Synthesis and use of anti-reverse mRBA cap analogues

Abstract

The ability to synthesize capped RNA transcripts in vitro has been of considerable value in a variety of applications. However, one-third to one-half of the caps have, until now, been incorporated in the reverse orientation. Such reverse caps impair the translation of in vitro-synthesized mRNAs. Novel cap analogues, such as P1-3′-deoxy-7-methylguanosine-5′P3-guanosine-5′triphosphate and P1-3′-O,7-dimethylguanosine-5′P3-guanosine-5′triphosphate, have been designed that are incapable of being incorporated into RNA in the reverse orientation. Transcripts produced with SP6 polymerase using “anti-reverse” cap analogues were of the predicted length. Analysis of the transcripts indicated that reverse caps were not formed. The in vitro translational efficiency of transcripts with the novel “anti-reverse” cap analogues was significantly higher than that of transcripts formed with conventional caps.

Description

[0003] In eukaryotes, the 5′ end of most mRNA is blocked, or “capped.” In addition, there are some other forms of RNA that are also capped. The cap contains a 5′-5′ triphosphate linkage between two nucleotides, and also contains methyl groups. The capping of RNA promotes its normal function in cells.

[0004] The ability to synthesize capped RNA molecules in vitro is therefore useful, because it allows workers to prepare RNA molecules that behave properly as mRNA transcripts in a variety of in vitro applications. Such applications include both research applications and commercial production of certain polypeptides in an in vitro translation system, for example the production of polypeptides containing an “unnatural” amino acid at a specific site.

[0005] The method most frequently used to make capped RNAs in vitro is to transcribe a DNA template with either a bacterial RNA polymerase or a bacteriophage RNA polymerase in the presence of all four ribonucleoside triphosphates and a cap dinucleotide such as m7G(5′)ppp(5′)G. The polymerase initiates transcription with a nucleophilic attack by the 3′-OH of the Guo moiety in m7GpppG on the α-phosphate of the next templated nucleoside triphosphate, resulting in the initial product m7GpppGpN. The alternative, GTP-initiated product pppGpN is suppressed by setting the ratio of m7GpppG to GTP between 5 and 10 in the transcription reaction mixture.

[0006] Synthetic RNAs may be synthesized by cell-free transcription of DNA templates. See R. Contreras et al., “Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes,” Nucl. Acids Res., vol. 10, pp. 6353-6362 (1982); J. Yisraeli et al., “Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases, pp.42-50 in J. Dahlberg et al. (Eds.), Meth. Enzymol., vol.180., pp.42-50 (1989); and D. Melton et al, “Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter,” Nucl. Acids Res., vol.12, pp. 7035-7056 (1984).

[0007] Capped RNAs thus produced are active in in vitro splicing reactions. See M. Konarska et al., “Recognition of cap structure in splicing in vitro of mRNA precursors. Cell, vol. 38, pp. 731-736 (1984); and I. Edery et al., “Cap-dependent RNA splicing in a HeLa nuclear extract,” Proc. Natl. Acad. Sci. USA, vol. 82, pp. 7590-7594 (1985).

[0008] Capped mRNAs are translated more efficiently than are non-capped mRNAs. See S. Muthukrishnan et al., “5′-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation,” Nature, vol. 255, pp. 33-37 (1975); L. Chu et al., “Paradoxical observations on the 5′ terminus of ovalbumin messenger ribonucleic acid,” J. Biol. Chem., vol. 253, pp. 5228-5231 (1978); E. Darzynkiewicz et al., “β-Globin mRNAs capped with m7G, m22.7G or m32.2.7G differ in intrinsic translation efficiency,” Nucl. Acids Res., vol. 16, pp. 8953-8962 (1988); and E. Darzynkiewicz et al., “Inhibition of eukaryotic translation by nucleoside 5′-monophosphate analogues of mRNA 5′-cap: Changes in N7 substituent affect analogue activity,” Biochem., vol. 28, pp. 4771-4778 (1989).

[0009] 5′-Unmethylated mRNAs have been reported to be translationally less active than 5′-methylated mRNAs. See G. Both et al., “Methylation-dependent translation of viral messenger RNAs in vitro,” Proc. Natl. Acad. Sci. USA, vol. 72, pp. 1189-1193 (1975).

[0010] E. Darzynkiewicz et al., “Chemical synthesis and characterization of 7-methylguanosine cap analogues,” Biochem., vol. 24, pp.1701-1707 (1985) reported the synthesis of derivatives of 7-methylguanosine 5′-phosphate that were modified in the ribose moiety by 2′-O or 3′-O-methylation, or by conversion to the 2′-deoxy or arabinosyl form, and reported that these derivatives retained cap analogue activity.

[0011] F. Sanger et al., “DNA sequencing with chain-terminating inhibitors,” Proc. Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467 (1977) reported a method for determining DNA nucleotide sequences with 2′,3′-dideoxy and arabinonucleoside analogues of normal deoxynucleoside triphosphates, in which the analogs act as specific chain-terminating inhibitors of DNA polymerase.

[0012] M. Kadokura et al. 1997, “Efficient synthesis of γ-methyl-capped guanosine 5′-triphosphate as a 5′-terminal unique structure of U6 RNA via a new triphosphate bond formation involving activation of methyl phosphorimidazolidate using ZnCl2 as a catalyst in DMF under anhydrous conditions,” Tetrahedron Lett., vol. 38, pp. 8359-8362 (1997) reported the synthesis of CH3pppG from GDP and the imidazolide of methyl phosphate in DMF, obtaining a yield of 39% in the absence of ZnCl2, and a yield of 98% in the presence of ZnCl2.

[0013] A. Pasquinelli et al., “Reverse 5′ caps in RNAs made in vitro by phage RNA polymerases,” RNA, vol. 1, pp.957-967 (1995) reported that bacteriophage polymerases also use the 3′-OH of the 7-methylguanosine moiety of m7GpppG to initiate transcription, demonstrating that approximately one-third to one-half of RNA products made with this cap analogue actually contain the “reverse cap” Gpppm7GpN. Such reverse-capped RNA molecules behave abnormally. The same authors reported that when reverse-capped pre-U1 RNA transcripts were injected into Xenopus laevis nuclei, they were exported more slowly than natural transcripts. Similarly, cytoplasmic reverse-capped U1 RNAs in the cytoplasm were not properly imported into the nucleus. The presence of a cap on mRNA strongly stimulates translation of an mRNA transcript into protein. To the knowledge of the present inventors, there have been no previous reports directly addressing whether, and at what rate, reverse-capped mRNAs are translated into protein. However, based on what is known about recognition of the cap structure by elF4E, one would expect reverse-capped mRNAs to be translated no more efficiently than uncapped RNAs.

