Dinucleotide monophosphate derivative, the nucleic acid derivative containing it, and the method of obtaining it

Abstract

Chemically modified 5' monophosphate oligonucleotides are disclosed, which have been used to precisely modify the 3' end of mRNA by enzymatic ligation. Also disclosed are methods for obtaining the modified oligonucleotides, methods for their enzymatic ligation to the 3' ends of mRNA, the use of the modified mRNAs in the context of their use in gene therapies, and their other potential applications.

Description

[0001] Dimicleotide monophosphate derivative, the nucleic acid derivative containing it, and the method of obtaining it

[0002] Summary of the invention

[0003] The invention relates to chemically modified 5' oligonucleotide monophosphates that have been employed to achieve precise modification of the 3' end of mRNA through enzymatic ligation. The main objects of the disclosed invention are messenger RNA (mRNA) molecules to the 3' end of which short oligonucleotide sequences containing modified subunits have been attached by enzymatic ligation to increase their stability and enhance their translational activity. Additionally, the methods for obtaining modified oligonucleotides, strategies for enzymatic ligation to the 3' ends of mRNAs, and the utilization of modified mRNAs in gene therapies and other potential applications are also disclosed.

[0004] Technical field

[0005] The presented invention relates to RNA molecules, methods for preparing chemically modified and stabilized mRNA molecules, the efficiency of translation in cells, and potential treatments based on mRNA technology.

[0006] Background art

[0007] One of the main obstacles to the development of synthetic mRNAs for therapeutic applications is their stability in the cellular environment. A multitude of chemical and structural modifications are being employed to enhance the efficiency of mRNA-based gene therapies. The objective of these modifications is to improve the stability and translational efficiency of mRNA molecules while modulating their immunogenicity, depending on the desired therapeutic outcome. The initial step in the complete enzymatic degradation of transcripts is deadenylation, which involves the removal of the poly(A) tail from the 3' end of cytoplasmic mRNA molecules. This process occurs via both the 3'— >5' and 5'— >3' pathways. This invention relates to methods for obtaining any RNA chain, primarily mRNA, containing modifications of the 3' end resulting in a significant increase in the resistance of ribonucleic acid to specific degradation by 3 '-exonucleases. The objective is to achieve the highest possible expression of therapeutic genes and proteins at the lowest possible dose of mRNA. The precursor RNA molecule can be efficiently obtained by chemical (i.e., solid-phase synthesis) or enzymatic methods (e.g., by in vitro transcription), depending on the desired RNA chain length. The solid-phase synthesis method enables the automated preparation of substantial quantities of oligoribonucleotide chains. The principal disadvantage of this method is that it enables preparing relatively short RNA sequences, with a maximum length of 60 nucleotides. It can thus be employed in the preparation of potential oligonucleotide therapeutics, but not protein-coding sequences. In contrast, in vitro transcription represents an effective approach for the synthesis of long RNA chains, including mRNA molecules. In vitro transcription results in small amounts of RNA, but theoretically with no upper limit on sequence length. Modification and stabilization of the RNA chain's 3' end can be achieved through enzymatic ligation with RNA Ligase 1 T4. The ligase's substrate specificity allows for the attachment of chemically modified oligonucleotides up to dozens of subunits to the 3' end of the precursor RNA.

[0008] The publication “Chemically Modified mocRNAs for Highly Efficient Protein Expression in Mammalian Cells”, ACS Chem. Biol. 2022, 17, 3352-3366 revealed the modification of the polyadenylated tail from the 3' end of mRNA using synthetic oligonucleotides attached to the mRNA chain by ligation. The ligation reaction was carried out in the presence of T4 RNA ligase I. Modifications ranged from three to six nucleotide residues, whereby the introduction of chemically modified oligonucleotides (ligation) at the 3'end of the RNA was found to protect poly(A) fragments from degradation, resulting in improved translation efficiency.

[0009] Concept of the invention

[0010] The aim of the invention is to propose a method and compounds for implementing the method which allow precise addition of modifications at the 3' end of RNA chains. It is particularly desirable that, due to a suitably designed structure of the 5'-phosphate oligonucleotides, uncontrolled polymerisation during ligation can be prevented. A particular aim of the invention is to propose mRNA modifications that would increase translation efficiency.

[0011] Unexpectedly, the stated goal has been achieved in the invention.

[0012] The object of the invention is a dinucleotide monophosphate derivative of Formula 1:

[0013]

[0014] Formula 1 wherein:

[0015] Z is a nucleotide base, independently selected from: A, G, C, U, T, hypoxanthine, xanthine, N7- methylguanine, N6-methyladenine, N2-methyladenine, dihydrouracil, 5-methylcytosine, 5- hydroxymethylcytosine, pseudouracil, methylpseudouracil, nicotinamide, but also 2-aminopurine, 2,6-diaminopurine, flavine, C8-substituted adenine, C8-substituted guanine, C7-substituted 7- deazaadenine, C7- substituted 7-deazaguanine, C5-substituted uracil, C5-substituted cytosine, di- i tricyclic nucleotide bases,

[0016] R1 is thiol, borane, selenol lub hydroxyl,

[0017] R2 is metyl, alkyl, metoxyl, alkoxyl, hydrogen atom, tert-butylodimethylosilyl, trisubstituted silyl group or together with a substituent bonded to the 4th carbon of pentose ring forms -CH2- group, or group -OR2 is replaced with fluorine atom, -SCH3 group or thioalkyl except compounds in which::

[0018] - both of Z is guanine, R1 is thiol or hydroxyl and R2 is a hydrogen atom,

[0019] - one of Z is guanine and the second Z is adenine, R1 is thiol or hydroxyl and R2 is a hydrogen atom,

[0020] - both of Z is adenine, R1 is thiol and R2 is a hydrogen atom. Preferably, the object of the invention is a dinucleotide monophosphate derivative of Formula 2:

[0021] Formula 2 wherein:

[0022] R1 is thiol or hydroxyl group,

[0023] R2 is methyl group.

[0024] Preferably, according to the invention, the dinucleotide monophosphate derivative was selected from among: compounds of Formula 1 wherein R1 is thiol group and R2 is methyl group or compounds of Formula 1 wherein R1 is hydroxyl group and R2 is methyl group or their isomers

[0025] Another object of the invention is a nucleic acid derivative containing at the 3' end a covalently attached dinucleotide monophosphate derivative according to the invention as defined above. Preferably, the nucleic acid is an RNA or DNA chain. Preferably, the nucleic acid is an mRNA containing a poly(A) tail and possibly a cap structure or analog thereof.

[0026] Another object of the invention is a method for obtaining a nucleic acid derivative according to the invention as defined above, characterized in that:

[0027] (a) A nucleic acid with a free hydroxyl group at the 3' end is contacted with a dinucleotide monophosphate derivative in accordance with the invention as previously defined. (b) An enzymatic ligation reaction is conducted in the presence of a ligase.

