The invention is related to RNA molecules containing novel caps of the 5’ mRNA terminus, an in vitro or cell culture method of protein or peptide synthesis, wherein said method involves translation of a RNA molecule, a RNA molecule for use in medicine and the use of new cap analogues in RNA capping.

The scientific publication “Decapping Scavenger Enzyme Activity Toward N2-Substituted 5' End mRNA Cap Analogues" Pietrow et al„ ACS Omega 2019 [1] presents the results of a study on the DcpS activity in humans (hDcps), Caenorhabditis elegans (CeDcpS) and Ascaris suum (AsDcpS) aimed at dinucleotide cap analogues modified at the N ² position of 7-methylguanosine. Three different alkyl substitutes were tested, wherein cap analogues with a chain longer than three carbon atoms were not hydrolysed by hDcpS and CeDcpS.

The scientific publication “Synthesis of N2-modified 7-methylguanosine 5 ’-monophosphates as nematode translation inhibitors” Piecyk et al„ Bioorganic & Medicinal Chemistry 2012 [2] discloses 5’- monophosphate analogues of 7-methyloguanosine containing one or two substituents at the N ² position. A preparative scale synthesis of 14 novel monoucleotide analogues of the 5’ RNA cap, modified at the N ² , has been achieved. The derivatives were tested as translation inhibitors on a parasitic roundworm Ascaris suum, in a cell-free system.

Another scientific publication “Triazole-containing monophosphate mRNA cap analogs as effective translation inhibitors” Piecyk et al., RNA 2014 [3] discloses the methodology of producing monophosphate cap analogues containing N ² -triazole and their biological evaluation as protein synthesis inhibitors. Five cap analogues with a heterocyclic ring separated from m ⁷ -guanine by one to three carbon atoms and/or additionally substituted with various groups containing a benzene ring have been synthesised. All the obtained compounds proved to be effective translation inhibitors with IC50 similar to that of a m ⁷ GpppG dinucleotide triphosphate. In addition, an example dinucleotide was made of the respective mononucleotide containing a benzyl-substituted 1,2,3-triazole and studied.

The scientific publication “How to find the optimal partner — studies of snurportin 1 interactions with UsnRNA 5’ TMG-cap analogues containing modified 2-amino group of 7-methylguanosine" by Piecyk et al„ Bioorganic & Medicinal Chemistry 2015 [4] discloses the synthesis and properties of 7-methylguanosine cap analogues with sub-substituents in the exocyclic 2-amine group in order to achieve better insight into how the TMG cap is adapted to cap binding pocket of snurportin. On the other hand, the scientific publication “Modified ARCA analogs providing enhanced translational properties of capped mRNAs” Kocmik et al., Cell Cycle 2018 [5] discloses dinucleotide cap analogues and the results of studies on them in a rabbit reticulocyte lysate (RRL) and in the HEK293 cell line obtained from kidneys of human embryos, in in vitro translation systems. The obtained data indicates that in both translation tests, the synthesised cap analogues included in the mRNA improved its translation properties compared to transcripts with a standard m7GpppG or ARCA cap. Additionally, the introduced modifications improved the stability of transcripts with a cap in the HEK.293 cells compared to transcripts with a regular or an ARCA cap.

Another scientific publication “Isoxazole-containing 5' mRNA cap analogues as inhibitors of the translation initiation process” Piecyk et al., Bioorganic Chemistry 2020 [6] discloses the synthesis of monophosphate analogues of the 5’ mRNA cap containing isoxazole formed in a cycloaddition reaction at the N ² position. The obtained analogues show the ability to inhibit cap-dependent translation in vitro and are characterised by a novel binding pattern using the isoxazole ring instead of guanine in n-electron interactions (stacking).

A technical issue the invention faces is to provide capped RNA molecules, which would be translated with high efficiency in cell-free systems or in cellular systems, whereby the caps should be preferentially incorporated in the RNA in the correct orientation. Another problem includes the use of such capped molecules in in vitro synthesis of a protein or a peptide. In particular, capped RNA molecules should display preferable biological properties compared to the m ⁷ GpppG and ARCA caps used as a standard. Another problem is the use of cap analogues in synthesis of RNA molecules modified such that their preferential incorporation into RNA in the correct orientation is ensured. Capped RNA molecules should also display improved stability under enzymatic hydrolysis conditions.

The subject of the invention is a RNA molecule containing a compound defined by formula (II) at the 5’ terminus where n is 1 to 10; m is 1 to 2;

R ₁ is selected from a group including a substituted aromatic ring, the aromatic ring being preferably phenyl, a substituted heteroaromatic ring, preferably a five-member heteroaromatic ring containing at least one nitrogen atom;

R ₂ is selected from a group including a hydrogen atom, a substituted aromatic ring or an unsubstituted aromatic ring, with the aromatic ring preferably selected from a group including phenyl or naphthyl;

R ₃ means a hydrogen atom or a methyl group;

X ₂ , X ₄ , X ₇ means: O-, S-, Se- or BH ₃ ;

X ₁ , X ₃ , X ₅ , X ₆ means: O, S, NH or CH ₂ ;

The base is selected from a group containing purine or pyrimidine derivatives.

In a preferable embodiment of the invention, the RNA molecule contains a compound defined by formula (II) at the 5’ terminus where n is 1 to 8, preferably 1 to 4; m is 1 to 2;

R ₁ is selected from a group including an aromatic ring, with the aromatic ring being preferably phenyl, the aromatic ring is preferably substituted with a halogen atom or an alkiloxy substitute, preferably a five-membered heteroaromatic ring containing at least one nitrogen atom, preferably the heteroaromatic ring is preferably substituted with a substituent selected from a group including the following substituent: alkiloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;

R ₂ is selected from a group including a hydrogen atom, an aromatic ring, wherein the aromatic ring is selected from a group containing phenyl or naphthyl; R ₃ means a hydrogen atom or a methyl group;

X ₂ , X ₄ , X ₇ means: O or S-;

X ₁ , X ₃ , X ₅ , X ₆ means: O, S, NH or CH ₂ ;

Base - is selected from a group including:

In another preferable embodiment of the invention, the RNA molecule contains at its S’ terminus a compound defined by the formula (II) where n is 1 to 4; m is 1 to 2;

R ₁ is selected from a group including a phenyl ring substituted with a halogen atom or an alkyloxy substituent, a substituted five-membered heteroaromatic ring containing at least one nitrogen atom, wherein the heteroaromatic ring is substituted with a substituent selected from a group including the following substituent: alkyloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;

R ₂ is selected from a group including a hydrogen atom or phenyl;

R ₃ means a hydrogen atom or a methyl group;

X ₂ , X ₄ , X ₇ means O-, S-, Se- or BH ₃ ; X ₁ , X ₃ , X ₅ , X ₆ means O or S;

Base - is selected from a group including:

In yet another preferable embodiment of the invention, the RNA molecule contains at its 5’ terminus a compound defined by the formula (II) where n is 1 to 4; m is 1;

R ₁ is selected from a group including a phenyl ring substituted with a halogen atom or an alkyloxy substituent, a substituted five-membered heteroaromatic ring containing at least one nitrogen atom, wherein the heteroaromatic ring is substituted with a substituent selected from a group including the following substituent: alkyloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;