[0014] Z. Peng et al., “Synthesis and application of a chain-terminating dinucleotide mRNA cap analog,” Org. Lett., vol. 4, pp. 161-164 (2002; published on the Web, December 2001; and including the supporting information for this article as reprinted from the journal's web site) reported the synthesis of a chain-terminating mRNA cap dinucleotide, 3′-O-Me-m7G(5′)pppG, and its use in the in vitro transcription of homogeneously capped RNA. Computer modeling was said to indicate that RNA capped with the compound would be a substrate for cap-dependent translation.

[0015] Because existing synthetically capped RNAs contain about one-third to one-half reverse caps, the overall translational activity of such a RNA preparation is reduced considerably. Other functional properties of the mRNA may also be affected. There is a previously unfilled need for a way to prepare capped RNA molecules in vitro, in which all or essentially all the caps have the proper orientation.

[0016] We have discovered and synthesized cap analogues that will not be incorporated into an mRNA molecule in the reverse orientation. In experiments in which we synthesized and tested two prototype “anti-reverse” cap analogues (ARCAs), we found that both were exclusively incorporated into mRNA molecules in the correct orientation. Furthermore, both behaved like natural RNA caps in interactions with the translational machinery. The resulting mRNAs were considerably more active translationally than are traditional in vitro-prepared RNAs containing a mixture of caps in both the correct and the reversed orientations.

[0017] Transcription by bacteriophage RNA polymerases in the presence of m7GpppG is initiated with a nucleophilic attack by the 3′-OH of either the m7Guo moiety or of the Guo moiety on the electrophilic α-phosphate of the first templated nucleoside triphosphate. We eliminated one of these two 3′-OH groups, so that the nucleophilic attack would cause incorporation only in the correct orientation. We have made two prototype ARCAs. In the case of P1-3′-deoxy-7-methylguanosine-5′P3-guanosine-5′triphosphate (FIG. 1, Compound 9, henceforth abbreviated m73′dGpppG), we substituted an —H for the 3′-OH. In the case of P1-3′-O,7-dimethylguanosine-5′P3-guanosine-5′triphosphate (FIG. 1, compound 10, henceforth abbreviated m27,O3′GpppG), we instead substituted a —OCH3 for the 3′-OH.

[0018] We also developed new coupling strategies to synthesize the prototype ARCAs. To avoid preparing imidazole derivatives from 7-methylated substrates, the activation of which can be difficult, we developed a new coupling strategy involving guanosine 5′-phosphorimidazolide and the modified 7-methylated nucleoside diphosphate. We obtained high yields by conducting the coupling reaction in the presence of ZnCl2 instead of Mn2+, and by using anhydrous dimethylformamide (DMF) instead of water as a solvent. See FIG. 1, depicting schematically the synthesis of “anti-reverse” cap analogs. (In FIG. 1, “ImGMP” refers to guanosine 5′-imidazolide monophosphate.)

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 depicts the synthesis of “anti-reverse” cap analogs.

[0020] FIG. 2 depicts an analysis of in vitro-synthesized RNAs by enzymatic digestion and anion exchange HPLC.

[0021] FIG. 3 depicts the inhibition of translation by ARCAs compared with m7GpppG and GpppG.

[0022] FIG. 4 depicts the translational activity of ARCA-capped mRNAs compared to that of other RNAs.

MATERIALS AND METHODS

[0023] Synthesis of Mono- and Dinucleotides

EXAMPLE 1

[0024] 3′-Deoxyguanosine 5′-monophosphate (Compound 3). 3′-Deoxyguanosine (Compound 1, commercial product from Sigma, 50 mg, 0.19 mmol) was stirred overnight with trimethylphosphate (2 mL) and phosphorus oxychloride (53 μL, 0.57 mmol) at 6° C. The reaction was quenched by adding 20 mL of water and neutralizing with 1 M NaHCO3. DEAE-Sephadex chromatography using a linear gradient of 0-0.9 M TEAB afforded Compound 3 (yield: 45 mg, 43%).

EXAMPLE 2

[0025] 3′-O-Methylguanosine 5′-monophosphate (Compound 4) was obtained by a procedure analogous to that for Compound 3, but instead starting with 59 mg of 3′-O-methylguanosine (Compound 2), which was prepared by the method of J. Kusmierek et al., “A new route to 2′(3′)-O-alkyl purine nucleosides,” Nucl. Acids Res. Special Publ. No. 4, pp. s73-s77 (1978) (yield: 80 mg, 69%).

EXAMPLE 3

[0026] 3′-Deoxyguanosine 5′-diphosphate (Compound 5). Compound 3 (55 mg, TEA salt, 0.1 mmol), imidazole (34 mg, 0.5 mmol) and 2,2′-dithiodipyridine (Aldrich, 44 mg, 0.2 mmol) were mixed in anhydrous DMF (1.2 mL) and TEA (14 μL). Triphenylphosphine (52 mg, 0.2 mmol) was added, and the mixture was stirred for 5 h at room temperature. The mixture was placed in a centrifuge tube, and sodium perchlorate (49 mg, anhydrous) dissolved in acetone (6 mL) was added. After cooling for 2 h in a refrigerator, the mixture was centrifuged and the supernatant was discarded. The precipitate was ground with a new portion of acetone, cooled and centrifuged again. The process was repeated once more, and the precipitate was dried in a vacuum desiccator over P4O10. The imidazolide thus obtained was dissolved in 1.2 mL of DMF, and 200 mg of triethylammonium phosphate was added. (The latter was prepared from TEA and phosphoric acid followed by drying over P4O10 in a desiccator to obtain a semicrystalline mass.) Finally, 80 mg of ZnCl2 were added, and the reaction mixture was stirred at room temperature for 6.5 h, poured into a beaker containing a solution of 250 mg EDTA in 15 mL water, and neutralized with 1 M NaHCO3. Chromatographic isolation on a DEAE-Sephadex column using a linear gradient of 0-1 M TEAB gave Compound 5 (yield: 41 mg, 66%).

EXAMPLE 4

[0027] 3′-O-Methylguanosine 5′-diphosphate (Compound 6) was obtained by a procedure analogous to that for Compound 5, except starting from 58 mg of Compound 4 (yield: 32 mg, 49%).

EXAMPLE 5

[0028] 3′-Deoxy-7-methylguanosine 5′-diphosphate (Compound 7). Compound 5 (34 mg, 0.055 mmol) was mixed with 1 mL of dimethylsulfoxide, 1 mL of DMF, and 100 μL of methyl iodide at room temperature. After 3 h the reaction mixture was treated with 30 mL of cold water and extracted three times with 10-mL portions of diethyl ether. After neutralization with NaHCO3, chromatographic separation of the aqueous phase on DEAE-Sephadex, using a linear gradient of 0 to 0.8 M TEAB, gave Compound 7 (yield: 10 mg, 28%).