[0028] (c) The resulting nucleic acid derivative, which has been covalently attached to the dinucleotide monophosphate derivative in accordance with the invention as previously defined, is then isolated.

[0029] It is advantageous to employ a ligase selected from the following enzymes in step (b): bacteriophage RNA T4 ligase 1, bacteriophage RNA T4 ligase 2, or bacteriophage DNA T4 ligase.

[0030] Detailed description

[0031] In the absence of a contrary definition, all terms used in this document shall be understood to have the same meaning as would be commonly understood by a person with the requisite skill and knowledge in the field to which the present invention pertains.

[0032] As used herein, terms “nucleic acids” and ‘nucleic acid chain’ refer to a polymeric compound composed of ribo- or 2'-deoxyribose-derived naturally occurring or synthetic nucleosides containing any nucleoside bases. These terms include non-canonical bonds and any polymer length. The terms include, but are not limited to, RNA or DNA, whether synthetic or natural, as well as nucleic acids consisting of or containing phosphorothioate, borane-phosphate, selenophosphate and phosphonate groups, morpholino nucleic acids (MNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) and treose nucleic acid (TNA).

[0033] As used herein, terms “nucleoside base”, ‘nitrogenous base’ refer to a specific part of nucleic acid molecules. These nucleoside bases include, among others, natural or synthetic purines (adenine, guanine, hypoxanthine, xanthine, N7-methylguanine, N6-metyloadenine, N2-methyladenine) and pyrimidines (cytosine, thymine, uracil, dihydrouracil, 5 -methylcytosine, 5- hydroxymethylocytosine, pseudouracil, methylpseudouracil, nicotinamide), and also 2- aminopurine, 2, 6- diaminopurine, flavine, C8-substituted adenine, C8-substituted guanine, C7- substituted 7-deazaadenine, C7-substituted 7-deazaguanine, C5-substituted uracil, C5 -substituted cytosine, di- i tricyclic nucleoside bases. Nucleoside bases can be involved in base pairing, nitrogenous base layering interactions, and other inter- or intramolecular interactions.

[0034] As used herein, terms “nucleic acid sequence,” “nucleotide sequence,” “nucleoside sequence,” “nitrogenous base sequence” and “sequence” refer to, but are not limited to, the sequence of nitrogenous bases in a nucleic acid chain. As used herein, terms “nitrogenous base pair” and “base pairing” refer to the basic unit of doublestranded nucleic acids consisting of two nitrogenous bases linked by hydrogen bonds. The term “base pairing” refers to the specific hydrogen bonding between bases, both canonical (Watson- Crick base pairing) and non-canonical (Wobble base pairing, Hoogsten base pairing).

[0035] As used herein, term “inter-nucleotide bond” refers to a covalent or non-covalent bond or multiple bonds that connect two monomers of a nucleic acid chain.

[0036] As used herein, terms “modification,” “modified,” and “modifying group” in the context of nucleic acids refer to any molecule that can be attached to a nucleic acid structure. This includes, but is not limited to, groups attached to a sugar, a nitrogenous base, an inter-nucleotide phosphate or a 5'-5'-phosphate bridge of a nucleic acid molecule.

[0037] As used herein, terms “messenger RNA” and “mRNA” refer to a transcript generated from a DNA template and encoding a peptide or protein. Typically, mRNA contains a 5'-UTR (5' non- translational region), a protein-coding sequence and a 3'-UTR (3' non-translational region). The mRNA can be further stabilized by attaching a cap structure at the 5' end and by polyadenylation at the 3' end.

[0038] As used herein, term “transcription” refers to the enzymatic synthesis of RNA from a complementary DNA template. Transcription can occur in the cells of living organisms, in cell systems or in an artificial configuration (in vitro). Transcription is carried out by RNA polymerase in a DNA-dependent manner, in the presence of natural or modified nucleoside triphosphates. As a result of the transcription reaction, the DNA sequence is copied into RNA. Multiple RNA molecules can be formed from a single DNA template.

[0039] As used herein, term “cap structure” refers to a 7-methylguanosine molecule attached via a 5'-5'- triphosphate bridge to a nucleoside, oligonucleotide or nucleic acid chain.

[0040] As used herein, terms “cap analog” and “5'-cap analog” refer to any molecule that contains a natural or modified 5'-5'-oligophosphate connecting natural or modified nucleosides, nucleotides or nucleic acids. The term also refers to a canonical or non-canonical structure, including but not limited to cap-0, cap-1, cap-2, TMG-cap, NAD-cap, FAD-cap, as well as other cap structures modified within a ribose, nitrogenous base or phosphate bridge. In the context of the invention, the cap analog can be attached to the 5' end of the RNA to enhance transcript stability. The cap analogue can be incorporated into the RNA strand co-transcriptionally during transcription in vitro if the mentioned cap analogue is present in the reaction mixture. The 5'-cap can also be generated post-transcriptionally using capping enzymes, such as the capping enzymes of Vaccinia virus.

[0041] As used herein, term “polyadenylation” refers to the addition of a polyadenylation sequence to the 3' end of a primary RNA transcript. The poly (A) sequence consists of multiple adenosine monophosphates. In eukaryotic cells, polyadenylation is part of the pre-mRNA maturation process, and more broadly, the overall process of gene expression.

[0042] As used herein, terms “poly(A) tail,” “polyadenylated sequence” and “poly(A) sequence” refer to sequences of adenosine monophosphates typically present at the 3' end of mRNA. In the context of the invention, the poly(A) tail is attached during the in vitro transcription process based on a DNA template containing repeated thymine bases in a strand complementary to the coding strand. In the context of the invention, the terms “A nucleotides” or “A” refer to the adenine base.

[0043] As used herein, term “complementary” refers to a nucleic acid or nucleic acids that can undergo sequence-dependent hybridization and form a stable duplex.

[0044] As used herein, terms “in vitro translation,” “in vitro protein synthesis” and “in vitro RNA expression” refer to the enzymatic production of polypeptides and proteins based on an RNA template in artificial systems. In vitro protein production systems refer to reaction mixture, cell lysates, cell cultures and tissue cultures, among others.

[0045] As used herein, terms “in vivo translation,” “in vivo protein synthesis” and “in vivo RNA expression” refer to the process of enzymatic production of polypeptides and proteins based on an RNA template in natural systems. In vivo protein production systems include, but are not limited to, whole organisms, organs, cell and tissue transplants of human, animal, plant, fungal or bacterial origin.

[0046] As used herein, term “therapeutic protein” refers to a polypeptide that can be administered to a patient to alleviate, reverse, delay the onset or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein may also have prophylactic properties and may be administered to delay the onset of a disease or reduce the severity of such a disease or pathological condition. Therapeutic proteins include, but are not limited to, antigens, antibodies and immunogenic proteins that induce an immune response in a patient, as well as fluorescent proteins, reporter proteins, protein hormones, membrane proteins, transcription factors, regulatory proteins, gene editing systems and gene regulatory systems.

