Synthesis of long RNA oligonucleotides using dimer nucleoside phosphoramidites

Abstract

The present invention provides methods for the preparation of homo and hetero dimer nucleotides and their use in the synthesis of long RNA oligomers (>150 nucleotides). All 16 combination of dimer nucleoside amidite block were used to achieve the synthesis of Long RNA. Each Dimer was linked by a phosphodiester bond. The synthesis of dimer nucleosides amidite blocks involves several key steps. Suitable nucleosides were selected based on the desired RNA sequence. Then, each nucleotide is chemically activated by Ethyl thio tetrazole to facilitate coupling with another nucleoside under controlled conditions to form the homo-dimer nucleoside. The coupling reaction results in the formation of a stable phosphodiester linkage between the two nucleosides, ensuring integrity during RNA synthesis. Phosphite triester linkage was oxidized to a stable phosphodiester bond using iodine in water or pyridine. Synthesized dimer block was purified using chromatography techniques to ensure high purity. This method significantly enhances the efficiency and accuracy of RNA synthesis, reduces the number of coupling steps, and improves the overall yield of the desired RNA product.

Description

[0001] Title of the Invention

[0002] Synthesis of Long RNA Oligonucleotides Using Dimer Nucleoside Phosphoramidites

[0003] Field of the Invention

[0004] The present invention relates to the field of nucleic acid chemistry and oligonucleotide synthesis. More particularly, the invention pertains to methods for the synthesis of long chain RNA oligonucleotides using homo- and hetero-dimer nucleoside phosphoramidite blocks, and to the dimer intermediates useful therein.

[0005] This invention relates to the field of nucleic acid synthesis, specifically to the preparation of homo- and hetero-dimer nucleoside amidite blocks and their use in the efficient synthesis of long RNA oligomers (>150 nucleotides) in 3 ’-5 ’direction. This invention is directed using experimental conditions that are adaptable for the synthesis of long RNA oligomers from about 100 to about 200 Mers.

[0006] Background of the Invention

[0007] Chemical synthesis of RNA oligonucleotides is a critical enabling technology for molecular biology, diagnostics, functional genomics, and therapeutic applications such as siRNA, sgRNA, mRNA vaccines, and antisense oligonucleotides. Conventional solid-phase RNA synthesis relies on stepwise coupling of monomeric nucleoside phosphoramidites. While this approach is effective for short oligonucleotides, it suffers from cumulative coupling inefficiencies, increased error rates, time-consuming and reduced overall yields as sequence length increases. The use of dimer nucleotide blocks offers a promising alternative to increase the efficiency and accuracy of RNA synthesis. This long synthetic oligonucleotide approach utilizes homo and hetero dimer nucleoside amidite.

[0008] Prior art:

[0009] 1. Jung et al. describes preparation of RNA via coupling reaction. Which is based on U. S. Apply No. 1. US2010-12775843. This publication describes coupling of nucleotide dimer or nucleotide trimer to a nucleoside attached to solid supports in order to synthesise SiRNA. Purity of SiRNAobtained was very low (75%) and coupling time of 10 minute is very long and is not practical for making good quality of oligonucleotides and such longer time invariably causes cleavage of oligonucleotide and DMT removal due to acidic nature of activation reagent. Further internucleotide protecting group was O -chlorophenyl which is very hard to remove selectively without causing damage to oligonucleotide.

[0010] 2. Jung et al. Preparation of oligoribonucleotide by solid phase synthesis method, which is based on U. S. Apply No. KR2007-115302. In this study the process involves coupling a solid support ribonucleoside single stranded nucleotide with ribonucleotide dimer or trimer, followed by stepwise coupling reaction and removal of a solid phase. This phosphoramidite method monomer and dimer phosphoramidite was used to prepare concentrated GFP-sense siRNA. This publication still has drawback which has described in prior art #1.

[0011] 3. Enya et al. chemical synthesis of diastereomeric diadenosine boranophosphates (ApbA) from 2’-0 (2-cyanoethoxymethyl) adenosine by the boranophoshotriester method. Bioorganic & Medicinal chemistry: (2008), 16(20), 9154-9160. In this study Diastereomerically pure diadenosine 3', 5' boranophosphates (ApbA) have been synthesized using the boranophosphotriester method from ribonucleosides. This study was directed towards making complexes with dimer blocks. There was no effort on attempt toward the long chain RNA Synthesis.