EXAMPLE 6

[0029] 3′-O, 7-Dimethylguanosine 5′-diphosphate (Compound 8) was obtained by a procedure analogous to that for Compound 7, except that the starting material was 66 mg of Compound 6 (yield: 64 mg, 95%).

EXAMPLE 7

[0030] P1-3′-Deoxy-7-methylguanosine-5′ P3-guanosine-5′triphosphate (Compound 9). GMP (purchased from Sigma, converted to the TEA salt, 29 mg, 0.05 mmol), imidazole (17 mg, 0.25 mmol), and 2,2′-dithiodipyridine (22 mg, 0.1 mmol, purchased from Aldrich) were mixed in anhydrous DMF (1.2 mL) and TEA (7 μL). Triphenylphosphine (26 mg, 0.1 mmol) was added, and the mixture was stirred for 5 h at room temperature. The mixture was placed in a centrifuge tube, and sodium perchlorate (25 mg, anhydrous) dissolved in acetone (6 mL) was added. The procedure for washing the precipitate with acetone and drying over P4O10 was the same as for Compound 5. The imidazolide of GMP thus obtained was dissolved in DMF (1.2 mL), and Compound 7 (10 mg, TEA salt, 0.015 mmol) was added. Next ZnCl2 (40 mg) was added. The mixture was stirred at room temperature overnight, poured into a beaker containing a solution of 125 mg of EDTA in 15 mL of water, and neutralized with 1 M NaHCO3. Chromatographic isolation as for Compound 5 gave Compound 9 (13 mg, 88% based on the amount of Compound 7 used).

EXAMPLE 8

[0031] P1-3′-O, 7-Dimethylguanosine-5′P3-guanosine-5′triphosphate (Compound 10) was prepared from GMP and Compound 8 (34 mg) by a procedure analogous to that for Compound 9 (yield: 23 mg, 78%).

EXAMPLES 9 & 10

[0032] The final products (Compounds 9 and 10) were converted to their Na+ salts by ion exchange on a small column of Dowex 50W×8 (Na+ form), followed by evaporation of the eluates to a small volume, precipitation with ethanol, and centrifugation to give amorphous white powders. Parameters from the 1H NMR spectra of Compounds 9 and 10 are shown in Tables 1 and 2 below.

EXAMPLE 11

7-Methylguanosine 3′,5′-diphosphate. Guanosine 3′,5′-diphosphate was methylated to make the chromatographic standard pm7Gp (FIG. 2) by the same procedure as used for Compound 7.

EXAMPLE 12

[0033] Column Chromatography

[0034] Both final products (Compounds 9 and 10, FIG. 1) and intermediate nucleotides (Compounds 3-8) were isolated from reaction mixtures by column chromatography on DEAE-Sephadex (A-25, HCO3− form) using a linear gradient of triethylammonium bicarbonate (TEAB), pH 7.5, in water. Fractions were collected, and products peaks (monitored at 260 nm) were pooled and evaporated to dryness, with ethanol added repeatedly to remove the TEAB buffer. The products were obtained as TEA salts.

[0035] The purity of intermediates and products was monitored at 260 nm by analytical HPLC using a Spectra-Physics SP8800 apparatus on a 25-cm LC-18-T reverse phase column (Supelco). The mobile phase was a linear gradient of methanol from 0 to 25% in 0.1 M KH2PO4, pH 6, over 15 min with flow rate of 1.3 mL/min.

[0036] Mono- and dinucleotides obtained by enzymatic digestion of in vitro-synthesized RNAs were analyzed by HPLC using a Waters 625 instrument with a 996 PDA detector on a 4.5×250 mm Partisil 10SAX/25 column (Whatman). The program for elution of nucleotides comprised water for the first 5 min; a linear gradient of 87.5 to 500 mM KH2PO4, pH 3.5, over 35 min; a linear gradient of 87.5 to 500 mM of KH2PO4 over 30 min; and isocratic elution at 500 mM KH2PO4 for 21 min—all at a flow rate of 1 mL/min.

EXAMPLE 13

[0037] Spectroscopy

[0038] 1 H NMR and 13C NMR spectra were recorded on a Varian UNITY plus 500 MHz instrument in dimethylsulfoxide-d6 (for nucleoside intermediates) or D2O (for mono- and dinucleotides). Absorption spectra were obtained on a Cary 3E spectrophotometer.

[0039] 1 H NMR spectra for compounds 9 and 10 were run at 25° C. at 1.4 mg/0.7 mL and 0.4 mg/0.7 mL in D2O, respectively. Conformations of the sugar moieties were derived from the vicinal 1H—1H coupling constants. Conformations of the phosphate groups were determined from the 1H—31P coupling constants.

EXAMPLES 14 & 15

[0040] In vitro Synthesis of RNA

[0041] Two lengths of RNA, either uncapped or capped with one of the cap analogues, were synthesized by in vitro transcription. The DNA template used for both lengths of RNA was pSP−luc+ (Promega), which contains an SP6 bacteriophage promoter and a sequence encoding luciferase. To generate the short RNAs (43 bases exclusive of the cap), the plasmid was digested with Ncol. To generate the long RNAs (1706 bases, containing the entire luciferase coding region), the plasmid was digested with EcoRl. A typical 20 μL in vitro transcription reaction contained 40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM DTT, 2 μg BSA, 20 units of RNasin (Promega), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.1 mM GTP, 1 mM cap analogue (GpppG, m7GpppG, m73′dGpppG, or m27,O3′GpppG), 0.2-1.0 μg DNA, and 20 units of SP6 polymerase (Promega). Reactions to synthesize the short RNAs also contained 28 pCi of [α-32P]ATP (ICN), and those to synthesize the long RNAs contained 0.8 pCi of [α-32P]CTP (ICN). Reaction mixtures were incubated for 60 min at 37° C., extracted with phenol and chloroform, and the solution was made 2 M in sodium acetate. The nucleic acids were then precipitated with 3 volumes of ethanol on dry ice for 5 min, and the mixture was centrifuged at 14,000 rpm for 30 min. The resulting pellet was dissolved in water, and the solution was made 0.2 M in sodium acetate. The nucleic acid was precipitated with 2.5 volumes of ethanol at 4° C. for 30 min, and the mixture was centrifuged at 14,000 rpm for 30 min. The pellet was allowed to air-dry and then dissolved in diethylpyrocarbonate-treated water.