R ₂ is selected from a group including a hydrogen atom or phenyl;

R ₃ means a hydrogen atom;

X ₂ , X ₄ , X ₇ means O- or S-;

X ₁ , X ₃ , X ₅ , X ₆ means O or S;

Base - is selected from a group including:

In another preferable embodiment of the invention, the RNA molecule contains at its 5’ terminus a compound defined by the formula (II) where n is 1 to 4; m is 1;

R ₁ is selected from a group including a phenyl ring substituted with a halogen atom or an alkyloxy substituent, a substituted five-membered heteroaromatic ring containing at least one nitrogen atom, wherein the heteroaromatic ring is substituted with a substituent selected from a group including the following substituent: alkyloxyaromatic, preferably 2,6-disubstituted with an alkoxy, phenyl or alkyl substituent;

R ₂ is selected from a group including a hydrogen atom or phenyl;

R ₃ means a hydrogen atom;

X ₂ , X ₄ , X ₇ means O ;

X ₁ , X ₃ , X ₅ , X ₆ means O;

Base - is selected from a group including: In the preferable embodiment of the invention, the aromatic ring is substituted in the para position, preferably with a halogen atom or an alkyloxy substituent.

In another preferable embodiment of the invention, the five-membered heteroaromatic ring is selected from a group including 1,2,3-triazole, isoxazole, thiazole. The heteroaromatic ring is preferably substituted in the 1-, 3- or 4- position.

In another preferable embodiment of the invention, the alkyloxyaromatic substituent is substituted in the 2- and 6- position with a methoxy substituent.

In yet another preferable embodiment of the invention, the halogen atom is selected from a group including a fluorine atom, a chlorine atom, a bromine atom or a iodine atom, preferably a chlorine atom.

In another preferable embodiment of the invention, R ₁ is selected from a group including

In yet another preferable embodiment of the invention, the compound defined by formula (II) is selected from a group including:

A) P1-N2-benzyl-7-methyloguanosine-P3-guanosine 5’,5’-triphosphate, b) P1-N2-p-methoxybenzyl-7-methylguanosino-P3-guanosine 5’, 5’-tri phosphate, c) P1 -N2-p-chlorobenzyl-7-methylguanosine-P3-guanosine 5’,5’-triphosphate, d) P1-N2-{1-[3-(2,6-dimethoxyphenoxy)propyl]-1H-1,2,3-triazol-4 -yl}methylene-7-methylguanosine-P3- guanosine 5’,5’-triphosphate, e) P1 -N2-[(3-phenylisoxazol-5-yl)methylene-7-methylguanosine-P3-g uanosine 5’,5’-triphosphate, f) P1-N ² -[3-(4-methylthiazol-2-yl)]propyl-7-methylguanosine-P3 -guanosine 5’,5’-triphosphate.

The subject of the invention also includes the use of the RNA module defined in the first subject of the invention in in vitro synthesis of a protein or a peptide. Another subject of the invention is a RNA molecule defined in the first subject of the invention, for use in medicine. The invention also includes the use of cap analogues defined by formula (II) in RNA capping.

N ² -modified cap analogues with formula (II) present very favourable biological properties compared to the m ⁷ GpppG and ARCA caps used as the standard. They show a much stronger ability to inhibit translation in RRL using ARCA-mRNA (ca. 15-4 stronger inhibition compared to the control cap m ⁷ GpppG). RNA capped in the IVT reaction (in vitro transcription) using compounds 1-6 and 7-15 show much better translation-related properties in a cell-free system for protein production made of rabbit reticulocytes (translation yields ca. twice higher than in the case m ⁷ GpppG were achieved, and in the case of the best compounds, translation yields exceeded that of m ⁷ GpppG by more than three times, for comparison, the same ratio for ARCA 3’ caps is 1.47). RNA capped using compounds 1-6 were also tested in HEK293 cells, where they showed the total protein expression by ca. 1.5-3 times higher compared to the standard m ⁷ GpppG-RNA; for comparison, in the case of ARCA 3’ caps this ratio is 1.46). All compounds were preferentially incorporated into the RNA in the correct orientation, and compounds 4 and 12 were incorporated similar to ARCA, only in the correct orientation. The characteristic feature of the invention is the use of dinucleotide cap analogues with formula (II) in the N ² position of the first base, displaying significantly improved properties compared to m ⁷ GpppG and ARCA commonly used in RNA capping reactions in RNA synthesis. In the case of analogues containing 2’-O- methyladenosine as the second nucleoside, a trinucleotide (i.e. 12-14) should be used, as the available polymerases do not incorporate dinucleotides in the capping reaction, where adenosine methylated at the sugar is the second nucleoside. Additionally, because of their varied properties, they offer the option of selecting the appropriate compound depending on the desired features of the obtained RNA.

In relation to the invention, ribonucleic acid (RNA) should be understood as a polynucleotide chain of any length.

In relation to the invention, a base should be understood as any substituted or unsubstituted purine or pyrimidine derivative. In particular, the base is selected from a group including adenine, guanine, xanthine, N ⁶ -(A ² -isopentenyl)adenine, acycloguanosine, hypoxanthine, uracil, cytosine, N ⁴ - methylcytosine, 6-carboxyuracil, 2-thiocytosine or barbituric acid, or their substituted derivatives. Example embodiments of the invention are presented in the figures, where:

Fig. 1 presents the efficacy of mRNA translation in HEK293 cells, including Fig. 1A presenting the translation level as a relative luciferase activity after normalisation to protein concentration, and Fig. 1B presents the level of total luciferase expression presented as the surface area under the curve, normalised to the value of expression obtained for m7GpppG-mRNA, wherein both graphs present the average values ± SD ( Standard Deviation) for at least two independent experiments, each one including triplicates;

Fig. 2 illustrates a comparison of IC50 values (concentration inhibiting the protein biosynthesis in 50%) of the cap analogues 1 -6 and of the control compound m ⁷ GpppG;

Fig. 3 presents the hydrolysis of a 5' mRNA capped with N ² -modified cap analogues, using the enzymes human Nudt 16 (panels 3A) and human Dcp2 (panel 3B);

Fig. 4 presents the hydrolysis level of a cap incorporated into mRNA using hNudt16.