[0012] 4. Janczyk et al. A new and convenient approach for the preparation of β- cyanoethyl protected trinucleotide phosphoramidites published on Organic & Biomolecular Chemistry: (2012), 10(8), 1510-1513. The method describes preparation of fully protected trinucleotide synthons for gene library synthesis and carrying out standard oligonucleotide synthesis having β-cyanoethyl groups at the phosphate residues without additional steps for deprotection and workup. This study utilized 2 ’-deoxy nucleoside trimer blocks having cyanoethyl phosphate internucleotide protecting 2 group. Thus, this study differs significantly from our invention leading to various chain Lenth of clean and high purity of medium and long chain RNA.

[0013] 5. Mueller et al. Method for producing protected trinucleotide using selective protection and deprotection steps. Report on patentability on wo2011061114, which is based on Germany. Apply No. W02010-EP67313. This patent addresses only the synthesis of deoxy nucleoside trinucleotide block using dimers. This process has different approach in which base protected group was acyl, 5’ DMT protecting group and 3’-TBDMS protecting group were used. The trimers were fully characterized. However, no further studies were carried out towards long chain DNA oligonucleotides. The challenges come in the oligonucleotide synthesis where the target is medium or long chain RNA synthesis.

[0014] 6. Honke et al; Preparation of N- glycosyl acid amide amino acid-nucleic acid derivatives and method for producing them. Report on patentability on WO2014157434, Al, which is based onJapan. Apply No. WO2014-JP58711. This patent application addresses synthesis of amino acid conjugates which are limited to dimer synthesis only. There is no teaching in this publication how to make medium and long chain macromolecule. Further this application is quite distinct and from the medium and long chain RNAs of our invention.

[0015] 7. Sekine et al. Process for deblocking the 2’ -hydroxyl groups of ribonucleosides Report on patentability on W02006095739, Al, which in turn is based on Japan. Apply No. WO2006 JP304399. This patent shows the process involves synthesis of 2’-O-cyanoethyl protected nucleoside and deblocking 2'-O-cyanoethyl groups of ribonucleosides or oligoribonucleotides. This study utilized 2’ cyanoethyloxy protecting group for stepwise RNA synthesis Subsequently, the step of deprotection of 2’-O-cyanoethyl group was carried out preferably using tetraalkylammonoium fluoride (tetra n-butyl) ammonium fluoride. However, it seems there are issues in the deprotection of 2 ’-cyanoethyloxy group and overall yield was approximately 15% of a 30-mer RNA. Thus, this approach does not seem to be attractive and practical.

[0016] 8. Chiba et al. Alkoxy phenyl derivative, protected nucleoside, protected nucleotide, method for production oligonucleotide, and method for removing substituent. Report on patentability on JP2020011932, A, which in turn is based on Japan. Apply No. JP2018-136466. According to Chiba et al, the invention relates to an alkoxy phenyl derivative, which can be synthesized using a liquid phase method faster than conventional methods. It also includes a protected nucleoside and a nucleotide, a method for producing an oligonucleotide using the same, and a method for selectively removing the alkoxy phenyl derivative moiety. Mostly, this study oligonucleotide with lipophilic protecting group either with 3’- hydroxyl of sugar backbone or internucleotide phosphate in order to a impart overall lipophilic character to the synthesized oligonucleotide with the specific purpose of solution phase oligonucleotide synthesis. The process was directed only towards large scale short chain length RNA. This study is much different than from our approach of rapid synthesis of medium and long chain RNA. Authors also tried to synthesize morpholino oligonucleotide with the solution phase approach. However, this approach was not directed towards rapid synthesis of medium to long chain RNA’s using efficient technology as dimer blocks disclosed in our invention.

[0017] 9. Damha et al. Block synthesis of oligoribonucleotides. Report on patentability on WO2012024776, Al, which in turn is based on Canada. Apply No. W02011-CA950. This invention describes a method for synthesizing ribonucleotides blocks (dimer, trimer, tetramer etc.) for RNA synthesis using block coupling reactions. This patent utilizes 2'-TIPS and TBDMS protecting groups, which are known to undergo 2'-3' migration. The process involves the use of adenine (A), thymine (T), and uracil (U) as 3 the nucleobases, resulting in the formation of dimers and trimers. However, the overall yield of these oligonucleotides was reported to be less than 95%, with the yield of trimer amidites ranging from 67% to 88%. Oligonucleotides of lengths between 4 and 100 mers were successfully synthesized. Notably, the yield and purity of the synthesized oligonucleotides were not explicitly specified in the patent. The purity ofoligonucleotides in the range of 11-mer to 30-mer was reported to be between 44.5% and 30%, which is considered suboptimal and not practical to purify full length RNA.