[0047] As used herein, term “therapeutic gene” refers to a nucleic acid (e.g., DNA, RNA, mRNA, plasmid vectors) containing an open reading frame in which the sequence of a therapeutic protein is encoded.

[0048] As used herein, terms “alcoholic precipitation of nucleic acids,” “nucleic acid precipitation” and “alcoholic precipitation” refer to the method of isolating nucleic acids. By alcohol precipitation, nucleic acids can be isolated from aqueous solutions and concentrated as a solid precipitate. Alcoholic precipitation is carried out by adding organic solvents (e.g. ethanol, isopropanol) and salts (e.g. sodium acetate, lithium chloride, ammonium acetate) to aqueous solutions of nucleic acids.

[0049] As used herein, terms “polyacrylamide gel electrophoresis” and “PAGE” refer to a method of separating biological macromolecules based on their electrophoretic mobility. In the context of PAGE performed under native conditions, electrophoretic mobility depends on the mass, conformation and charge of the macromolecule. Under denaturing conditions, electrophoretic mobility depends on the composition and mass of the macromolecule.

[0050] As used herein, terms “RP-HPLC” (reversed-phase high-performance liquid chromatography) and “HPLC” refer to an established method for the separation, identification, quantification and isolation of individual components of a mixture of substances. The HPLC method is widely used in biochemistry, analytical chemistry and chemistry. Basicly, an HPLC system consists of a pump with an eluent tank containing the mobile phase, a sample delivery system, a separation column containing the stationary phase and a detector. Sample components interact differently with the stationary and mobile phases through ionic and hydrophobic interactions. The components are characterized by their elution rate and can be collected separately for further use. HPLC analysis and purification of nucleic acids is typically carried out using a porous reversed-phase resin as the stationary phase and a mixture of aqueous and organic solutions as the mobile phase.

[0051] As used herein, term “ligase” refers to a nucleic acid-modifying enzyme that catalyzes the intramolecular and intermolecular formation of phosphodiester bonds between the 5'-phosphate end and the 3 '-hydroxyl end of a nucleic acid strand. In some cases, the ligase is capable of ligating the 3'-end of the acceptor polynucleotide to the 5'-end of the donor polynucleotide. A bond can also be formed between DNA, RNA, cDNA, PNA, LNA and / or other modified nucleoside molecules or any combination thereof. Ligase can mean, for example, one of the following enzymes: bacteriophage RNA ligase 1 T4, bacteriophage RNA ligase 2 T4 or bacteriophage DNA ligase T4.

[0052] The invention relates to chemically modified 5 '-monophosphate oligonucleotides that have been used to precisely modify the 3' end of RNA by enzymatic ligation. The invention in question was developed in the context of potential therapeutic applications of the aforementioned modified RNA molecules. Accordingly, in order to prove the feasibility of applications in gene therapies, the main objects of the disclosed invention are messenger RNA (mRNA) molecules, to the 3' end of which short oligonucleotide sequences containing modified nucleotide subunits were attached by means of enzymatic ligation [Fig. 1], The goal of such chemo-enzymatic modification is to achieve better stability and increased translational potential of mRNA molecules. To test the conditions of the ligation protocol and the applicability of the modification method on a broader set of RNA precursors, the modification was also carried out on an oligonucleotide analog of the poly(A) tail. The main mRNA precursor, i.e. the mRNA encoding the Gaussia luciferase reporter protein (GLuc), can be prepared by enzymatic synthesis using in vitro transcription. An oligonucleotide precursor consisting of 12 adenosine (Al 2) subunits can be prepared by solid-phase synthesis using commercially available phosphoramidites. Dinucleotide 5 '-monophosphates were also obtained by solid-phase synthesis. Chemical modifications in the form of a thiophosphodiester bond (PS) and methylation of the 2'-OH ribose group were introduced [Fig. 1 A], The dinucleotides used in the enzymatic ligation reactions had 2'-O-methyladenosine or 2'-O-methylguanosine as the terminal subunit at the 3' end, and the structures of some of them were further enriched with a thiophosphodi ester bond (pApAm and pApsAm isomer DI and D2, pGpGm and pGpsGm isomer DI and D2, respectively). Commercially available phosphoramidites of natural nucleosides and nucleosides with chemical modifications within the ribose were used in the synthesis process. Unnatural phosphodiester bonds were obtained by introducing an additional sulfurylation step in the synthetic cycle. Chemically modified dinucleotide 5 '-monophosphates are attached to the 3' end of RNA chains by enzymatic ligation involving RNA T4 Ligase 1 [Fig. IB], The abbreviated names of the mRNAs modified at the 3' end are shown in Table 1. The modified ribonucleic acids (A12-pApAm, A12-pApsAm D1 / D2, A12-pGpGm, A12-pGpsGm D1 / D2,A1, A2, A3, Gl, G2, G3) resulting from the ligation process are purified using high-performance liquid chromatography (HPLC) to obtain high-purity products and, in the case of mRNA products, to reduce their immunogenicity.

[0053] The exonucleolytic digestion experiment can be used to distinguish between modified and unmodified RNA molecules [Fig. 2], The invention encompasses protecting potential mRNA- based therapeutics from deadenylation in the cellular environment. Therefore, digestion experiments involving a specific enzyme, i.e., CN0T7 deadenylase, were performed. CN0T7 is the catalytic subunit of the CCR4-Not protein complex responsible for removing the poly(A) tails of cytoplasmic mRNAs. The enzymatic degradation experiment can be used to confirm the efficiency of the ligation process and the resistance of the modified mRNA molecules to enzymatic removal of their poly(A) tails. The 3'-modified mRNA molecules showed significantly enhanced enzymatic stability and resistance to deadenylation and remained full length throughout the digestion experiment. At the same time, the unmodified control mRNA was shortened by the length of the poly(A) tail in less than one hour. Similar results can be observed for all 3'-modified oligonucleotides, which remained stable under experimental conditions, while the unmodified Al 2 molecule was completely degraded by CN0T7 deadenylase activity. The electrophoretic mobility of nucleic acids is mainly affected by their size, as well as their conformation and charge. Using polyacrylamide gel electrophoresis under denaturing conditions, it is possible to separate by mass and identify RNA molecules that remain full length or become shortened after 3' exonucleolytic digestion by CN0T7.

[0054] To investigate the translational potential of modified mRNAs, protein expression experiments can be performed in vitro, both in cell lysates and cell lines, as well as in vivo experiments in more complex organs and model organisms. Experiments conducted using cell lysate systems, such as rabbit reticulocyte lysates (RRL system), can allow direct determination of the translation efficiency of synthetic mRNAs under specific experimental conditions. However, the RRL system lacks the metabolic and structural complexity of living cell lines and organisms. Therefore, translation efficiency results obtained from the RRL system can be considered as preliminary indicators of the potential usefulness of a given synthetic mRNA in the field of gene regulation, vaccine development, regenerative medicine, etc. In the RRL system, the 3'-modified mRNA translated at a level similar to or slightly higher than the unmodified control mRNA [Fig. 3], Due to the short duration of the experiment (1-2 hours) and the simplified nature of the RRL system, the increased enzymatic stability of the modified transcripts translates little into their overall translational potential. Instead, it can be said with a high degree of confidence that the presence of chemically modified subunits at the 3' end of mRNA molecules does not interfere with their interaction with the translational machinery present in the RRL system.