EXAMPLE 16

[0042] Enzymatic Digestion of RNAs

[0043] The short RNAs were digested with 67 U RNase T2 (Life Technologies) in 15 μL of 0.14 M sodium acetate, pH 4.6, at 37° C. for 60 min. In some cases, the RNAs were subjected to a two-step digestion instead. The first digestion was with 10 U TAP (tobacco acid pyrophosphatase) (Epicentre Technologies) in 5 μL of 50 mM sodium acetate, pH 6.0, 1 mM EDTA, 0.1% β-mercaptoethanol, and 0.01% Triton X-100 at 37° C. for 60 min. The digestion was continued for 60 min at 37° C. with 67 U RNase T2 in a final volume of 16 μL of 0.12 M sodium acetate, pH 4.6. Samples were analyzed without further treatment by anion exchange HPLC as described above.

EXAMPLE 17

[0044] Cell-Free Translation

[0045] A micrococcal nuclease-treated RRL (rabbit reticulocyte lysate) system was used for in vitro translation as described in A. Cai et al., “Quantitative assessment of mRNA cap analogs as inhibitors of in vitro translation,” Biochemistry, vol.38, pp. 8538-8547 (1999). In some cases, the mRNA used in this system was natural rabbit globin mRNA, and protein synthesis was measured by incorporation of [3H]Leu into a trichloroacetic acid-precipitable form. In other cases, the mRNA was luciferase mRNA (the long form), synthesized in vitro as described above, and protein synthesis was assayed by measuring luciferase activity using beetle luciferin (Promega) as a substrate, and a Monolite 2010 luminometer to measure light emission.

[0046] The ability of cap analogues to inhibit cell-free translation in the RRL system programmed with globin mRNA was measured as described in Cai et al. (1999). Data were fit by least squares minimization to a theoretical rate equation. The concentrations of cap analogue solutions were measured by UV absorption at pH 7.0 using the following parameters for λ and εM, respectively: GpppG, 251 nm, 25.5×103; m7GpppG, m73′dGpppG, or m27,O3′GpppG, 255 nm, 22.6×103.

Results

[0047] 13 C NMR and UV spectra for intermediates were in good agreement with the predicted structures (data not shown). The 1H NMR assignments of protons in both prototype ARCAs confirmed their chemical structures (Table 1). Two sets of sugar 1H signals in each spectrum pointed to dinucleotides. The presence of methyl signals at 4.068 ppm (Compound 10) and 4.027 (Compound 9), together with disappearance of the H(8) resonances due to exchange for solvent deuterium, testified to the presence of 7-methylguanine. In the case of Compound 10, the additional methyl group was observed at 3.483 ppm, accompanied by a characteristic upfield shift of the H3′ signal. Lack of the 3′-hydroxyl in Compound 9 gave the characteristic “deoxy” pattern of H3′/H3″ at 2.086-2.148 ppm, with further scalar couplings to H4′ and H2′.

TABLE 1
1H NMR chemical shifts in ppm versus internal sodium
3-trimethylsilyl-[2,2,3,3-D4]-propionate
	m73′dGpppG	m27, O3′GpppG
	(Compound 9)	(Compound 10)
	m73′dG	G	m27, O3′G	G
	H8	—a	8.016	—a	7.990
	H1′	5.796	5.776	5.864	5.785
	H2′	4.587	4.650	4.682	4.687
	H3′	2.148	4.473	4.109	4.473
	H3″	2.086	—	—	—
	H4′	4.728	4.346	4.428	4.339
	H5′	4.460	4.26b	4.384	4.278
	H5″	4.196	4.26b	4.219	4.239
	CH3	4.027	—	4.068 (N7)	—
				3.483 (3′O)
	adeuterated
	bsignal overlapping

[0048] Table 2 provides NMR information concerning conformational parameters. These data reflected populations of the N form in the NS dynamic equilibrium of the sugar ring, populations of the +sc (gauche-gauche) conformer about C4′-C5′, and populations of the ap (gauche′-gauche′) conformer of the phosphate group. The 7-substituted Guo moieties showed the characteristic preference for the N conformer, up to 100% in the case of m27,O3Guo, as opposed to Guo, in which the S conformer dominates. The preference for +sc was also more pronounced in the 7-substituted guanosines. The conformation of the Guo moiety of ARCAs was similar to that of Guo in normal caps, in which about 64% has been reported to be in the S form (36% N) and about 63% in the +sc form. Thus, m27,O3′Guo and m73′dG both displayed conformational features that were characteristic of m7Guo rather than of Guo.

TABLE 2
1H—1H and 1H-31P coupling constants in Hz (±0.2), and conformer
populations (±5%) in the dynamic equilibria N S of the sugar ring,
and about C4′-C5′ (% + sc) and C5′-O5′ (% ap) bonds
	m73′dGpppG	m27, O3′GpppG
	(Compound 9)	(Compound 10)
	m73′dG	G	m27, O3′G	G
	J(1′,2′)	0.0a	6.2	4.0	6.3
	J(2′,3′)	4.5	5.2	5.0	5.1
	J(2′,3″)	0.0a	—	—	—
	J(3′,3″)	14.2	—	—	—
	J(3′,4′)	10.4	3.7	5.1	3.6
	J(3″,4′)	5.1	—	—	—
	J(4′,5′)	3.0b	4.0b	3.0	4.1
	J(4′,5″)	2.7	4.0b	2.6	4.2
	J(5′,5″)	11.6	b	11.5	11.8
	J(5′,P)	5.0	6.0b	4.4	5.4
	J(5″,P)	5.8	6.0b	5.9	6.5
	J(4′,P)	1.0b	1.0b	1.0b	1.0b
	% N	100	37	56	36
	% + scc	80	55b	80	54
	% apd	72	66b	74	66
	aless than the line width, ˜1 Hz
	bapproximate value
	c+synclinal, i. e., O5′ in gauche orientation to O4′ and C3′
	dantiperiplanar, i.e., P5′ in trans orientation to C4′

EXAMPLE 18

[0049] Synthesis of ARCA-Capped RNA Transcripts

[0050] We tested the prototype ARCAs in an in vitro transcription system. A template DNA was first generated by digesting the plasmid pSP−luc+ with EcoRl. The theoretical size of an RNA transcript from this template should be 1706 bases, which was consistent with the approximate size of the products that was observed by electrophoretic migration from reactions carried out in the presence of [α-32P]ATP and either GpppG, m7GpppG, m73′dGpppG, or m27,O3′GpppG. (data not shown). Samples were run on a 1% agarose gel containing 0.12 M formaldehyde in 0.4 M 3-(N-morpholino)propanesulfonic acid, pH 7.0, 0.1 M sodium acetate, and 0.01 M EDTA at 70 mA for 5 h. A Phosphorlmage was obtained with a Molecular Dynamics Storm 860 instrument. Standards used for comparison were rabbit 28S rRNA, 18S rRNA, and β-globin mRNA.

[0051] In six separate experiments, the yield of RNA product in the presence of ARCAs was not significantly different from the yield in the presence of m7GpppG.