[0018] 10. Dubensky et al. Compositions and method for activating “stimulator of interferon gene”-depending signalling. Report on patentability on WO2014189805, Al which in turn is based on U. S. Apply No WO2014-US38525. The invention offers active cyclic-di-nucleotide (CDN) immune stimulators that activate DCs via STING, a cytoplasmic receptor. This study addresses only cyclic dinucleotide and does not relate to our invention which involves medium and long chain oligonucleotide synthesis.

[0019] 11. Matthew Hassler et al. RNA synthesis via dimer and trimer phosphoramidite block coupling, Tetrahedron Letters 52 (2011) 2575-2578. The use of dimer and trimer phosphoramidite blocks in the solid-phase synthesis of oligoribonucleotides is outlined in this publication. In this study, the researchers utilized 2'-TBDMS, 2'-TIPS, and cyanomethyl as internucleotide phosphate groups to synthesize dimers and trimers and using DCI activator. They also employed a levulinyl group to protect the 3' hydroxyl, but during the deprotection of the 3' position, a migration of the protecting group from the 2'- to the 3'-position occurred, resulting in approximately 10% of the migrated product. This migration represents a significant drawback. The synthesis produced 18-mer oligonucleotides using monomers, dimers, and trimers; however, the results were inconsistent, as evidenced by the presence of multiple peaks in the dimer and trimer oligonucleotide products. In our approach of using 2’ -Tom protected monomers and dimers blocks the migration of TOM protecting group does not occur. Thus, our technology disclosed, is highly desirable approach for RNA synthesis.

[0020] 12. Anzhe Shi, A et al. practical dinucleotide phosphoramidite chemistry for de novo DNA synthesis via block coupling. Tetrahedron Letters. 2024, volume142, 1555106. This invention presents a denovo method for producing fully protected dimer amidites using a methyl phosphate and a β cyanoethyl phosphoramidite in a sustainable manner. Their study does not employ solidphase synthesis techniques and is primarily focused on the synthesis of 2’ -deoxy dinucleotides, using in the de novo synthesis in DNA not for RNA synthesis. Additionally, the yield and purity of the synthesized products were not reported.

[0021] 13. Synthesis of dimer and oligonucleotide ligation method using dimer block Report on patentability on JPH08507752A, which in turn is based on Japan. Apply No. JP6516255A. Using innovative dimer blocks and simplified chemistries, the invention offers ways to synthesize 2’-deoxy dimers blocks (dinucleoside derivatives with modified internucleoside links) and ways to construct oligonucleotides with modified internucleotide linkages using dimer blocks. Their study focuses on the synthesis of 2’-deoxy dinucleotides and 2’- deoxy oligonucleotides using an enzyme ligation method. The oligonucleotide products were limited to a maximum length of 25 nucleotides. This approach is distinct from our invention of medium to long chain defined sequence synthetic RNA.14. Daniel C. et al. Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages. Nucleic Acids Res; 2000 May 1; 28(9): e40. There is a description of a triester method for creating deoxynucleoside phosphorodithioate dimers. The researchers have developed modified oligonucleotides for use as siRNA. They employed both traditional and novel synthetic methodologies to create chiral phosphorothioate (PS) backbones. Additionally, the authors 4 synthesized 100-mer sgRNAs, each comprising a 20-mer tracrRNA with a 3'-propargyl protecting group and a 79-mer crRNA functionalized with sodium azide. The tracrRNA and crRNA were then conjugated via click chemistry. However, the purity and yield of the synthesized oligonucleotides were not reported. So, the value and utility of the approach remains unknown. It is important to note that our approach of dimer block coupling reaction and leading to medium to long chain oligonucleotide remain as unique, novel and practical process.

[0022] 15. Johnston et al, Investigations into the synthesis of a nucleotide dimer via mechanochemical phosphoramidite chemistry. R. Soc.open Sci; 8:201703. The study explored using a special method called liquid-assisted mechanochemistry to combine a nucleoside phosphoramidite with a partially protected nucleoside that has a 5’ -OH group. Their study is focused to the synthesis of short 2’ -deoxy dimer synthesis. Our invention leads to medium to long chain defined sequence high purity RNA.

[0023] RNA synthesis presents additional challenges compared to DNA synthesis due to the presence of the 2'-hydroxyl group, which necessitates temporary protection. Commonly used 2'-O-protecting groups such as TBDMS or TIPS may undergo migration or require harsh deprotection conditions, leading to strand cleavage or reduced purity in long RNA products.

[0024] Block coupling strategies employing dinucleotide or trinucleotide units have been proposed to reduce the number of coupling cycles. However, prior art methods typically suffer from drawbacks such as low coupling efficiency, long reaction times, difficult deprotection of internucleotide protecting groups, limited scalability, or restriction to short RNA or DNA sequences. In particular, prior methods have not reliably enabled high-purity synthesis of defined-sequence RNA oligonucleotides exceeding 100 nucleotides.