Example 1. General synthetic procedure for dinucleotide, tri- or tetraphosphate cap analogues modified at the N ² position

To 0.645 mg (1 mmoL) of guanosine-5’-diphosphate in a triethylammonium salt, previously dried over P ₂ O ₅ and suspended in 5 mL of anhydrous DMF imidazole (340 mg, 5 mmoL), 2,2’-dithiopyridine (440.6 mg, 2 mmoL), triethylamine (140 μL) and finally triphenylphosphine (525 mg, 5 mmoL) were added. The mixture was left for 8 h at room temperature and then the reaction was terminated by adding 440 mg (4 mmoL) of sodium perchlorate dissolved in 30 mL of acetone. The precipitated precipitate was centrifuged, washed with small amounts of acetone several times and dried over P ₂ O ₅ . The presence of imidazole GDP derivative was confirmed using mass spectrometry: m/z (M+H)+: 493.9483. 258 mg (0.5 mmoL) of imidazole derivative of 7-methylguanosine 5’-diphosphate, 0.6 mmoL 7-methylguanosine 5’- monophosphate (compounds 1-9) or 5’-diphosphate (compounds 10, 11, 15) modified at the N ² position (obtained according to [2] and [13]) and 0.324 mg (2.4 mmoL) ZnCl ₂ were combined, 7 mL DMF was added to the mixture, which was left and mixed intensively for 24 h. The reaction was terminated by adding aqueous EDTA solution (3 mmoL, 1.14 g in 15 mL of water). The reaction mixture was separated on a DEAE-Sephadex A-25 column, in a 0-1,0 M linear TEAB gradient (the total solution volume was 2 L). The fractions containing the product (measurement of radiation absorption at X=254 nm) were combined, evaporated and buffer traces were removed by evaporating several time with ethanol. Dinucleotide cap analogues were purified using high-performance liquid chromatography (HPLC) in a reversed phase system. A SUPERCOSILTM LC-18-T, 25 cm x 4.6 mm, 5 μm column, flow rate 1 mL/min, solvent system: (A) 0.05M ammonium acetate pH=5.9 (B) 0.05M ammonium acetate pH=5.9:MeOH (1:1); method: linear gradient 0-20 min, 0-100% B. P1 -N2-benzyl-7-methylguanosine-P3-guanosine 5’,5’-triphosphate (1)

30 mg (0.032 mmoL), 54%, 1H NMR (400 MHz, D ₂ O): δ 8.95 (s, 1 H, H-8 bz ² m ⁷ G), 7.97 (s, 1H, H-8 G), 7.41-7.27 (m, 5H, Ph), 5.87 (d, J = 2.84 Hz, 1H, H-1' bz ² m ⁷ G), 5.73 (d, J = 5.85 Hz, 1 H, H-1’G), 4.62- 4.48 (m, 4H, H-2’ bz ² m ⁷ G, H-2’G, CH ₂ Ph), 4.45-4.41 (m, 2H, H-3’ bz ² m ⁷ G, H-3’G), 4.34-4.32 (m, 2H, H- 4’ bz ² m ⁷ G, H-4’G), 4.29-4.26 (m, 1H, H-5’ bz ² m ⁷ G), 4.24-4.17 (m, 3H, H-5” bz ² m ⁷ G, H-5’ G, H-5”G), 3.99 (s, 3H, N7-CH3); 31 P NMR (162 MHz, D ₂ O) δ 11.67 (2P, Pa, y), 23.22 (1P, Pβ); HRMS (ES+) m/z: (M+H)+: 893.1415, calculated for C28H36N10O18P3+: 893.1416.

P1 -N2-p-methoxybenzyl-7-methyloguanosino-P3-guanosine 5’,5’-triphosphate (2)

28 mg (0.030 mmoL), 49%, 1 H NMR (400 MHz, D ₂ O): δ 8.97 (s, 1H, H-8 (p- CH ₃ Obz) ² m ⁷ G), 7.99 (s, 1H, H-8 G), 7.36-7.34 (m, 2H, Ph), 6.94-6.91 (m, 2H, Ph), 5.90 (d, J = 2.80 Hz, 1 H, H-1' CH ₃ Obz ² m ⁷ G), 5.74 (d, J = 5.84 Hz, 1H, H-1’G), 4.59 (t, J = 6.57 Hz, 1 H, H-2’ G), 4.54-4.45 (m, 3H, H-2’ CH ₃ Obz ² m ⁷ G, CHiPh), 4.44-4.40 (m, 2H, H-3’ CH ₃ O bz ² m ⁷ G, H-3’ G), 4.38-1.34 (m, 2H, H-4’ CH ₃ Obz ² m ⁷ G, H-4’ G), 4.29-4.18 (m, 4H, H-5’ and H-5’ CH ₃ Obz ² m ⁷ G, H-5 and H-5’ G), 4.00 (s, 3H, N7-CH3), 3.76 (s, 3H, CH ₃ O); 31 P NMR (162 MHz, D ₂ O) δ 11.65 (2P, Pa, y), 23.25 (1P, Pβ); HRMS (ES+) m/z: (M+H)+: 923.1531, calculated for C29H38N10O19P3* : 923.1520. P1 -N2-p-chlorobenzyl-7-methylguanosine-P3-guanosine 5’,5’-triphosphate (3)

27 mg (0.028 mmoL), 47%, 1 H NMR (400 MHz, D ₂ O): 6 8.97 (s, 1H, H-8 (p-Clbz) ² m ⁷ G), 8.03 (s, 1H, H- 8 G), 7.38-7.33 (m, 4H, Ph), 5.87 (d, J = 3.07 Hz, 1 H, H-1' (p-Clbz) ² m ⁷ G), 5.74 (d, J = 5.58 Hz, 1 H, H-1' G), 4.61-4.56 (m, 2H, H-2’ G, H-2’ (p-Clbz) ² m ⁷ G), 4.52 (m, 2H, CH ₂ Ph), 4.43-4.41 (m, 2H, H-3’ (p- Clbz) ² m ⁷ G, H-3’ G), 4.36-4.32 (m, 2H, H-4’ p-Clbz) ² m ⁷ G, H-4’ G), 4.28-4.27 (m, 1 H, H-5’ (p- Clbz) ² m ⁷ G), 4.23-4.17 (m, 3H, H-5” (p-Clbz) ² m ⁷ G, H-5’ G, H-5” G), 4.01 (s, 3H, N7- CH3), 31 P NMR (162 MHz, D ₂ O) δ 11.62 (2P, Pa, y), 23.31 (1 P, Pβ); HRMS (ES+) m/z: (M+H)+: 927.1034, calculated for C28H35CIN10O18P3+: 927.1026.

P1-N2-{1-[3-(2,6-dimethoxyphenoxy)propyl]-1H-1,2,3-triazo l-4-yl}methylene-7- methylguanosine-P3-guanosine 5*,5’-triphosphate (4)

OH OH

29 mg (0.026 mmoL), 43%, ammonium salt; 1H NMR (400 MHz, D ₂ O) δ 8.80 (s, 1 H, H8), 8.09 (s, 1H, H8), 8.00 (s, 1H, triazole), 6.90 (t, 1 H, J=8.817), 6.45 (d, 2H, J=8.457), 5.80 (d, 1H, J=2.09), 5.77 (d, 1H, J=6.25), 4.65-4.57 (m, 6H, H2’, H2’, CH ₂ -triazole-CH ₂ -), 4.49-4.47 (m, 2H, H3’, H3’), 4.45-4.42 (m, 2H, H4’, H4’), 4.39-4.30 (m, 4H, H5’, H5’, H5”, H5”), 3.94 (s, 3H, CH3), 3.87-3.75 (m, 2H, CH ₂ -CH ₂ -CH ₂ - Ph), 3.66 (s, 6H, 2xOCH3), 2.32-2.29 (m, 2H, triazole-CH ₂ -CH ₂ -CH ₂ -Ph); 31 P NMR (162 MHz, D ₂ O) δ - 11.76 (2P, Pa,y), -23.13 (1P, Pβ); CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ D ₂ O HRMS (ES+) m/z: (M+H)+: 1078.0549, calculated for C35H47N13O21P3+: 1078.7429. P1 -N2-[(3-phenylizoxazol-5-yl)methylene-7-methylguanosine-P3-g uanosine 5’, 5’- triphosphate (5)