EXAMPLE 19

[0052] Analysis of Cap Orientation in ARCA-Capped RNA Transcripts

[0053] The structure of the ARCAs was designed to prevent incorporation into RNA in the reverse orientation. We verified this property experimentally by digesting RNAs capped with ARCAs with RNase T2 and TAP. To obtain a higher proportion of radioactivity in the cap versus the internal positions, a shorter DNA template was produced by cleaving pSP−luc+ with Ncol instead of EcoRl. This digestion was expected to yield an RNA product of 43 bases (plus the cap). The size of this product was confirmed by polyacrylamide gel electrophoresis in Tris/borate/EDTA/urea (data not shown).

[0054] RNase T2 digests RNA with no base specificity. Thus, it was expected to generate primarily 3′-NMPs from this RNA. Those nucleotide residues that were located 5′ to an A residue would acquire a 32P-labeled 3′-phosphate by nearest-neighbor transfer. The pyrophosphate bonds in the cap, however, are not susceptible to RNase T2. Since the first nucleotide residue after the cap in the synthetic RNA is an A, the α-phosphate of [α-32P]ATP would be transferred to the cap following RNase T2 digestion. Thus, for RNAs initiated in the normal orientation with m7GpppG, the product was m7GpppGp* (where p* denotes radioactive 32p). The RNase T2-digestion products expected from RNAs initiated with GTP or with the four cap analogues in either normal or reverse orientations are shown in Table 3.

TABLE 3
Predicted and observed cap structures from in vitro-synthesized
mRNAs after enzymatic digestion
	Possible 5′ end labeled	Product
	digestion products	observed
Cap	Orien-	Possible transcription	RNase	RNase T2 +	RNase T2 +
dinucleotide	tation1	products	T2	TAP	TAP
None	N/A	pppGP*AP(NP)40C	pppGp*	pGp*	100%
GpppG	N/A	GpppGp*Ap(Np)40C	GpppGp*	pGp*	100%
m7GpppG	Normal	m7GpppGp*Ap(Np)40C	m7GpppGp*	pGp*	67%
	Reverse	Gpppm7Gp*Ap(Np)40C	Gpppm7Gp*	pm7Gp*	33%
m73′dGpppG	Normal	m73′dGpppGp*AP(NP)40C	m73′dGpppGp*	pGp*	100%
	Reverse	Gpppm73′dGp*Ap(Np)40C	Gpppm73′dGp*	pm73′dGp*	0%
m27, O3′GpppG	Normal	m27, O3′GpppGp*Ap(Np)40C	m27, O3′GpppGp*	pGp*	100%
	Reverse	Gpppm27, O3′Gp*Ap(Np)40C	Gpppm27, O3′Gp*	pm7, O3′Gp*	0%
1“Normal” orientation means that the 3′-OH of Guo in the structure m7G(5′)ppp(5′)G (or its analogues) is attached to the first nucleotide residue in the RNA chain by a 3′-5′ phosphodiester linkage. “Reverse” orientation means that the 3′-OH of m7Guo is the point of attachment.
2Radioactive atoms (32P) are indicated by *.

[0055] The RNase T2-digestion products of normal and reverse m7GpppG-capped RNAs (M7GpppGp* and Gpppm7Gp*, respectively) have identical masses and charges; they would therefore be expected to elute from an anion exchange column at nearly the same time. However, TAP digestion of normal and reverse-capped mRNAs should yield two alternate labeled products, pGp* and pm7Gp*, that differ in both charge and mass, because the m7 group confers a positive charge on G. The nucleotides pm73′dGp* and pm27,O3′Gp* have the same charge as pm7Gp*. Thus, although RNase T2 digestion alone would not be expected to distinguish between normal and reverse orientations, the combination of RNase T2 and TAP should do so (see Table 3).

[0056] RNA was synthesized from the short DNA template in the presence of: (1) [α-32P]ATP; (2) the other three NTPs (nonradioactive); and (3) either GpppG, m7GpppG, m73′dGpppG, m27,O3′GpppG, or no cap analogue. The products were digested with RNase T2 and resolved by anion exchange HPLC. FIG. 2 depicts an analysis of the in vitro-synthesized RNAs by enzymatic digestion and anion exchange HPLC. The mRNAs were generated by transcription of Ncol-digested pSP−luc+ with [α-32P]ATP and either no cap dinucleotide (panels A, B), GpppG (panels C, D), m7GpppG (panels E, F), m73′dGpppG (panels G, H), or m27,O3′GpppG (panels I, J). Aliquots of 5 to 13 ng of RNA were digested with RNase T2 (left panels), or with both RNase T2 and TAP (right panels). Nucleotides and caps were separated on a Partisil 10SAX/25 column developed with a gradient of potassium phosphate, pH 3.5. Fractions of 1 mL were collected, and their Cerenkov radiation was determined with a Beckman LS 6500 scintillation counter. The elution times of the following standard compounds, detected by UV absorption, are also shown: 3′-CMP; 3′-UMP; 3′-AMP; 3′-GMP; 5′-GDP; 5′-GTP; 3′,5′-GDP (pGp); 3′,5′-m7GMP (pm7Gp); and GpppG.

[0057] Uncapped RNA yielded primarily 3′-NMPs (Panel A, 20-30 min) with a small amount of material that may have been the partially degraded product ppGp* (Panel A, 76 min). The expected product pppGp* was not observed. Due to its high negative charge, that species may not have eluted from the column. Its presence, however, is likely since RNase T2 plus TAP digestion yielded pGp* (Panel B, 56 min) where none had existed previously (compare Panel A).

[0058] In the case of GpppG-capped RNAS, RNase T2 alone yielded a structure eluting at 89 min (FIG. 2, Panel C), likely GpppGp* (the presence of a second phosphate ester reduces the charge relative to pppGp*). The minor peak at 77 min may have been the partially degraded product ppGp*. Consistent with these assignments, both compounds disappeared following TAP digestion, coinciding with the appearance of a new peak corresponding to pGp* at 56 min (FIG. 2, Panel D). As expected, no pm7Gp* (42 min) was formed.

[0059] The major, highly-charged RNase T2-resistant product from m7GpppG-capped RNA eluted at 73 min (FIG. 2, Panel E), likely m7GpppGp*. This compound eluted earlier than the peak at 89 min in Panel C, tentatively assigned the structure GpppGp*, because of the additional positive charge. The minor peak at 77 min may be the reverse cap GPPPm7Gp*, suggesting that the proximity of the 3′-P to the positive charge of the m7G ring may influence the charge on the P. These assignments are strengthened by the fact that TAP digestion converted these products to two labeled compounds that eluted earlier, pGp* (56 min) and pm7Gp* (42 min) (FIG. 2, Panel F). The ratio of pGp* to pm7Gp* suggest that they were derived from the 73- and 77-min peaks of Panel E, respectively.