[0055] More complex protein expression experiments can be performed on cell lines. However, it is important to control for significant differences in translation mechanisms, mRNA metabolism and transfection efficiency occurring in different cell lines. To this end, the modified mRNA can be transfected into cells of several lines differing in morphology and origin. To test the applicability and, to a limited extent, the versatility of the invention, the 3 '-modified mRNA was transfected into three different cell lines (A549, JAWSS II, HEK293) [Fig. 4], The Gaussia luciferase protein is excreted from the cells into the cell culture medium, allowing accurate determination of protein production within days without the need for cell lysis. This is particularly beneficial for testing the stability and viability of mRNA-based therapeutics. Depending on the cell line, the amount of reporter protein produced by expression of modified mRNA was at levels up to 2.5 times higher than for unmodified mRNA. In addition, in some cases the modified mRNA showed longer survival compared to the control mRNA, with noticeable amounts of protein produced at the time point when expression derived from the unmodified mRNA was almost completely stopped. The results indicate that the introduction of short chemically modified sequences at the 3' end of the mRNA by enzymatic ligation is beneficial for the delivery of therapeutic genes and proteins.

[0056] Figures and tables and their brief descriptions

[0057] In order to better explain the essence of the invention, its exemplary realizations are illustrated by the following figures:

[0058] Table 1 shows the full names and their abbreviated forms corresponding to each mRNA modified at the 3' end.

[0059] Figure 1 shows the strategy proposed according to the invention for the modification of the 3' end and enzymatic evaluation of the mRNA encoding Gaussia luciferase (GLuc). (A) 5'- phosphorylated dinucleotides containing chemical modifications to increase stability and reduce susceptibility to enzymatic degradation. (B) Schematic showing the ligation protocol for attaching chemically modified dinucleotides to the 3' end of GLuc mRNA, followed by evaluation of ligation efficiency through degradation experiments with CN0T7 and PAGE analysis.

[0060] Figure 2 shows the results of PAGE analysis of modified Gaussia luciferase (GLuc) mRNA molecules, along with unmodified control GLuc mRNA, after an enzymatic degradation experiment with CN0T7. All modified mRNAs remained stable and full-length after incubation with CN0T7, while unmodified GLuc was shortened by 180 nucleotides after removal of the poly(A) tail. PAGE conditions: 7% PAA, AA:MBAA 19: 1 , 7 M urea, 1 x TBE. After analysis, the gel was stained with SYBR Gold.

[0061] Figure 3 shows the results of the translation efficiency assay of 3'-modified Gaussia luciferase mRNA molecules in rabbit reticulocyte lysates (RRL). Translation efficiency values were calculated based on the linear dependence of luminescence on mRNA concentration. Data are presented as mean ±SD, normalized to the value calculated for the unmodified control.

[0062] Figure 4 shows the results of the translation efficiency assay of 3'-modified Gaussia luciferase mRNA molecules in cell lines: (A) A549; (B) JAWS II; (C) HEK 293. Left: Gaussia luciferase activity in cell culture medium at 16, 40, 64 and 80 hours after transfection. Data presented as mean ± SD values obtained from 3 independent experiments, each consisting of 3 independent transfections. Right side: total expression of Gaussia luciferase for 4 days. Total expression values obtained for modified mRNAs were normalized to the value corresponding to the unmodified control mRNA. Data are presented as mean ± SD values.

[0063] Figure 5 shows the HR spectrum of MS (-) ESI obtained for the pApAm compound.

[0064] Figure 6 shows the spectrum obtained for the DI and D2 isomers of the pApsAm compound.

[0065] Figure 7 shows the HR MS (-) ESI spectrum obtained for the compound pGpGm.

[0066] Figure 8 shows the HR MS (-) ESI spectrum obtained for the compound pGpsGm.

[0067] Figure 9 shows the spectrum obtained for the compound pApATBDMS

[0068] Figure 10 shows the result of electrophoretic analysis of the ligation products obtained in Example 5. Example 1. Preparation of dinucleotide monophosphate derivatives according to the invention.

[0069] Solid phase synthesis

[0070] Solid-phase oligonucleotide syntheses were performed on an AKTA Oligopilot plus 10 instrument (GE Healthcare) using a PrimerSupport™ column packed with solid support suspended in anhydrous acetonitrile. In the coupling process, an acetonitrile solution containing a 5 -fold molar excess of phosphoramidite relative to the solid support occupancy was recirculated through the column for 15 minutes. Sulfurization of the phosphate group of selected subunits was carried out in a 0.05M solution of EDITH (3-ethoxy-l,2,4,-dithiazolidin-5-one) in acetonitrile. A 3% solution (v / v) of dichloroacetic acid in toluene was used for detritylation, a 0.05M solution of iodine in pyridine for oxidation, a 20% solution (v / v) of 1 -methylimidazole in acetonitrile was used as CapA and a 40% (v / v) solution of acetic anhydride and 40% (v / v) pyridine as CapB. After the last synthesis cycle, the oligonucleotides attached to the support were washed with a 20% (v / v) solution of diethylamine in acetonitrile to remove the 2-cyanoethyl protecting groups. Finally, the deposits were washed with anhydrous acetonitrile and dried with argon.

[0071] All oligonucleotides were cleaved from the support, with simultaneous removal of nitrogenous base protecting groups, using a 1: 1 (v / v) solution of ammonia (30%) and methylamine (40%). After a 1-hour incubation at 50 °C with simultaneous shaking (350 rpm), the solution was filtered through a syringe filter to remove solid support. The support was washed twice with water to wash out the synthesis products. Excess ammonia was removed from the solution by evaporation under reduced pressure. The solution was then dried by freeze-drying. The 2'-O-TBDMS protecting group was removed by dissolving the oligonucleotides in DMSO with triethylamine trihydrofluoride and incubating at 65 °C for 3 hours. Then 3M sodium acetate solution and 1- butanol were added and the mixture was cooled at -18 °C for 30 minutes. The precipitate in the form of the sodium salt of the oligonucleotide was then centrifuged, washed with a 70% ethanol solution and freeze-dried after re-centrifugation.