4.7 mg (0.047 mmoL), 25%, ammonium salt; 1 H NMR (600 MHz, D ₂ O) m7G: δ 9.03 (s, 1 H, H8), 7.69 (d, 2H, Ph), 7.48-7.43 (m, 3H, Ph), 6.80 (s, 1 H, isoxazole), 5.97 (d, 1H, J= 2.98 Hz, H-1’), 4.74-4.75 (m, 2H, NH-CH ₂ ), 4.53 (t, 1 H, J= 3.62 Hz, H-2’), 4.43-4.40 (m, 2H, H-3’, H-4’), 4.26-4.24 (m, 2H, H-5’, H-5”), 4.04 (s, 3H, N7-CH3); G: δ 7.92 (s, 1H, H8), 5.69 (d, 1H, J=5.62 Hz, H-1’), 4.59 (t, 1 H, J=5.15 Hz, H-2’), 4.43-4.40 (m, 1H, H-3’), 4.36-4.35 (m, 1 H, H-4’), 4.26-4.24 (m, 1H, H-5’), 4.20-4.18 (m, 1 H, H-5”); 31 P NMR (243 MHz, D ₂ O): -12.05 (2P, Pa,y), -23.64 (1 P, Pβ); HRMS (ES+) m/z: (M+H)+: 960.14746, calculated for C31H37N11O19P3+: 960.14745.

P1 -N2-[3-(4-methyltiazol-2-yl)]propyl-7-methylguanosine-P3-gua nosine 5’, 5’- triphosphate (6)

6 mg (0.063 mmoL), 48%, ammonium salt; 1 H NMR (600 MHz, D ₂ O) m7G: δ 9.01 (s, 1H, H8), 7.00 (s,1H, thiazole), 5.92 (d, 1H, J=3.12 Hz, H-1’), 4.57 (t, 1 H, J=4.73 Hz, H-2’), 4.47-4.42 (m, 2H, H-3’, H- 4’), 4.33-4.31 (m, 1H, H-5’), 4.29-4.21 (m, 1 H, H-5’), 4.06 (s, 3H, N7-CH3), 3.54-3.46 (m, 2H, NCH ₂ ), 3.10 (t, 2H, CH ₂ -CH ₂ -CH ₂ ), 2.31 (s, 3H, CH3 thiazole), 2.15-2.11 (m, 2H, CH ₂ -CH ₂ -CH ₂ ); G: δ 8.01 (s, 1H, H8), 5.78 (d, 1 H, J=5.93 Hz, H-1’), 4.63 (t, 1 H, J=5.19 Hz, H-2’), 4.47-4.42 (m, 1H, H-3’), 4.40-1.38 (m, 1 H, H-4’), 4.29-4.21 (m, 2H, H-5’, H-5’). 31 P NMR (243 MHz, D ₂ O): -12.02 (2P, Pa, _Y ), -23.64 (1 P, Pβ); HRMS (ES+) m/z: (M+H)+: 943.14677, calculated for C28H39N11O18P3S*: 943.14808. P1 -N2-{1-[2-(2,6-dimethoxyphenoxy)propyl]-1 H-1,2,3-triazol-4-yl}ethylene-7- methylguanosine-P3-guanosine 5’,5’-triphosphate (7)

8 mg (0.074 mmoL), 18 %, ammonium salt; 1 H NMR (600 MHz, D ₂ O) δ 8.91 (s, 1 H, H8), 7.99 (s, 1H, H8), 7.917 (s, 1H, triazole), 6.99 (t, 1 H, J=8.42 Hz, Ph), 6.60 (t, 2H, J=.8.47, Ph), 5.86 (d, 1 H, H-1’, J=3.08), 5.79 (d, 1 H, H-1’, J=6.26), 4.66 (t, 1 H, H-2’), 4.66 (t, 1 H, H2’), 4.60 (t, 2H, triazole), 4.53-4.51 (m, 1 H, H2’), 4.49-4.48 (m, 1 H, H3’), 4.42 (t, 1 H, H3’), 4.40-4.36 (m, 2H, H4’, H4’), 4.34-4.29 (m, 2H, H5’, H5’), 4.25-4.22 (m, 2H, H5”, H5”), 3.91 (s, 3H, CH3), 3.78 (s, 6H, OCH3), 3.80-3.75 (m, 2H, CH ₂ - Ph), 3.76-3.63 (m, 4H, NH-CH ₂ -CH ₂ -triazole), 2.25-.2.21 (m, 2H, CH ₂ -CH ₂ -CH ₂ -Ph). 31 P NMR (243 MHz, D ₂ O): -14.70 (2P, Pa,y), -26.21 (1P, Pβ); HRMS (ES+) m/z: (M+H)+: 1078.22217, calculated for C36H49N13O21P3 ⁺ : 1078.22169.

P1 -N2-{1-[2-(2,6-dimethoxyphenoxy)propyl]-1 H-1,2,3-triazol-4-yl}butylene-7- methylguanosine-P3-guanosine 5*,5’-triphosphate (8)

21 mg (0.018 mmoL), 32 %, ammonium salt; 1 H NMR (600 MHz, D ₂ O) δ 8.80 (s, 1 H, H8), 8.10 (s, 1H, H8), 7.99 (s, 1H, triazol), 6.91 (t, 1 H, J=8.82 Hz, Ph), 6.45 (t, 2H, J=8.46, Ph), 5.80 (d, 1H, H-1’, J=2.09), 5.77 (d, 1H, H-1’, J=6.25), 4.65-4.57 (m, 6H, H2’, H2’, CH ₂ ,-triazole-CH ₂ ), 4.49-4.47 (m, 2H, H3’, H3’), 4.45-4.42 (m, 2H, H4’, H4’), 4.39-4.30 (m, 4H, H5’, H5’, H5”, H5”), 3.94 (s, 3H, CH3), 3.87-3.75 (m, 2H, CH ₂ -CH ₂ -CH ₂ -Ph), 3.66 (s, 6H.OCH3). 31 P NMR (243 MHz, D ₂ O): -15.65 (2P, Pa, y), -26.39 (1P, Pβ); HRMS (ES+) m/z: (M+H)+: 1120.26900, calculated for C38H53N13021 P3 ⁺ : 1120.26863. P1 -N2-{1-[2-(2,6-dimethoxyphenoxy)propyl]-1 H-1,2,3-triazol-4-yl}methylene-7- benzylguanosine-P3-guanosine 5’,5’-triphosphate (9)

