanying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m7G cap and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 angstroms apart. The structures of the stacked complexes were determined. In particular, stacking between m(2,2,7)3G (which has no free amino group for hydrogen bonding) and the indole ring is weaker and quite different from that between m7G and m(2,7)2G and indole. With the dinucleotide cap-analogues, only the m7G component stacks with the indole ring, without disruption of intramolecular stacking. In contrast to numerous earlier reports, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has been shown that the positively charged (cationic) form of m7G stacks much more efficiently with the indole ring than the zwitterionic form resulting from dissociation of the guanine ring N1H (pKa approximately 7.3).

Fluorescence and NMR studies of intramolecular stacking of mRNA cap-analogues.

Intramolecular stacking of a series of new synthesized dinucleotide mRNA cap analogues has been investigated in aqueous buffers by means of fluorescence and 1H-NMR at various pH and temperatures, and compared with that for 7-methylguanosine(5')ppp(5')guanosine (m7GpppG), as well as its hypermethylated derivative m(3)2,2,7GpppG. Thermodynamic parameters for intramolecular self-association stabilized by stacking were established by temperature-dependent fluorescence quenching, taking into account collisional deactivation of the excited states. Relative orientations of the stacked bases in the cap analogues were determined with the aid of a program GEOSHIFT (Stolarski et al., Biochim. Biophys. Acta (1996) 1293, 97), based on ring-current anisotropy. 1D-soft-TOCSY experiments were applied to extract the exact values of vicinal coupling constants, and hence to resolve solution conformation of the cap molecules. Stacking interaction has been discussed in detail in terms of the cap structural features, e.g., types of bases and length of the 5',5'-phosphate bridges, and regarding the interactions stabilizing intramolecular stacking.

Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.

The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG. The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM). Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.

Kinetics of deamination of cytosine nucleosides with etherified sugar hydroxyls.

The kinetics of deamination of derivatives of the therapeutically important 1-beta-D-arabinofuranosylcytosine with etherified (methylated) sugar hydroxyls has provided additional direct evidence for involvement of the 2'-hydroxyl in intramolecular catalysed deamination. In the case of 1-beta-D-lyxofuranosylcytosine, the kinetics of deamination of its 2'-O-methyl and 3'-O-methyl derivatives pointed to similar involvement of the "up" 2'-OH in intramolecular catalysed deamination. Participation by the 3'-OH, which is also in the "up" position, was excluded. A qualitative correlation was shown to exist between the electron density distributions on C(4) and C(6) of the cytosine rings in cytosine, 1-methylcytosine and cytidine, and their relative susceptibilities to deamination.

Preparation of O'-METHYL derivatives of 9-beta-D-xylofuranosyladenine.

Treatment of the therapeutically important 9-beta-D-xylofuranosyladine in strongly alkaline medium with dimethyl sulphate led principally to etherification of sugar hydroxyls and, to a minor extent, to formation of products with a methylated exocyclic amino group. The various O'-methyl derivatives of xylofuranosyladenine were fractionated on a strongly basic ion exchange column, and isolated in pure form. Also isolated was 9-beta-D-xylofuranosyl-N6-methyladenine and its 2'-O-methyl derivative. The products were identified from their 1H NMR spectra, for which extensive data are tabulated. The susceptibilities of the various derivatives to calf intestinal adenosine deaminase were examined in relation to those of other adenine nucleosides; in particular, 5'-O-methylation led to total loss of substrate properties for the riboside, arabinoside and xyloside of adenine.

Base stacking of simple mRNA cap analogues. Association of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole and purine derivatives in aqueous solution.

Equilibrium constants for the association of different ionic forms of 7,9-dimethylguanine, 7-methylguanosine and 7-methylguanosine 5'-monophosphate with indole, caffeine and various methylated adenines have been determined by distributing the latter compounds between an organic solvent and aqueous solutions of the 7-methylguanine derivatives. The data are compared to those obtained for the association of unsubstituted purine with the same cosolutes. The stacking affinity of both cationic and zwitterionic forms of the 7-methylguanine ring correlates with the ring polarizability rather than the polarizing power of the cosolute. The cationic species stacks usually more efficiently. The chemical nature of the N9-substituent has only a moderate influence on the base-stacking properties.

Association of nucleosides and their 5'-monophosphates with a tryptophan containing tripeptide, Trp-Leu-Glu: the source of an overestimation by fluorescence spectroscopy.

Association of 7-methylguanosine 5'-monophosphate with a tryptophan containing tripeptide, Trp-Leu-Glu, has been studied by fluorescence titration using two different geometries of detection, viz. right angle and front surface geometry. The applicability of these two techniques to determine the stability constant of the nucleotide-peptide adduct is discussed. Evidence is presented that fluorescence titration based on right angle detection may lead to considerable overestimation of the strength of interaction.

A fluorescence spectroscopic study on the binding of mRNA 5'-cap-analogs to human translation initiation factor eIF4E: a critical evaluation of the sources of error.

Equilibrium constants for the association of human protein translation initiation factor eIF4E with two mRNA 5'-cap analogs, namely 7-methylguanosine 5'-triphosphate and P1-(7-methylguanosine-5') P3-(guanosine-5') triphosphate, and with guanosine 5'-monophosphate have been redetermined by the fluorescence quenching method taking the inner filter effect of the cap-analog into account. It has been shown that neglecting the latter correction may lead to either underestimation or overestimation of the association constant obtained by applying the Eadie-Hofstee plot: the reasonably firm binding of 7-methylated cap-analogs becomes underestimated, while the weak binding of non-methylated nucleotides becomes overestimated.

Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues.

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence delta Fmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.

Assignment of reovirus mRNA ribosome binding sites to virion genome segments by nucleotide sequence analyses.

All ten reovirus genome RNA segments were radiolabeled at their 3'-termini by incubation with RNA ligase and 32pCp. The extent of radiolabeling was similar for each of the double-stranded RNAs in the genome segment mixture. Radioactivity was equally distributed between the separated plus and minus strands indicating that the 5'-cap in plus strands did not block 3'-end-labeling of minus strands. The 3'-termini of the four S and three M segments included the common sequences: ...U-A-G-C in minus strands and ...U-C-A-U-C in plus strands. By comparing the minus strand 3'-sequences with 5'-sequences of reovirus mRNAs, small-size genome segments S2, S3 and S4 were correlated with the previously sequenced initiation fragments s46, s45 and s54 derived from small class mRNAs. Medium-size genome segments M1, M2 and M3 similarly were correlated with fragments m30, m52 and m44, respectively. The N-terminal amino acid sequences deduced from the mRNA nucleotide sequences can now be assigned to the nascent chains of particular reovirus proteins.