[0060] With the ARCA m73′dGpppG, an RNase T2-resistant product was observed at 78 min, likely m73′dGpppGp* (FIG. 2, Panel G). It eluted at nearly the same time as the compound thought to be m7GpppGp* (78 min versus 77 min for Panels G and E, respectively). Note that where there were two peaks in this region for RNA synthesized with m7GpppG (Panel E), there was only one peak for RNA synthesized with the ARCA (Panel G), consistent with the inability of the ARCA to be incorporated in the reverse orientation. After digestion with TAP, the peak at 78 min disappeared and a new one appeared at the elution time of pGp* (Panel H, 56 min). The fact that no pm7Gp* appeared at 42 min with the ARCA (Panel H), while it did with m7GpppG (Panel F), is further proof that the ARCA was incorporated in only a single orientation.

[0061] The products observed upon digestion of RNA synthesized with the second ARCA, m27,O3′GpppG (FIG. 2, Panels I and J), eluted almost the same as those that had been obtained with the m73′dGpppG-capped RNA—again, consistent with the expectation that the ARCA should be incorporated in only one orientation.

EXAMPLE 20

[0062] Competitive Inhibition of Protein Synthesis by ARCAs

[0063] One measure of the interaction between a cap analogue and the translational machinery is competitive inhibition of protein synthesis. The binding of cap analogues to elF4E, measured in vitro with purified components, and the resulting competitive inhibition of protein synthesis in a cell-free translation system have been correlated for several different cap analogue structures. We separately tested GpppG, m7GpppG, and the two ARCAs for their ability to compete with natural globin mRNA for recognition by the translational machinery, and thereby to inhibit translation in an RRL system. GpppG did not act as an inhibitor, and in fact slightly stimulated protein synthesis at low concentrations. The two ARCAs, on the other hand, were equally as inhibitory as m7GpppG. FIG. 3 depicts the inhibition of translation by ARCAs compared with m7GpppG and GpppG. Natural rabbit globin mRNA was translated at 5 μg/mL in the RRL system, and globin synthesis was detected by incorporation of [3H]Leu into protein. The following cap analogues were included during translation at the indicated concentrations: GpppG, circles; m7GpppG, squares; m73′dGpppG, triangles; and m27,O3′GpppG, diamonds.

[0064] One may compare cap analogues as inhibitors quantitatively, by fitting a theoretical curve to observed translation data. The value of the dissociation constant, Kl, for the cap analogue•elF4E complex was varied to obtain the best least-squares fit. FIG. 3 depicts such curves for m7GpppG, m73′dGpppG, and m27,O3′GpppG, with corresponding Kl values of 27.8±12.6, 27.8±7.1, and 14.3±1.9 μM, respectively. Although it appeared in this experiment that the m27,O3′GpppG compound was more inhibitory, in a replicate of this experiment the Kl values for the ARCAs did not differ statistically from those of m7GpppG.

EXAMPLE 21

[0065] Translation of ARCA-Capped mRNAs

[0066] Because one-third to one-half of m7GpppG was incorporated into RNA in the reverse orientation, because the novel ARCAs were incorporated exclusively in the normal orientation, and because the ARCAs were recognized to the same extent as m7GpppG in the translational inhibition experiment, we predicted that a homogeneous population of in vitro-synthesized ARCA-capped mRNAs should be more active translationally than m7GpppG-capped mRNAs. We tested this prediction using luciferase mRNAs that were either uncapped, capped with GpppG, capped with m7GpppG, or capped with one of the two ARCAs.

[0067] FIG. 4 depicts the translational activity of the ARCA-capped mRNAs. Luciferase mRNAs were synthesized in vitro using SP6 RNA polymerase in the presence of all four NTPs, and either no cap analogue (circles), GpppG (squares), m7GpppG (diamonds), m27,O3′GpppG (inverted triangles) or m73′dGpppG (triangles). The RNAs were translated for 60 min in the RRL system, and luciferase activity was measured in triplicate by luminometry (RLU, relative light units). Translational efficiency for each mRNA was estimated from the slopes of the curves of luciferase activity versus mRNA concentration.

[0068] The fact that all the m7G-containing mRNAs were translated more efficiently than the uncapped or GpppG-capped mRNAs indicated that the RRL system we used was highly dependent on the presence of a cap on the mRNA. As shown in FIG. 4, we found that the m27,O3′GpppG-and m73′dGpppGp-capped mRNAs were more efficient in translation than m7GpppG-capped mRNA. In six experiments employing four separate batches of in vitro-synthesized mRNAs, the mRNAs produced with the novel ARCAs were consistently more active than those produced with m7GpppG.

[0069] Pasquinelli et al. (1995) found that the extent of reverse capping varied between 28% and 48%, depending on the pH of the in vitro-transcription reaction. In the experiments whose results are shown in FIG. 2 and summarized in Table 3, the extent of reverse capping was approximately 33%. Assuming that the novel ARCAs and normal caps stimulate translation to the same degree, an assumption that seems likely based on the inhibition data (FIG. 3), we predicted that the (homogeneous) preparation of ARCA-capped mRNA should be more active than the (heterogeneous) preparation of m7GpppG-capped mRNAs, a prediction that was consistent with our experimental observations.

[0070] These results showed that the novel ARCAs behaved very similarly to normal cap analogues, except that they were not incorporated into RNAs in the reverse orientation, and that they can cause substantially higher translational activity. The modifications at the 3′-O-position of m7Guo did not appear to substantially alter conformation (Table 2) or interaction with translational machinery (FIGS. 3 and 4). The ARCAs have the advantage of being incorporated into RNA exclusively in the normal orientation, but have no apparent disadvantages. To our knowledge, the degree to which m7G is incorporated in place of G in internal positions of a synthetic RNA chain by bacteriophage polymerases has not been rigorously determined. Regardless of the level of such misincorporation, the ARCAs should be essentially incapable of donating m7G either internally or at the 5′-end. A different type of ARCA, e.g., m42,2,7,O3′GpppG or m32,2,73′dGpppG, would be useful for in vitro synthesis of U-type snRNAs with 100% normal cap orientation.

EXAMPLE 22

[0071] An Arabinose-Derived ARCA

[0072] Anti-reverse mRNA cap analogues may also be derived from arabinose, for example: 1

[0073] For example, where n=1, this ARCA may be synthesized starting with 9-β-D-arabinofuranosylguanine, which is commercially available or which may be prepared by the method of Ikehara et al., “Studies of nucleosides and nucleotides. XLVIII. Purine cyclonucleosides. 29. A new method for the synthesis of 9-β-arabinofuranosylguanine (Ara G),” J. Carbohydr. Nucleosides Nucleotides, vol. 3, pp. 149-159 (1976), in place of the 3′-deoxyguanosine in Example 1.