3.8 mg (0.0032 mmoL), 29.1%, ammonium salt; 1 H NMR (400 MHz, DMSO-d6) N2bz7G: δ 9.41 (s, 1H, H8), 8.03 (s, 1H, triazole), 7.56 (d, 2H, Ph), 7.41-7.35 (m, 4H, Ph), 6.57-6.55 (m, 2H, Ph), 6.18 (d, 1H, H- 1’), 5.57-5.52 (m, 2H, CH ₂ benzyl), 4.71-4.69 (m, 1 H, H-2’), 4.65-4.56 (m, 2H with 3H, CH ₂ CH ₂ triazole), 4.54-4.51 (m, 1H, H-3’), 4.45-4., 39 (m, 1H with 2H, H-4’), 4.35-4.26 (m, 2H z 4H, 5H’, 5H”), 3.86-3.73 (m, 2H, -CH ₂ -O-Ph), 3.60 (s, 6H, CH3-O-Ph), 2.30-2.26 (m, 2H, CH ₂ CH ₂ CH ₂ -OPh)4; G: δ 8.09 (s, 1H, H8), 5.80 (d, 1H, H-1’), 4.65-4.56 (m, 1H with 3H, H-2’), 4.50-4.47 (m, 1 H, H-3’),), 4.45-4.39 (m, 1 H with 2H, H-4’), 4.35-4.26 (m, 2H with 4H, 5H’, 5H”), 31P NMR (162 MHz, DMSO-d6) δ -14.70 (2P, Pa.y), -26.07 (1 P, Pβ); HRMS(ES+) m/z: (M+H)+:1154.25308, calculated for C41H51N13O21P3+: 1154.25298.

P ¹ -N ² -benzyl-7-methylguanosine-P ⁴ -guanosine 5’,5’-tetraphosphate (10)

4.9 mg (0.05 mmoL), 7%, ammonium salt; 1H NMR (600 MHz, D ₂ O) δ 8.02 (s, 1H, H8), 7.45-7.31 (m, 5H, Ph), 5.97 (d, 1H, J=3.2, H1’), 5.81 (d, 1 H, J=6.33, H1’), 4.71-4.69 (m, 1 H, H2’), 4.64-4.55 (m, 3H, H2’, NH-CH ₂ Ph), 4.52-4.51 (m, 1H, H3’), 4.44-4.42 (m, 1H, H3’), 4.41-4.37 (m, 2H, H4’, H4’), 4.40-4.24 (m, 4H, 5’, 5’, 5”, 5”), 4.06 (s, 3H, CH3). 31P NMR (243 MHz, D ₂ O): -14.49 (2P, Pa, ₅ ), -26.14 (2P,P _β , _Y ); HRMS (ES+) m/z: (M+H)+: 973.1084, calculated for C28H37N10O21P4+: 973.1079

P ¹ -N ² -(p-chlorobenzyl)-7-methylguanosine-P ⁴ -guanosine 5’,5’-tetraphosphate (11)

4.9 mg (0,0095 mmoL), 20 %, ammonium salt; 1H NMR (600 MHz, D ₂ O) δ 8.02 (s, 1 H, H8), 7.38-7.35 (m, 4H, Ph), 5.96 (d, 1 H, J=8.3, HT), 5.79 (d, 1 H, J=2.45, HT), 4.70-4.68 (m, 1 H, H2’), 4.59-4.50 (m, 4H, H-2’, H3’, NH-CH ₂ -Ph), 4.38 (m, 3H, H4’, H4’, H3’), 4.33-4.24 (m, 4H, H5’, H5’, H5”, H5”), 4.06 (s, 3H, CH3). 31P NMR (243 MHz, D ₂ O): -14.48 (2P, Pa, _δ ), -26.23 (2P, P _β , _Y ); HRMS (ES+) m/z: (M+H)+: 1007.0701, calculated for C28H36CIN10O21P4+: 1007.9873.

P1-N2-benzyl-7-methylguanosine-P2-thiophosphate-P3-guanos ine 5’,5’-triphosphate

(15) ammonium salt; mixture of diastereoisomers, 1H NMR (400 MHz, D ₂ O) δ: 8.01; 7.99 (s, 2X ₁ H, H8), 7.45-7.31 (m, 2X ₅ H, Ph), 5.94-5.92 (2xd, 2XiH, HT), 5.80-5.79 (2xd, 2XiH, HT), 4.70-4.25 (m, 20H with H2’, H3’, H4’, H5’, H5” and 4H with CH ₂ Ph), 4.06; 4.06 (2xs, 2X ₃ H, CH3) 31 P NMR (162 MHz, D ₂ O) δ -15.4 to 1-15.56 (2X ₂ P, Pa, y), +26.91; 26.83 (2XiP, Pβ-SH); HRMS: (ES+) m/z: (M+H)+: 909.11922, calculated for C28H36N10O17S1P3+ : 909.11880 Trinucleotides 12, 13, 14

Chemical reagents, including the fully protected 2'-O-methyladenosine phosphoramidite, were obtained from commercial sources. P-imidazolide of N2-modified N7-methylguanosine 5KI-diphosphate was obtained using the method according to prior publications ([2] and Jemielity J„ et. al. RNA 2003;9(9):1108-22). N2-isobutyryl-2',3'-isopropylidenoguanosine was synthesised according to the previously disclosed protocols (Eisenfuhr A., et al. Bioorganic & medicinal chemistry vol. 11,2 (2003): 235-49).

General procedure used to obtain trinucleotide cap analogues containing Am

The dinucleotide (pAmpG) synthesis was performed in a solution, using phosphotriester chemistry. The conjugation reaction was performed using 1 eq. of 5'-O-DMT-2'-O-methyl-3'-O-phosphoramide and 1 eq. of protected guanosine in the presence of 0.40 M 5-(benzylthio)-1-H-tetrazole in acetonitrile. The reaction was performed over 4 hours at room temperature, under argon atmosphere. After 4h, the mixture was cooled to 4°C and 0.1 M iodine in pyridine was added and mixed for 1 hour at room temperature. The reaction mixture was extracted with dichloromethane and washed with brine. The obtained organic layer was dried, evaporated and purified using flash chromatography on silica gel, using gradient elution (0— >5% methanol in dichloromethane). The purified compound was dissolved in 20% aqueous TFA solution and mixed at RT for 4 hours. The mixture was evaporated under vacuum and evaporated 6 times with methanol. The raw nucleotide was crystallised from diethyl ether. The precipitate was filtered, washed with diethyl ether and dried in a vacuum dessicator over phosphorus pentaoxide. In the last stage, the dinucleotide was phosphorylated at the 5'-OH position using the standard Yoshikawa method, according to the previously described protocols ([2], [3]). The obtained product had its protection removed with ammonia, was evaporated and purified using ion exchange chromatography on DEAE-Sephadex (A-25, HCO ₃ - form) using a linear gradient of triethylammonium bicarbonate (TEAB), pH 7.5 in water. The fractions containing the desired product have been combined, evaporated and lyophilised to obtain the TEA salt of the product as white powder.

The triethylammonium salt of pAmpG (1.0 eq.), imidazole derivative of N2-modified diphosphate (2.0 eq.) and anhydrous ZnCl ₂ (25 eq.) were dissolved in anhydrous DMF. The mixture was mixed at RT for 24 hours and the reaction was then stopped by adding aqueous EDTA solution. The product was isolated using ion exchange chromatography on DEAE Sephadex (gradient elution using 0-1.2 M TEAB) and purified using semi-preparative RP HPLC (SUPELCOSIL™ LC-18-DB, gradient elution 0-50% MeOH in 0.05 M ammonium acetate buffer pH 5,9), to obtain the ammonium salt of trinucleotide cap analogues after evaporation and repeated lyophilisation. The reaction yield varied between 10 and 40%.