[0072] An synthesis Ribo A300 CPG 1000 / 110 solid support (34 mg, 10 pmol) was placed in a synthesis column and suspended in acetonitrile. Phosphoramidite rA (Bz) CE (642 mg, 650 pmol) was dissolved in anhydrous acetonitrile to obtain a solution of 0.2 M. Synthesis, cleavage from solid support and purification of the oligonucleotide were carried out according to the general procedure. The deprotected oligonucleotide was purified using semi-preparative RP HPLC (method C). The collected eluate was lyophilized several times. 20 mg (5.2 pmol) of compound An was obtained as a white powder. pApAm synthesis

[0073] The solid support 2'-OAc CNA CPG 1000 / 110 (176 mg, 5 pmol) was placed in a synthesis column and suspended in acetonitrile. Phosphoramidite rA (Bz) CE (74 mg, 75 pmol) and Bis(2- cyanoethyl)-N,N-diisopropyl phosphoramidite (21 mg, 75 pmol) were dissolved in anhydrous acetonitrile to obtain solutions of 0.2 M. Synthesis, cleavage from solid support and purification of the oligonucleotide were carried out according to the general procedure. The deprotected oligonucleotide was purified using semi-preparative RP HPLC. The collected eluate was lyophilized several times. 2.1 mg (3.0 pmol) of the pApAmcompound was obtained as a white powder. The yield after purification on RP-HPLC was 60%. HR MS (-) ESI measured m / z (M-H)’ : 689.12535, calculated for C2iH27NioOi3P2’ 689.1240 [Fig. 5], pApsAm D1 / D2 synthesis

[0074] The solid support 2'-OAc CNA CPG 1000 / 110 (176 mg, 5 pmol) was placed in a synthesis column and suspended in acetonitrile. Phosphoramidite rA (Bz) CE (74 mg, 75 pmol) and Bis(2- cyanoethyl)-N,N-diisopropyl phosphoramidite (21 mg, 75 pmol) were dissolved in anhydrous acetonitrile to obtain solutions of 0.2 M. Synthesis, cleavage from solid support and purification of the oligonucleotide were carried out according to the general procedure. Sulfurization of the phosphate group was carried out by recirculating 0.05 M EDITH solution through the column for 10 minutes. The deprotected oligonucleotide was purified using semi-preparative RP HPLC. The collected eluate was lyophilized several times. 1.1 mg (1.5 pmol) of pApsAmDI compound and 1.4 mg (2.0 pmol) of pApsAmD2 compound were obtained as a white powder. The total yield after purification on RP-HPLC was 70%. HR MS (-) ESI measured m / z (M-H)': for pApsAmDI 705.10189, for pApsAmD2 705.10226 calculated for C21H27N10O12P2S- 705.1011 [Fig. 6], pGpGm synthesis

[0075] The solid support 2'0Me-G (dmf) RNA CPG 1000 / 110 (167 mg, 5 pmol) was placed in a synthesis column and suspended in acetonitrile. Phosphoramidite Ac-G-CE (70 mg, 75 pmol) and Bis(2- cyanoethyl)-N,N-diisopropyl phosphoramidite (21 mg, 75 pmol) were dissolved in anhydrous acetonitrile to obtain solutions of 0.2 M. Synthesis, cleavage from solid support and purification of the oligonucleotide were carried out according to the general procedure. The deprotected oligonucleotide was purified using semi-preparative RP HPLC. The collected eluate was lyophilized several times. 1.6 mg (2.2 pmol) of the pGpGm compound was obtained as a white powder. The yield after purification on RP-HPLC was 44%. HR MS (-) ESI measured m / z (M-H)’ : , calculated for C21H27N10O15P2- 721.1138 [Fig. 7], pGpsGm D1 / D2 synthesis

[0076] The solid support 2'0Me-G (dmf) RNA CPG 1000 / 110 (176 mg, 5 pmol) was placed in a synthesis column and suspended in acetonitrile. Phosphoramidite Ac-G-CE (70 mg, 75 pmol) and Bis(2- cyanoethyl)-N,N-diisopropyl phosphoramidite (21 mg, 75 pmol) were dissolved in anhydrous acetonitrile to obtain solutions of 0.2 M. Synthesis, cleavage from solid support and purification of the oligonucleotide were carried out according to the general procedure. Sulfurization of the phosphate group was carried out by recirculating 0.05 M EDITH solution through the column for 10 minutes. The deprotected oligonucleotide was purified using semi-preparative RP HPLC. The collected eluate was lyophilized several times. 0.7 mg (0.9 pmol) of pGpsGmDI compound and 0.9 mg (1.2 pmol) of pGpsGmD2 compound were obtained as a white powder. The total yield after purification on RP-HPLC was 31%. HR MS (-) ESI measured m / z (M-H)': for pGpsGmDI 737.09273, for pGpsGmD2 737.09284 calculated for C21H27N10O12P2S- 737.0909 [Fig. 8],

[0077] Example 2. Preparation of a nucleic acid derivative containing at the 3' end a covalently attached dinucleotide monophosphate derivative according to the invention.

[0078] In vitro transcription

[0079] The mRNA encoding Gaussia luciferase was obtained using plasmid pJET_T7_GLuc_31 A, which was previously linearized by digestion with the restriction enzyme Aarl (ThermoFisher Scientific). The plasmid was obtained by cloning the T7 promoter sequence and the Gaussia luciferase coding sequence into pJET_T7_Gluc_31A. The T7 promoter sequence was cloned and inserted, along with the Gaussia luciferase coding sequence, into pJET_luc_31A. The in vitro transcription reaction (120 pl) was incubated at 37 °C for 3 h in transcription buffer (40 mM Tris-HCl pH 7.9, 20 mM MgCb RNase inhibitor, 1 mM DTT, and 2 mM spermidine) with 0.125 mg / ml T7 RNA polymerase, 1 U / pl RiboLock, 4 mM ATP / UTP / CTP, 2 mM GTP, 4 mM m7GpppAmpG (cap 1 analog), 0.002 U / pl inorganic pyrophosphatase, and 40 ng / pl DNA template. After 3h of incubation, 1 U / pl DNase was added to the reaction mixture and incubation was continued for another 30 min at 37 °C. The reaction was then diluted 2x and mRNA was purified using Monarch RNA Cleanup Columns T2050 (New England BioLabs). Transcript quality was assessed on a native 1% agarose 1 x TBE gel, and sample concentration was determined spectrophotometrically.

[0080] Final purification on RP HPLC was performed on an Agilent Technologies Series 1200 HPLC instrument using an RNASep™ Prep - RNA column (ADS Biotec) at 55oC. A linear gradient of buffer B (0.1 M tri ethylammonium acetate pH 7.0 and 50% acetonitrile) from 20% to 25% in buffer A (0.1 M triethylammonium acetate pH 7.0) over 20 minutes at a flow rate of 0.9 ml / min was used. mRNA was then precipitated from the eluate using 1.2 volumes of isopropanol with 0.3 M NaOAc (pH 5.2) and 1 pl / ml glycogen. The mRNA pellet was washed twice with 70% ethanol. The mRNA pellet was then dried under reduced pressure and dissolved in RNase-free water. The concentration of the final sample was determined spectrophotometrically, and its quality was assessed on a 1% agarose gel in 1 x TBE.