[0074] Although the examples described above employed particular cap analogs, other analogs will also work in practicing the invention, for example:

2
1. m27,3′OGpppG:	X = OH,	Y = OCH3,	n = 1;
2. m73′dGpppG:	X = OH,	Y = H,	n = 1;
3. m27,2′OGpppG:	X = OCH3,	Y = OH,	n = 1;
4. m72′dGpppG:	X = H,	Y = OH,	n = 1;
5. m72′,3′didGpppG:	X = H,	Y = H,	n = 1;
6. m37,2′O, 3′OGpppG:	X = OCH3,	Y = OCH3,	n = 1;
7. m7et3′OGpppG:	X = OH,	Y = OC2H5,	n = 1;
8. m7et2′OGpppG:	X = OC2H5,	Y = OH,	n = 1;
9. m27,3′OGppppG:	X = OH,	Y = OCH3,	n = 2;
10. m73′dGppppG:	X = OH,	Y = H,	n = 2;
11. m27,2′OGppppG:	X = OCH3,	Y = OH,	n = 2;
12. m72′dGppppG:	X = H,	Y = OH,	n = 2;
13. m72′,3′didGppppG:	X = H,	Y = H,	n = 2;
14. m37,2′O, 3′OGppppG:	X = OCH3,	Y = OCH3,	n = 2;
15. m7et3′OGppppG:	X = OH,	Y = OC2H5,	n = 2;
16. m7et2′OGppppG:	X = OC2H5,	Y = OH,	n = 2.

[0075] Note that both 2′ and 3′ modifications may be used. These compounds may, for example, be synthesized in a manner generally analogous to the syntheses described above. For example, the synthesis of alternative 3 in the above list (X═OCH3, Y═OH, n=1) may be conducted in a manner similar to that described in the above Examples, starting by replacing the 3′-O-methylguanosine with 2′-O-methylguanosine in Example 2, the synthesis of the latter of which is also described in Kusmierek et al. (1978).

[0076] Likewise, the synthesis of alternative 4 in the above list (X═H, Y═OH, n=1) may be conducted in a manner similar to that described in the above Examples, starting with 2′-deoxyguanosine 5′-diphosphate, which is commercially available, in Example 3 in lieu of 2′-deoxyguanosine 5′-diphosphate.

[0077] The synthesis of alternative 5 may be conducted in a similar manner, starting with 2′,3′-dideoxyguanosine (which is commercially available) in lieu of 3′-deoxyguanosine in Example 1.

[0078] The synthesis of alternative 6 may be conducted by modifying the procedure of Kusmierek et al. (1978) by using a large excess of methylation reagent to prepare the starting material to use as otherwise described in the Examples, starting with Example 2.

[0079] The synthesis of alternative 7 may be conducted by modifying the procedure of Kusmierek et al. (1978) by using diazoethane (instead of diazomethane) as alkylating reagent to prepare the starting material to use as otherwise described in the Examples, starting with Example 2.

[0080] The synthesis of alternative 8 may be conducted by modifying the procedure of Kusmierek et al. (1978) by using diazoethane (instead of diazomethane) as alkylating reagent to prepare the starting material to use as otherwise described in the Examples, starting with Example 2.

[0081] The synthesis of alternatives 9-16 (n=2) may be conducted as otherwise described in the above Examples, or in the above syntheses of alternatives 3-8 (n=1), as appropriate, but using GDP instead of GMP in the steps as otherwise described in Example 7. Likewise, analogues with higher values of n may be prepared using guanosine triphosphate, guanosine tetraphosphate, guanosine pentaphosphate, etc. in lieu of GMP in the steps otherwise described in Example 7.

[0082] We expect that 2′,3′-dideoxy- and 2′,3′-dimethyl cap analogs will function in the present invention. We also expect that introducing additional phosphate groups into the phosphate bridge (creating, e.g., dinucleotide tetraphosphates or even penta-, hexa-, or heptaphosphates (n=3, 4, or 5 in the above structure))—will produce compounds that may be more effective than the triphosphates. Other possible substituents X and Y include OCH2CH3. If Y is OH, then it is preferred that X is neither H nor OH.

[0083] The “non-methylated” guanosine in the ARCA may be replaced with another nucleoside, e.g., uridine, adenosine, or cytosine: 3

[0084] wherein the moiety B is selected from the group consisting of 4

[0085] The synthesis of the ARCAs with these alternative nucleosides may be conducted as otherwise described in the above Examples, or in the above syntheses of alternatives 1-1 6, as appropriate, but using AMP, ADP, ATP, UMP, UDP, UTP, CMP, CDP, CTP, etc. in lieu of GMP in the steps otherwise described in Example 7.

[0086] Another alternative is to replace the 7-methyl group with another substituent, such as C1 to C4 substituted or unsubstituted alkyl, C6 to C8 substituted or unsubstituted aryl, or C1 to C4 substituted or unsubstituted alkoxy, as illustrated in the examples below:

5
[a] et7m3′OGpppG:	X = OH,	Y = OCH3,	Z = C2H5	n = 1;
[b] et73′dGpppG:	X = OH,	Y = H,	Z = C2H5	n = 1;
[c] bn7m3′OGpppG:	X = OH,	Y = OCH3,	Z = CH2C6H5	n = 1;
[d] bn73′dGpppG:	X = OH,	Y = H,	Z = CH2C6H5	n = 1;
[e] et7m3′OGpppG:	X = OH,	Y = OCH3,	Z = C2H5	n = 2;
[f] et73′dGpppG:	X = OH,	Y = H,	Z = C2H5	n = 2;
[g] bn7m3′OGpppG:	X = OH,	Y = OCH3,	Z = CH2C6H5	n = 2;
[h] bn73′dGpppG:	X = OH,	Y = H,	Z = CH2C6H5	n = 2;
[i] pFbn7m3′OGpppG:	X = OH,	Y = OCH3,	Z = CH2C6H4pF	n = 1;
[j] pFbn73′dGpppG:	X = OH,	Y = H,	Z = CH2C6H4pF	n = 1;
[k] pClbn7m3′OGpppG:	X = OH,	Y = OCH3,	Z = CH2C6H4pCl	n = 1;
[l] pClbn73′dGpppG:	X = OH,	Y = H,	Z = CH2C6H4pCl	n = 1;
[m] pFbn7m3′OGpppG:	X = OH,	Y = OCH3,	Z = CH2C6H4pF	n = 2;
[n] pFbn73′dGpppG:	X = OH,	Y = H,	Z = CH2C6H4pF	n = 2;
[o] pClbn7m3′OGpppG:	X = OH,	Y = OCH3,	Z = CH2C6H4pCl	n = 2;
[p] pClbn73′dGpppG:	X = OH,	Y = H,	Z = CH2C6H4pCl	n = 2;

[0087] The synthesis of alternatives [a] and [b] above may be carried out, for example, as otherwise described in the Examples above, starting with Example 5 or 6, and replacing the 100 μL of methyl iodide with 100 μL of ethyl iodide.