HRMS:

12: (ES+) m/z: (M+H)+: 1236.21098 calculated for C39H50N15O24P4+ : 1236.20981

13: (ES+) m/z: (M+H)+: 1270.17113 calculated for C39H49N15O24CI1 P4+ : 1270.17084

14: (ES+) m/z: (M+H)+: 1303.21658 calculated for C42H51N16O25P4+ : 1303.21562

Example 2. Capped RNA synthesis via transcription in vitro

2.1 Synthesis of luciferase coding mRNA

2.1.1. mRNA for experiments in RRL

A PCR product containing a sequence encoding firefly luciferase and the SP6 promotor sequence (for cap analogues: no. 1, 2, 3, 4, 5, 6) or T7 (for cap analogues: no. 4, 7, 8, 9, 10, 11, 12, 13, 14, 15) was used as the dsDNA template for the in vitro transcription (IVT) reaction. The PCR product had been purified using a NucleoSpin® Gel PCR Clean-up kit (Macherey-Nagel) before being added to the reaction. The newly synthesised cap analogues according to the invention, in particular those described in example 1, were added to the transcription reaction, in a molar ratio cap:GTP 5:1, which enabled the cap to be incorporated during the synthesis of the RNA product. The transcription reaction contained: the transcription buffer, 25 ng/μL of the dsDNA matrix, 0.5 mM ATP/CTP/UTP, 0.1 mN GTP, 0.5 mM of the dinucleotide cap analogue, 0.5 U/μL of the Ribolock ribonuclease inhibitor and 1 U/μL of the RNA SP6 polymerase (Thermo Fisher Scientific) or the RNA T7 polymerase (Thermo Fisher Scientific). The IVT reaction mixture was incubated for 1 h at 37°C, followed by an addition of 0.025 U/μL DNaze I (Thermo Scientific) which was incubated with the IVT reaction mixture for 20 min at 37°C in order to remove the DNA template. The reaction mixture was purified using NucleoSpin® RNA Clean-Up (Macherey-Nagel) according to the instructions of the manufacturer. Transcript integrity was checked using non-denaturing 1% agarose gel and the concentration was determined using spectrophotometry.

2.1.2. mRNA for experiments in HEK293 cells mRNA for HEK293 cells was obtained in an in vitro transcription reaction (IVT) using dsDNA of the PCR product containing the sequence coding firefly luciferase as a template and the SP6 promotor sequence (for cap analogues: no. 1, 2, 3, 4, 5, 6) or of the T7 promotor (for cap analogues: no. 4, 7, 8, 9, 10, 11, 12, 13, 14, 15). Before adding to the reaction, the PCR product had been purified using a NucleoSpin® Gel PCR Clean-up kit (Macherey-Nagel). The newly synthesised cap analogues according to the invention, in particular those described in example 1, were added to the transcription reaction, in a molar ratio cap:GTP 5:1, which enabled the cap to be incorporated during the synthesis of the RNA product. The transcription reaction included: the transcription buffer, 25 ng/μL of the dsDNA template, 0.5 mM ATP/CTP, 0.5 mM 4*UTP, 0.1 mM GTP, 0.5 mM of the dinucleotide cap analogue, 0.5 U/μL of the Ribolock ribonuclease inhibitor and 1 U/μL of the RNA SP6 polymerase (Thermo Fisher Scientific) or 2.5 U/μL of the RNA T7 High polymerase (NEB). The IVT reaction mixture was incubated for 1 h at 37°C for the SP6 polymerase or 2 - 4 h at 50°C for the T7 High polymerase, followed by addition of 0.025 U/μL DNaze I (Thermo Scientific) and incubated with the reaction mixture for 20 min at 37°C in order to remove the DNA template. The poliA tail was added to the transcripts in a reaction including the poliA buffer, 1 mM ATP, 0.1 U/μL of the poliA (NEB) polymerase and 0.2 U/μL of the Ribolock ribonuclease inhibitor (Thermo Scientific). The reaction was performed for 0.5 h at 37°C. In order to remove free phosphates from the obtained transcripts, the IVT were treated with alkaline phosphatase (FastAP) for 15 min. at 37°C. The reaction mixture contained the FastAP buffer, 0.033 U/μL FastAP and 0.5 U/μL of the Ribolock ribonuclease inhibitor (Thermo Scientific). The reaction mixture was purified using NucleoSpin® RNA Clean-Up (Macherey-Nagel) according to the instructions of the manufacturer. Transcript integrity was checked using non-denaturing 1% agarose gel and the concentration was determined using spectrophotometry. 2.2 Synthesis of short capped RNAs

The dsDNA template containing a sequence of the SP6 promotor or a sequence of the T7 promotor was prepared through hybridisation of the respective two complementary oligonucleotides. An oligonucleotide set with the SP6 G+1 promotor sequence: (SEQ. 1) i (SEQ.

2); an oligonucleotide set with the T7 A+1 promotor sequence: (SEQ.

3) (SEQ. 4) and an oligonucleotide set with the T7 G+1 promotor sequence: (SEQ. 5) (SEQ. 6). The hybridisation reaction was performed in a 10 mM Tris buffer pH 7.4 with added 1 mM MgCl ₂ , 100 mM NaCI and 25 |1M of the oligonucleotides, for 2 minutes at 95°C in a water bath, followed by cooling to 3O-35°C for 1 h and quickly down to 25°C. The thus prepared template was used in the IVT reaction with simultaneous capping (the cap:GTP ratio of 10:1) using compounds denoted as (II). The reaction was performed overnight at 37°C, in 20 pl containing: 200 x diluted dsDNA template, transcription buffer, 0.5 mM ATP/CTP/UTP, 0.125 mM GTP, 1.25 mM cap analogue with formula (II), 0.5 U/μL of the Ribolock ribonuclease inhibitor and 1 U/μL of the RNA SP6 polymerase (Thermo Fisher Scientific). The dsDNA template has been removed by 30 minute digestion using DNase I (Thermo Fisher Scientific) at 37°C. The obtained product was purified using a Oligo CleanUp & Concentration kit (Norgen Biotek), according to the instructions of the manufacturer. In order to obtain a product with a homogenous 3' terminus, the DNazyme was used (SEQ. 7). The reaction mixture containing 30 moles of DNazyme per 400 ng RNA in 50 mM Tris pH 7.4 and 50 mM MgCl ₂ was incubated for 1 h at 37°C. Next, the DNazyme was digested using DNase I (Thermo Fisher Scientific) for 30 minutes at 37°C, and the homogenous transcripts 24 or 25 nucleotides long and terminated with the respective cap analogue (no. 1 to 6) and (7, 8, 9, 12, 15), according to example 1, was purified using a Oligo CleanUp & Concentration kit (Norgen Biotek), according to the instructions of the manufacturer. RNA with any desired length may be obtained using a similar method.