[0081] Enzymatic ligation involving mRNA

[0082] Dinucleotide 5'-phosphates (pApAm, pApsAmD1 / D2, pGpGm, pGpsGmD1 / D2) were attached to the 3' end of Gaussia Luciferase mRNA using T4 RNA Ligase 1 using the following enzymatic ligation protocol: reaction mixture (5 pl) containing 0.5 pl of lOx concentrated buffer (final buffer concentration: 50 mM Tris / HCl, 10 mM MgCb, 1 mMDTT, pH 7. 5), 0.5 pl DMSO, 1 mM ATP, 1.2 U / pl RiboLock RNase inhibitor, 2U / pl T4 RNA Ligase 1, 15% PEG800, 1.5 pg purified mRNA and 81 pM selected dinucleotide, incubated for Ih at 24 °C. The reaction was stopped by 10-fold dilution with water, and mRNA products were purified using Monarch RNA Cleanup Columns T2030 (New England BioLabs). The quality of the modified mRNA was assessed on a native 1% agarose l x Tris-Borate-EDTA (TBE) gel, and sample concentration was determined spectrophotometrically. Yields after ligation and purification were typically -70%.

[0083] Example 3. In-vitro stability of nucleic acid derivatives containing a dinucleotide monophosphate derivative covalently attached at the 3' end according to the invention.

[0084] Enzymatic digestion involving CNOT7 deadenylase

[0085] The degradation reaction of the poly(A) tail (deadenylation) of Gaussia luciferase mRNA was carried out according to the following protocol: the reaction mixture (5 pl) in buffer (20 mM Tris, 20 mM DTT, 10 mM MgCh, 100 mM KC1, pH 8.0) containing 60 ng of the selected mRNA and 0.2 pg / pl of CNOT7 was incubated at 37 °C for 40 minutes. The reaction was stopped with EDTA (to a final concentration of 12 mM). The reaction products were separated electrophoretically on a 7% urea polyacrylamide gel. The results of the experiment are shown in Fig. 2.

[0086] The 3'-modified mRNA molecules showed significantly increased enzymatic stability and resistance to deadenylation and remained full length throughout the digestion experiment.

[0087] Example 4. Increased translational efficiency of nucleic acid derivatives containing a dinucleotide monophosphate derivative covalently attached at the 3' end according to the invention.

[0088] Studies of translation efficiency of 3'-modified Gaussia luciferase mRNA molecules in rabbit reticulocyte lysates (RRL)

[0089] Translation efficiency experiments were performed in an RRL system (Promega). 4 pl of RRL was diluted with 5 pl of buffer (40 pM amino acid mixture Acysteine, 40 pM amino acid mixture Amethionine, 2 mM MgCL, 380 mM KO Ac). The mixture of lysates was incubated in a 96- well plate for 1 hour at 30°C with gentle agitation (250 rpm), using 9 pL of RRL and buffer mixture per well. After incubation, 1 pl of Gaussian luciferase-encoding mRNA solution at the specified concentration (0.25, 0.125, 0.0625 or 0.0312 ng / pl) was added to each well. The reaction was incubated again for 1 hour at 30oC. The reaction was stopped by placing the plate at -80 °C. The plate was brought back to room temperature before measuring Gaussian luciferase activity. To detect the luminescence of the product of the luciferase-catalyzed reaction, 50 pl of a 10 ng / ml solution of h-coelenterazine (NanoLight) in PBS buffer was added to 10 pl of thawed translation mixture. Luminescence was measured using a Synergy Hl microplate reader (BioTek). Translation efficiency experiments were performed in triplicate for each mRNA, including a control mRNA lacking the 3' end modification. Each mRNA concentration point was also measured in triplicate.

[0090] Studies of translational potential in cell lines

[0091] Cells of the A549 (ATCC CCL-185) and HEK 293 (ATCC CRL-1573) lines were cultured in DMEM cell medium (Gibco) supplemented with 10% FBS (fetal bovine serum), GlutaMAX supplement (Gibco) and 1% penicillin and streptomycin, at 37 °C in an atmosphere of 5% CO2. On the other hand, cells of the JAWS II line (ATCC CRL-11904) were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS, sodium pyruvate (Gibco), 1% penicillin and streptomycin, and 5 ng / ml granulocyte-macrophage colony-stimulating factor GM-CSF (PeproTech), also at 37 °C in an atmosphere of 5% CO2. In all experiments, cells passaged 5-25 times were used. On the day of transfection, cells were seeded in 96-well plates at 104 cells per 100 pl of medium per well, without the addition of antibiotics. The transfection mixture consisted of 10 pl OptiMem medium (Gibco) supplemented with 5 ng / pl mRNA and 0.3 pl lipofectamine (MessengerMAX Transfection Reagent; Invitrogen). Transfection lasted 16 h. Subsequent time points were tested by taking the medium from above the cells in a given well and replacing it with fresh solution. The medium was collected every 24 hours for 4 days after transfection. Gaussian luciferase activity was tested by adding 50 pl of coelenterazine solution in PBS (10 ng / ml) to 10 pl of the medium in which transfected cells were cultured. The luminescence of the product of the luciferase-catalyzed reaction was then measured on a Synergy Hl microplate reader (BioTek).

[0092] The results of the experiment are shown in Fig. 3 and 4. The results indicate that the presence of chemically modified subunits at the 3' end of mRNA molecules does not interfere with their interaction with the translation machinery present in the RRL system. At the same time, it was found that the amount of reporter protein formed as a result of expression of mRNA modified according to the invention was at a level up to 2.5 times higher than that of unmodified mRNA.

[0093] The results obtained confirm that the introduction of short chemically modified sequences at the 3' end of mRNA by enzymatic ligation is beneficial for the delivery of therapeutic genes and proteins.

[0094] Example 5. Other variants of dinucleotide monophosphate derivatives according to the invention, their synthesis and properties.