[0088] The synthesis of alternatives [c] and [d] above may be carried out, for example, as otherwise described in the Examples above, starting with Example 5 or 6, and replacing the 100 μL of methyl iodide with 100 μL of benzyl bromide. See generally M. Jankowska et al., “Synthesis and properties of new NH2 and N7 substituted GMP and GTP 5′-mRNA cap analogues,” Collect Czech. Chem. Commun., vol. 58, pp. S1 38-S141 (1993).

[0089] The synthesis of alternatives [e] through [h] above may be carried out, for example, as otherwise described in the Examples above, starting with Examples 5 through 8, and replacing the 100 μL of methyl iodide with 100 μL of ethyl iodide or benzyl bromide, as appropriate, and replacing GMP with GDP.

[0090] The synthesis of alternatives [i] through [p] above may be carried out, for example, as otherwise described in the Examples above, starting with Examples 5 through 8, and replacing the 100 μL of methyl iodide with 100 μL of p-chlorobenzyl chloride or p-fluorobenzyl chloride, as appropriate, and replacing GMP with GDP when appropriate. See Jankowska et al. (1993).

[0091] Another possible modification is a methyl or other substitution at the N2 position: 6

[0092] where, R may, for example, be H, CH3, CH2C6H5, CH2COC6H5, CH2CH2CH2OH, CH2CH═CH2, or another substituent, such as C1 to C4 substituted or unsubstituted alkyl, or C6 to C8 substituted or unsubstituted aryl. Such modifications may, for example, be made at the beginning of the synthetic route, in the initial synthesis of the nucleoside, prior to carrying out the other steps of the synthesis. For example, N2,3′-O-dimethylguanosine 5′-monophosphate may be obtained by a procedure analogous to that for Compound 3, but instead starting with N2,3′-O-dimethylguanosine, which may be prepared by introduction at the beginning of methyl groups into the N2 position of guanosine by the method of J. Boryski et al., Nucleosides Nucleotides, vol. 4, pp. 595 ff (1985); or Sekine et al., “A convenient method for the synthesis of N2,N2-dimethylguanosine by reductive C—S bond cleavage with tributyltin hydride,” J. Org. Chem., vol.56, pp.1224-1227(1991) See also J. Boryski, “Application of the 1,N-2-isopropenoguanosine system for synthesis of novel N-2-substituted derivatives of guanosine and acyclovir,” Coll. Czech. Chem. Commun., vol. 55 (special issue), pp. 85-88 (1990). rally, compounds in accordance with the present invention will the following: 7

[0093] wherein the substituents R, X, Y, and Z are as previously described, and the moiety B is selected from the group consisting of 8

[0094] Miscellaneous

[0095] The complete disclosures of all references cited in this specification are hereby incorporated by reference. Also incorporated by reference are the complete disclosures of the following publications of the inventors' own work, which are not prior art to the present application: J. Stepinski et al., “Synthesis and properties of mRNAs containing the novel ‘anti-reverse’ cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl(3′-deoxy)GpppG,” RNA, vol. 7, pp. 1486-1495 (2001); E. Darzynkiewicz et al., “New ‘anti-reverse’ 5′-mRNA dinucleotide cap analogues (ARCA),” Abstract POTH-035,27th Meeting of the Federation of European Biochemical Societies (Lisbon, Portugal, Jun. 30-Jul. 5, 2001); J. Stepinski et al., “Synthesis and properties of ‘anti-reverse’ cap analogues,” Abstract, 6th Meeting of the RNA Society (Banff, Canada, May 29-Jun. 3, 2001); and J. Stepinski et al., “Preparation and properties of mRNAs capped with the novel ‘anti-reverse’ dinucleotide cap analogues,” Abstract P-13, 4th West Coast Meeting on mRNA Stability and Translation (Seattle, Wash., Oct. 14-16, 2001). In the event of an otherwise irreconcilable conflict, however, the present specification shall control.

Claims

1. A composition comprising

2. A composition as recited in claim 1, wherein X is OH, Y is H, and n is 1.

3. A composition as recited in claim 1, wherein X is OH, Y is OCH3, and n is 1.

4. An RNA molecule whose 5′ end incorporates a composition as recited in claim 1.

5. An RNA molecule whose 5′ end incorporates a composition as recited in claim 2.

6. An RNA molecule whose 5′ end incorporates a composition as recited in claim 3.

7. A method for synthesizing an RNA molecule as recited in claim 4 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

8. A method for synthesizing an RNA molecule as recited in claim 5 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

9. A method for synthesizing an RNA molecule as recited in claim 6 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

10. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 4 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

11. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 5 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

12. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 6 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

13. A composition comprising

14. A composition as recited in claim 13, wherein X is OH, Y is H, and n is 1.

15. A composition as recited in claim 13, wherein X is OH, Y is OCH3, and n is 1.

16. An RNA molecule whose 5′ end incorporates a composition as recited in claim 13.

17. An RNA molecule whose 5′ end incorporates a composition as recited in claim 14.

18. An RNA molecule whose 5′ end incorporates a composition as recited in claim 15.

19. A method for synthesizing an RNA molecule as recited in claim 16 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

20. A method for synthesizing an RNA molecule as recited in claim 17 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

21. A method for synthesizing an RNA molecule as recited in claim 18 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

22. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 16 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

23. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 17 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

24. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 18 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

25. A composition comprising

26. An RNA molecule whose 5′ end incorporates a composition as recited in claim 25.

27. A method for synthesizing an RNA molecule as recited in claim 26 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

28. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 26 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

29. A composition comprising

30. An RNA molecule whose 5′ end incorporates a composition as recited in claim 29.

31. A method for synthesizing an RNA molecule as recited in claim 30 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

32. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 30 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.

33. A composition comprising

34. An RNA molecule whose 5′ end incorporates a composition as recited in claim 33.

35. A method for synthesizing an RNA molecule as recited in claim 34 in vitro; said method comprising reacting ATP, CTP, UTP, GTP, a composition as recited, and a polynucleotide template; in the presence an RNA polymerase; under conditions conducive to transcription by the RNA polymerase of the polynucleotide template into an RNA copy; whereby some of the RNA copies will incorporate the composition as recited to make an RNA molecule as recited.

36. A method for synthesizing a protein or a peptide in vitro, said method comprising translating an RNA molecule as recited in claim 34 in a cell-free protein synthesis system, wherein the RNA molecule comprises an open reading frame, under conditions conducive to translating the open reading frame of the RNA molecule into the protein or peptide encoded by the open reading frame.