2.3 Electrophoretic analysis of 24 or 25 nucleotide-long oligonucleotides capped with modified cap analogues

Oligonucleotides capped with compounds 1-6 and 7, 8, 9, 12, 15 were subjected to a 30-minute incubation with hNudt16 and separated into 15% polyacrylamide gel with added 7M urea, where C indicates a product with a cap which did not hydrolyse and D means an oligonucleotide without a cap. Oligonucleotides capped with compounds 1-6 were checked in a reaction with hDcp2. The uncapped product at time “0” is a result of incomplete capping during the IVT reaction; Fig. 6 presents the hydrolysis level of a cap incorporated into mRNA using hNudt16. The graphs presents the percentage share of mRNA which underwent cap hydrolysis during a 30-minute reaction with hNudt16. The hydrolysis level was calculated on the basis of densitometric analysis, as a percentage loss of the capped band after enzyme addition. The data presents average values ± SD from at least 3 independent experiments.

Example 3. Biological studies

3.1 Translation yield in RRL

Differently capped mRNA, according to example 2.1, were added to the reaction mixture of a cell-free translation system from rabbit reticulocyte lysate (RRL). The translation reaction was carried out for 60 minutes and its product, luciferase, was measured using luminometry, after adding a specific substrate (Table 1A, B). The study included three control mRNA capped with m ⁷ GpppG, m ^73O GpppG (ARCA 3’)

2 and ApppG. The translation yield of a transcript containing an ARCA cap was 1.47 times (for the first studied group, Table 1A) and 1.51 times (for the second studied group, Table 1B) higher than in the case of a transcript containing m ⁷ GpppG, as was expected and according to previously published literature data [5], On the other hand, the control mRNA capped with ApppG was translated at a very low level, which means that the conditions for monitoring the cap-dependent translation were selected correctly. Among the mRNAs tested in group one three transcripts with analogues 1, 3, 5 can be identified, which showed the highest translational yield, more than three times higher than that obtained for m ⁷ GpppG-RNA (Table 1A). The remaining three transcripts had a slightly lower translation yield, but it was still 2.37 to 2.88 times higher than the control m ⁷ GpppG-RNA (Table 1A). In the second group of the studied analogues, mRNA with analogues 12, 13, 14 were characterised by the highest translation yield, achieving results ca. 3.2 - 3.7 times higher than m ⁷ GpppG-mRNA (Table 1B). The mRNA with analogue 11 also achieved a very high translation level, ca. 2.9 times higher than m ⁷ GpppG- mRNA. The other mRNA with the following analogues 7, 8, 9, 10, 15 showed higher translation levels in the range from ca. 2.5 to 2.7 times higher than m ⁷ GpppG-mRNA (Table 1B).

3.2 Translation properties in HEK293T cells

The following section presents the results of studies on the translation efficiency of mRNA with incorporated, synthesised cap analogues defined by the formula (II) in a more complex environment, namely the HEK293 cells. mRNA with modified caps and a sequence coding firefly luciferase was obtained as described in the example 2.1.2 in an in vitro transcription reaction and transfected into HEK293 in identical amounts. The yield of translations of the modified mRNA was measured 6, 12, 24 and 48 hours after transfection as relative luciferase activity (Fig. 1A). The relative, total luciferase expression has been defined as the surface area under the curve and normalised to a value obtained for mRNA with the m ⁷ GpppG cap (Fig. 1B and table 1). The highest result for the total protein expression was achieved for 3 (increased by a factor of 3.09 ± 0.5), which was one of the three mRNA achieving the highest results in the RRL test. The RNA group capped with compounds 1, 2, 4, 5 achieved very similar results (2.2 ± 0.27, 2.36 ± 0.49, 2.51 ± 0.54 and 2.0 ± 0.28 respectively), the total protein expression in this group increased 2.0-2.5 times compared to the standard m ⁷ GpppG-RNA. The following results were obtained for cap analogues 4, 7, 8, 11, respectively: 1.88 ± 0.4, 1.66 ± 0.07, 1.6 ± 0.26, 1.39 ± 0.35 and 1.25 ± 0.3.

Table 1. Translation properties of mRNA capped with newly synthesised cap analogues in the RRL system and the HEK293 cells. The level of total luciferase expression in HEK293 cells was presented as the surface area under the curve normalised to the value obtained for mRNA capped with m ⁷ GpppG. The data presents average values ± SD from at least two independent experiments, each consisting of threereplicates.

3.3 Inhibitory potential

Next, the ability of cap analogues defined by formula (II) to inhibit cap-dependent translation in the RRL was determined. In order to obtain the IC50 value, the yield of ARCA-mRNA luciferase translation was measured in the presence of increasing concentrations of the given cap analogue.

The obtained values are summarised in Table 2. The best inhibiting properties were displayed by compounds 5 and 1 (IC50 0.57 and 0.6 respectively). Compounds 2 and 3 were slightly weaker inhibitors, with IC50 values equal to 0.887 and 0.977. The compounds 4 and 6 were the weakest inhibitors in this group, although with still 6.8 and 4.7 times stronger inhibition compared to the control m ⁷ GpppG (Fig. 2) Table 2. ARCA-mRNA luciferase translation inhibition in the RRL system.

3.1 Susceptibility of capped oligonucleotides to hydrolysis with Nudt16 and Dcp2

Next, the susceptibility of 24- or 25-nt long nucleotides containing modified caps to hydrolysis by the following human enzymes was evaluated: Nudt16 and Dcp2. Capped oligonucleotides (C) show slower migration on an RNA separation gel than uncapped products (D) (Fig. 3). Ca. 15-30% of uncapped product always remains at time 0, which is a result of incomplete capping during the IVT reaction, the yield of which is always lower than 100%. After 30 minutes of reaction, hDcp2 cleaved m ⁷ GDP from each of the studied RNAs with a 100% yield (Fig. 3B), whereas the level of cap hydrolysis using hNudt16 varied between the studied samples (Fig. 3A). In all the cases, replacing the GpppG cap with a different, studied cap analogue resulted in improved stability of the oligonucleotide (Fig. 3 and Table 3). The hydrolysis level for RNA with the analogue 4 (9.8 ± 5.0) and analogue 12 (3.0 ± 1.0) was comparable with the level achieved for transcripts with an ARCA cap (8.7 ± 4.6 and 6.35 ± 2.47), while in the case of analogues 1, 2, 3, 5, 6, 7, 8, 9 it varied between 11.5 ± 1.5 and 33.3 ± 12.4% (Fig. 6 and Table 3).

Table 3. Hydrolysis of a cap incorporated into mRNA using human Nudt16. The table presents the percentage of mRNA, which underwent cap hydrolysis during a 30-minute reaction with Nudt16, calculated on the basis of densitometric analysis as a percentage loss of the cap after enzyme addition. The data represents average values ± SD from at least 3 independent experiments.

The human Nudt16 enzyme displays specific, hydrolytic activity towards substrates including a nucleoside diphosphate connected to another X molecule. Previous studies of the Inventors proved that this enzyme preferentially hydrolyses GppG molecules. At higher concentrations, it can also hydrolyse GpppG substrates and GpppG connected to RNA chains, despite the fact that these are very unlikely to be its specific substrates. On the other hand, compounds with the same structure but methylated in the N7 position are very weakly hydrolysed or not hydrolysed at all by Nudt16 ([7], [8]).