[0095] The following derivatives were also obtained: pApAiBDMs: compound obtain as a mixture of 2’ and 3’ isomers (ratio 2:1) and pApA, purity of compound was determined by HPLC and it was 90%. HR-MS ESI: m / z calc, for C26H39N10O13P2S? [M-H] : 789.19480, found: 789.19499. Results are presented on Fig. 9. pAmpAm: 'H NMR (500 MHz, D2O, 25°C): d = 8.52 (s, 1H), 8.49 (s, 1H), 8.33 (s, 1H), 8.22 (s, 1H), 6.19 (d, VH,H=4.6HZ, 1H), 6.09 (d, VH,H=5.1HZ, 1H), 4.93 (m, 1H), 4.68 (m, 1H), 4.48 (m, 1H), 4.46 (m, 1H), 4.44 (m, 1H), 4.40 (m, 1H), 4.25 (ddd, VH,H=11 6Hz, VH_H=3.8HZ, VH_H=2.6HZ, 1H), 4.19 (ddd, VH,H=11.7HZ, VH,H=3.9HZ, VH,H=3.9HZ, 1H), 4.12 (m, 2H), 3.50 (s, 3H), 3.46 (s, 3H) ppm;31P NMR (202 MHz, D2O, 25°C): d = 1.04 (s, IP), -0.12 (s, IP) ppm. HR-MS ESI: m / z calc, for C22H29NIOOI3P2’ [M-H] : 703.13963, found: 703.13989. pCpAm: 'H NMR (500 MHz, D2O, 25°C): d = 8.51 (s, 1H), 8.32 (s, 1H), 8.03 (d, VH_H=7.8HZ, 1H), 6.22 (d, VH_H=4.6HZ, 1H), 6.18 (d, VH,H=7.8Hz, 1H), 5.84 (d, VH,H=4.8HZ, 1H), 4.69 (m, 1H), 4.60 (m, 1H), 4.47 (m, 1H), 4.38 (m, 2H), 4.32 (m, 1H), 4.24 (m, 1H), 4.15 (m, 1H), 4.11 (ddd,2JH,H=11.8Hz, VH_H=4.3HZ, VH,H=2.2HZ, 1H), 4.06 (ddd,2JH.H= 1 1.8HZ, VH,H=4.3HZ, VH_H=2.2HZ, 1H), 3.50 (s, 3H) ppm;31P NMR (202 MHz, D2O, 25°C): d = 0.93 (s, IP), 0.09 (s, IP) ppm. HR-MS ESI: m / z calc, for C2oH27N8Oi4P2’ [M-H] : 665.11274, found: 665.11312. pGpAm: 'H NMR (500 MHz, D2O, 25°C): d = 8.53 (s, 1H), 8.39 (s, 1H), 8.18 (s, 1H), 6.21 (d, VH.HM.4HZ, 1H), 5.79 (d, VH_H=4.6HZ, 1H), 4.76 (m, 1H, overlapped with HDO), 4.73 (m, 1H), 4.69 (m, 1H), 4.44 (m, 2H), 4.39 (m, 1H), 4.32 (m, 1H), 4.19 (m, 1H), 4.16 (m, 1H), 4.10 (ddd, VH,H=11.8HZ, VH_H=4.7HZ, VH,H=3.2HZ, 1H), 3.51 (s, 3H) ppm;31P NMR (202 MHz, D2O, 25°C): d = 1.09 (s, IP), 0.12 (s, IP) ppm. HR-MS ESI: m / z calc, for C2iH27NioOi4P2- [M-H] : 705.11889, found: 705.11937. pApAF: 'H NMR (500 MHz, D2O, 25°C): d = 8.48 (s, 1H), 8.36 (s, 1H), 8.32 (s, 1H), 8.20 (s, 1H), 6.38 (d, VH.FM7.0HZ, 1H), 5.99 (d, VH,H=4.5HZ, 1H), 5.36 (dd, VH,F=52.0HZ, VH_H=4.2HZ, 1H), 4.79 (m, 1H, overlapped with HDO), 4.77 (m, 1H, overlapped with HDO), 4.74 (m, 1H), 4.51 (m, 1H), 4.46-4.38 (m, 2H), 4.22 (m, 1H), 4.17 (ddd, VH,H=11.4HZ, VH_H=4.0HZ, VH,H=2.4HZ, 1H), 4.12 (m, 1H) ppm;31P NMR (202 MHz, D2O, 25°C): d = 1.09 (s, IP), 0.12 (s, IP) ppm. HR-MS ESI: m / z calc, for C2oH24FNioOi2P2’ [M-H] : 677.10399, found: 677.10429. pApALNA: ‘HNMR (500 MHz, D2O, 25°C): d = 8.51 (s, 1H), 8.30 (s, 1H), 8.14 (s, 1H), 8.13 (s, 1H), 6.06 (d, VH,H=3.9HZ, 1H), 5.99 (s, 1H), 4.92 (m, 1H), 4.87 (m, 1H), 4.61 (s, 1H), 4.58 (m, 1H), 4.53 (s, 1H), 4.40 (dd,2JH,H=12.4HZ, VH,H=5.1HZ, 1H), 4.36 (dd, VH,H=12.4HZ, VH,H=4.3HZ, 1H), 4.25 (m, 1H), 4.16 (m, 1H), 4.13 (d,2JH,H=8.5HZ, 1H), 4.08 (d,2JH,H=8.5HZ, 1H) ppm;31P NMR (202 MHz, D2O, 25°C): d = 1.07 (s, IP), 0.31 (s, IP) ppm. HR- MS ESI: m / z calc, for C21H25N10O13P2" [M-H]": 687.10833, found: 687.10867. pApAsMe: ’HNMR (500 MHz, D2O, 25°C): d = 8.55 (s, 1H), 8.51 (s, 1H), 8.33 (s, 1H), 8.23 (s, 1H), 6.21 (d, VH,H=8.1HZ, 1H), 6.04 (d, VH,H=5.2HZ, 1H), 4.82 (m, 1H, overlapped with HDO), 4.77 (m, 1H, overlapped with HDO), 4.69 (dd, VH,H=5.5HZ, VH,H=2.4HZ, 1H), 4.52 (m, 1H), 4.44 (m, 1H), 4.29 (ddd,2JH,H=H .4HZ, VH,H=3.5HZ, VH,H=3.5HZ, 1H), 4.21 (ddd,2JH,H=11.4HZ, VH.HM.1HZ, VH,H=4.1HZ, 1H), 4.14 (m, 2H), 4.02 (dd, VH,H=8.1HZ, VH,H=5.5HZ, 1H), 1.96 (s, 3H) ppm;31P NMR (202 MHz, D20, 25°C): d = 1.07 (s, IP), 0.18 (s, IP) ppm. HR-MS ESI: m / z calc, for C21H27N10O12P2S- [M-H]": 705.10113, found: 705.10179.

[0096] General procedure of synthesis

[0097] Synthesis of dinucleotides was performed using AKTA Oligopilot plus 10 synthesizer (GE Healthcare) on Unylinker PrimerSupport 5G (352 pmol / g) solid support in 2 step synthesis. In the each coupling step, 5.0 equivalents of appropriate 5'-O-DMT-3'-O-phosphoramidite (1st step: AmBz, AFBz, ALNABz or ASMedmf; 2nd step: AmBz, CPivOMAc, GPivOMipr-PAC or APivOMPAC) or biscyanoethyl phosphoramidite and 0.30 M 5-(benzylthio)-l-H-tetrazole in acetonitrile were recirculated through the column for 15 min. Synthesis of pApATBDMS was performed on 5'-O-DMT-2'-O-TBDMS-rABz-3’ PrimerSupport 5G (299 pmol / g) using 5.0 equivalents of 5'-O-DMT-2'-O-PivOM-6-N-PAC-adenosine-3'-O-phosphoramidite. A solution of 3% (v / v) dichloroacetic acid in toluene was used as a detritilation reagent and 0.05 M iodine in pyridine for oxidatition. After the last cycle of synthesis, dinucleotides, still on the solid support, were treated with 20% (v / v) diethylamine in acetonitrile to remove 2-cyanoethyl protecting groups. Finally, the solid support was washed with acetonitrile and dried with argon. The product was cleaved from the solid support and deprotected with AMA (methylamine / ammonium hydroxide 1:1 v / v; 35 °C, 24 h), evaporated to remove all volatile amines then isolated by ion-exchange chromatography on DEAE Sephadex (gradient elution 0-0.9 M TEAB) to afford after evaporation triethylammonium salt of dinucleotide. The final compounds were additionally purified using semi-preparative RP-HPLC using a Gemini (Phenomenex) NX-C18 RP HPLC column (150 x 10 mm, 5 pm, 110 A, flow rate 5.0 ml / min) with UV detection at 254 nm and isolated from the eluate by repeated freeze-drying. The scales and yields for particular dinucleotides are summarized in Table 1.

[0098] Table 1. Results of dinucleotides synthesis.

[0099] Ligation

[0100] Enzymatic ligation reactions of modified dinucleotide 5'-phosphates (pApAtNA, pGpAm, pApAsMe, pAmpAm, pCpAm, pApAiBDMs) to model RNA (Cap-1-RNA35) were carried out with the T4 RNA Ligase 1 enzyme and the solutions provided by the manufacturer (ThermoFisher Scientific). Cap-1-RNA35 (3.2 pl, 9 pM) was transferred to an Eppendorf tube. 10x Tris-HCl buffer (1.0 pl, 50 mM Tris / HCl, 10 mM MgCb, 1 mM DTT, pH 7.5) was added and mixed thoroughly using an automatic pipette. To the mixture was added 1 pl of DMSO, an aqueous solution of ATP (1.0 pl, 1 mM), an aqueous solution of DTT (0.33 pl, 3.3 mM), an aqueous solution of the corresponding dinucleotide 5 '-monophosphate (2.1 pl, 9 mM), 1.2 U / pl of RNase inhibitor RiboLock, 1 U / pl of T4 RNA Ligase 1 and H2O MQ to the final volume of the mixture (10 pl). The tube with the contents was incubated for 16 h at 20 °C. The reaction was terminated by a 5-fold dilution ofeoMQ. The product was isolated using the Monarch® RNA Cleanup Kit (New England BioLabs), and dinucleotide attachment to the RNA was verified by electrophoretic analysis on a 15% polyacrylamide gel (PAA / TBE, 50 min, 13 W). RNA was stained with SybrGold dye and visualized on a GelDoc GO Imaging System (Fig.10). The concentration of RNA in the sample was determined spectrophotometrically. Fig.10 shows the result of electrophoretic analysis.

[0101] Summary

[0102] The dinucleotide 5 '-monophosphates (pApAtNA, pGpAm, pApAsMe, pAmpAm, pCpAm, pApAiBDMs) obtained by solid-state synthesis were tested for their suitability for precise modification of the 3' end of RNA by enzymatic ligation reactions. The use of short oligonucleotide sequences for this purpose represents a potentially simple and versatile approach for efficiently making point modifications to ribonucleic acid chains, regardless of their sequence or length. Such modifications could be crucial in the creation of new molecular tools, studies of gene expression regulation, or the development of novel therapeutic approaches. In experiments designed to verify the ability to incorporate nucleotide subunits into RNA strands, a series of reactions involving the enzyme T4 RNA Ligase 1 and a short fragment of capped RNA (Cap-1- RNA35) were carried out. The reactions were carried out under conditions that guaranteed the stability of the oligonucleotides, while maintaining high enzyme activity. The final product was isolated each time using commercially available RNA cleanup kits (Monarch® RNA Cleanup Kit, New England BioLabs) and then performing electrophoretic analysis on a 15% polyacrylamide gel, thus verifying the attachment efficiency. For each of the analogs tested, attachment of a single dinucleotide to the 3' end of the RNA was observed, as evidenced by the appearance of a slower migrating band on the gel and the disappearance or low intensity of the band originating from the oligonucleotide substrate.

Claims

Claims1. Dinucleotide monophosphate derivative of formula 1 :Formula 1 wherein:Z is a nucleotide base, independently selected from: A, G, C, U, T, hypoxanthine, xanthine, N7- methylguanine, N6-methyladenine, N2-methyladenine, dihydrouracil, 5-methylcytosine, 5- hydroxymethylcytosine, pseudouracil, methylpseudouracil, nicotinamide, but also 2-aminopurine, 2,6-diaminopurine, flavine, C8-substituted adenine, C8-substituted guanine, C7-substituted 7- deazaadenine, C7- substituted 7-deazaguanine, C5-substituted uracil, C5-substituted cytosine, di- i tricyclic nucleotide bases,R1 is thiol, borane, selenol lub hydroxyl,R2 is metyl, alkyl, metoxyl, alkoxyl, hydrogen atom, tert-butylodimethylosilyl, trisubstituted silyl group or together with a substituent bonded to the 4th carbon of pentose ring forms -CH2- group, or group -OR2 is replaced with fluorine atom, -SCH3 group or thioalkyl except compounds in which:- both of Z is guanine, R1 is thiol or hydroxyl and R2 is a hydrogen atom,- one of Z is guanine and the second Z is adenine, R1 is thiol or hydroxyl and R2 is a hydrogen atom,- both of Z is adenine, R1 is thiol and R2 is a hydrogen atom.

2. Dinucleotide monophosphate derivative of Formula 2:Formula 2 wherein:R1 is thiol group of hydroxyl group,R2 is methyl group.

3. Dinucleotide monophosphate derivative according to claim. 2, characterized in that it is selected from: the compound of formula 2 where R1 denotes a thiol group and R2 denotes a methyl group or the compound of formula 2 where R1 denotes a hydroxyl group and R2 denotes a methyl group or their isomers.

4. A nucleic acid derivative containing at the 3' end a covalently attached dinucleotide monophosphate derivative as defined in claim 1-3.

5. Nucleic acid derivative according to claim. 4, characterized in that the nucleic acid is an RNA or DNA chain.

6. Nucleic acid derivative according to claim. 4, characterized in that the nucleic acid is an mRNA containing a poly(A) tail and possibly a cap structure or analog thereof.

7. Method for obtaining a nucleic acid derivative as defined in claim. 4, characterized in that:(a) a nucleic acid having a free OH group at the 3 'end is contacted with a dinucleotide monophosphate derivative as defined in claims 1-3,(b) an enzymatic ligation reaction is carried out in the presence of a ligase,(c) the obtained nucleic acid derivative having at the 3 'end a covalently attached dinucleotide monophosphate derivative as defined in claim 1-3 is isolated.

8. Method according to claim 7, characterized in that step (b) uses a ligase selected from the following enzymes: bacteriophage RNA ligase 1 T4, bacteriophage RNA ligase 2 T4 or bacteriophage DNA ligase T4.

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