RNA polymerases (SP6, T7) may initiate the transcription reaction in the presence of m ⁷ GpppG analogues and their derivatives both by an attack of the 3’-OH group of guanosine, as well as of m ⁷ G. This results in a mixture of transcripts with m ⁷ GpppG-RNA and Gpppm ⁷ G-RNA at the 5’ terminus. The latter product is inactive in terms of translation and significantly decreases the quantity of the obtained, heterologous protein from RNA preparation. The problem of inverse incorporation of cap analogues was solved as a result of chemical modification of the 3’-O or 2’-O position of m ⁷ G. The analogues containing such modifications are known as ARCA ( anti reverse cap analogue) and are incorporated only in the correct orientation.

Incubation of RNA capped with GpppG, m ⁷ GpppG and ARCA analogues with the Nudt16 enzyme showed significant differences in the quantities of the obtained product after hydrolysis of the triphosphate bond.

In the case of GpppG-RNA it was observed that the uncapped product comprised 86.2 ± 7.9% of the obtained reaction product. It should be noted here that observing 100% of reaction progress is difficult, as both the SP6 and the T7 polymerase, in addition to the main transcription process, also synthesises slight quantities of longer and shorter RNA as a result of adding a nucleotide or nucleotides not coded in the template at the 3’ terminus ([9]; [10]). These products are visible in the background both in the position of the capped and uncapped product and to a certain, slight degree, make the densitometric analysis more difficult.

Incubation of RNA capped with an ARCA analogue with the Nudt1 enzyme showed that the uncapped product comprised only 8.7 ± 4.6% of the reaction product. On the other hand, it was observed after incubation of m ⁷ GpppG-RNA with Nudt16 that the uncapped fraction comprises 45.7 ± 6.8% of the product. These results were in agreement with the expectations. Nudt16 is hydrolytically active towards unmethylated GpppG-RNA, which became almost completely uncapped. Because the ARCA analogue is incorporated in the correct orientation only, the unhydrolysed, methylated part of the analogue is available to the Nudt16 enzyme. This explains the very poor progress of the decapping reaction.

RNA capped with m ⁷ GpppG is a mixture of two products m ⁷ GpppG-RNA and Gpppm ⁷ G- RNA, only one of which is hydrolysed by Nudt16. It was thus observed that only half of the material in this sample underwent hydrolysis. This means that the Nudt16 enzyme is a tool, which allows evaluation of the presence of unmethylated RNA in the prepared preparations. Indirect conclusions regarding the orientation of the cap analogue incorporation can also be drawn from the obtained data.

It was also decided to explore the course of the hydrolysis reaction with the Nudt16 enzyme, run on RNA capped using the newly synthesised cap analogues 1-6. The compound 4 showed a very slow progress of the decapping reaction. The uncapped fraction comprised 9.8 ± 5.0%, which is comparable with the product quantity obtained for ARCA-RNA. The obtained result allows an assumption that the compound has been incorporated only in the correct orientation, similar to ARCA. Interesting results were obtained in the case of RNA capped with the compounds 1, 2, 3, 5 and 6. The decapping level for RNA containing those analogues varied between 21.2% ± 9.1 and 33.3% ± 12.4. This data indicates the positive influence of cap analogue modification on the incorporation into RNA by the used SP6 polymerase.

The second series of experiments explored the progress of hydrolysis reaction with the enzyme Nudt16 of RNA capped with the analogues : 4, 7, 8, 9, 12, 15. Among these transcripts, we observed a very slow progress of hydrolysis of analogues 15 and 12 . In the case of 15, this observation is related to the presence of S in the triphosphate bridge, which prevents the hydrolysis of the P-P bond. The trinucleotide 12 is incorporated only in one correct orientation and thus, no progress of the Nudt16 hydrolysis reaction has been observed. Compounds 8 and 4 are distinguished among the other, studied compounds, for which a much slower hydrolysis progress than in the case of m ⁷ GpppG is observed. The decapping level for these compounds varied between 11.5 and 13.5%, while in the case of ARCA it was 6.35%. Compounds 9 and 7 displayed a slightly slower progress of hydrolysis compared to m ⁷ GpppG. The decapping level was 17.8%, 29.6% compared to m ⁷ GpppG with the value of 49.6%.

The obtained, experimental data are in agreement with prior observations ([116]). The impact of the presence of various labelled (within the phosphate) modifications of nucleotides on the yield of their incorporation into RNA was examined. It was shown that incorporation of nucleotides containing modifications in the form of 2'-deoxy- and 2'-O-methyl- or N ² -methylguanosine into RNA is difficult in the case of the T7 RNA polymerase. In order to achieve it, the polymerase requires a point mutation at the Y639F position, often combined with an increased concentration of the modified analogue and a decreased concentration of unmodified NTPs. The point mutation Y639F loosens the substrate specificity of the T7 RNA polymerase. Tyr639 is located within the active centre and is able to detect incorrect geometry in the substrate structure.

Tyr639 thus plays a specialised role in controlling the correct structure of the incorporated substrate ([12]). Both Tyr639 and the nearest amino acids are strictly conserved in the sequence of both polymerases T7 and SP6 (Tyr639 is marked with an arrow, and the nearby fragment with a black frame in the figure in the annex). It is thus unsurprising that dinucleotide analogues containing 2'-deoxy and 2'- O-methyldeoxyrybose on one side are incorporated into the RNA chain only on the other side of the unmodified nucleobase. The described results also indicate that modifications of the N ² position have positive impact on the orientation of dinucleotide incorporation. In the case of some substituents, this modification is enough for the analogue to be incorporated in the correct orientation only, similar to ARCA. Bibliography:

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[2] Piecyk K, Davis RE, Jankowska-Anyszka M. “Synthesis of N ² -modified 7-methylguanosine 5’-monophosphates as nematode translation inhibitors” Bioorganic & Medicinal Chemistry 20 (2012) 4781-4789

[3] Piecyk K, Lukaszewicz M, Darzynkiewicz E, Jankowska-Anyszka M. “ Triazole-containing monophosphate mRNA cap analogs as effective translation inhibitors”, RNA 2014, 20:1539-1547

[4] Piecyk K, Niedzwiecka A, Ferenc-Mrozek A, Lukaszewicz M, Darzynkiewicz E, Jankowska-Anyszka M. ‘‘How to find the optimal partner — studies of snurportin 1 interactions with U snRNA 5' TMG-cap analogues containing modified 2-amino group of 7-methylguanosine”, Bioorganic & Medicinal Chemistry 23 (2015) 4660—4668

[5] Kocmik I., Piecyk K., Rudzinska M., Niedzwiecka A., Darzynkiewicz E., Grzela R., Jankowska- Anyszka M. “Modified ARCA analogs providing enhanced translational properties of capped mRNAs”, 2018 Cell Cycle 17(13):1624- 1636.

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[13] Kowalska J., Lewdorowicz M., Zuberek ]., Grudzien-Nogalska E, Bojarska E., Stepinski ]., Rhoads R.E., Darzynkiewicz E, Davis R.E., Jemielity J„ “Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bifid tightly to elF4E and are resistant to the decapping pyrophosphatase DcpS” RNA. 2008 Jun; 14(6); 1119-1131 List of sequences

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