[0025] Accordingly, there remains a need for an improved, practical, and scalable method for synthesizing long RNA oligonucleotides with high coupling efficiency, reduced synthesis time, and high final purity.

[0026] Ob jects of the InventionSome of the objects of the present disclosure, which at least one embodiment herein satisfies are listed herein below.

[0027] It is an object of the present disclosure to provide a novel and efficient method with enhanced accuracy for synthesizing long RNA sequences with reduced number of individual pair steps required using homo and hetero dimer nucleoside amidite blocks.

[0028] Further objects of the invention include:

[0029] reducing the total number of coupling cycles required for RNA synthesis;

[0030] improving coupling efficiency and sequence fidelity;

[0031] enabling synthesis of RNA oligonucleotides exceeding 100 nucleotides in length.

[0032] Summary of the Invention

[0033] The present invention provides a method for synthesizing RNA oligonucleotides in the 3'-to-5' direction using pre-formed homo- and hetero-dimer nucleoside phosphoramidite blocks. Each dimer block comprises two ribonucleosides linked through a protected internucleotide phosphate linkage and bearing a 3 '-phosphoramidite functionality for coupling.

[0034] The invention utilizes 2'-0-T0M (triisopropylsilyloxymethyl) protection of ribose hydroxyl groups and labile base-protecting groups such as N-acetyl, which together allow high coupling efficiency and mild deprotection conditions.

[0035] Using preformed dimer nucleoside phosphoramidite, this approach reduces the number of individual pair steps required, increasing overall RNA synthesis efficiency and accuracy. Precise control of sequence assembly is achieved by either using homo dimers (two of the same nucleoside base), or hetero dimers (two different nucleoside base). Thus, improving the fidelity of synthesized RNA. The process involves the addition of dimer segments to a growing RNA chain immobilized on a rigid substrate, coupled with an appropriate cap shielding step to ensure proper extension. This approach provides a synthesis framework, which is not only faster but reduces the possibility of errors and maximizes the quality of RNA obtained. Long sequenced RNAs have broad applications, In CRISPR-Cas9 Guide RNA Synthesis Synthetic long RNA oligonucleotides are used to produce sgRNA, which is crucial for guiding the Cas9 enzyme to the specific DNA sequence that needs to be edited. Synthetic RNA allows for precisecustomization of the guide RNA sequences, enhancing the efficiency and accuracy of gene editing. Synthetic mRNA can also be designed to produce therapeutic proteins in cells, providing a temporary yet effective treatment for various genetic disorders. Like, In the recent development of mRNA vaccines, such as those for COVID-19, highlights the potential of synthetic long RNA in inducing an immune response without the need for live virus. Synthetic siRNA (small interfering RNA) and shRNA (short hairpin RNA) are used to silence specific genes, providing insights into gene function and potential therapeutic pathways. Synthetic RNA oligonucleotides can be used in CRISPR Activation to upregulate the expression of target genes, which can be useful in studying gene function and in developing treatments.

[0036] The present invention provides methods for the preparation of homo and hetero dimer nucleotides and their use in the synthesis of long RNA oligomers (>150 nucleotides). This method significantly enhances the efficiency and accuracy of RNA synthesis, reduces the number of coupling steps, and improves the overall yield of the desired RNA product.

[0037] Synthesizing long RNA oligonucleotides often face challenges such as low efficiency, increased synthesis errors, and instability due to the presence of labile 2'-OH groups. The 2'-0-T0M (N-Ac) protection strategy offers improved stability but can be further optimized by using dimer blocks. This invention provides a refined method for synthesizing long RNA oligonucleotides using 2'-0-T0M (N-Ac) nucleoside amidite dimer blocks, enhancing the overall synthesis process.

[0038] The Synthesis of Long RNA Oligonucleotide using homo- or hetero-dimer nucleoside amidite blocks comprising:

[0039] 1. Preparation of protected monomeric nucleosides

[0040] Starting Materials; i.e. [Compound II; a-dl & [Compound III; a-dl: 2'-0-T0M (N-Ac) phosphoramidite and 2'-0-T0M protected ribonucleosides with N-acetyl (N-Ac) protection.

[0041] 2. Synthesis of dimer nucleoside phosphoramidite blocks: Homo- and hetero-dimer nucleosides are synthesized by activating a 3 ’ -phosphoramidite group of a first nucleoside, coupling it with a 5 ’-hydroxyl group of a second nucleoside, and subsequently oxidizing the resulting phosphite triester to form a stable phosphodiester linkage. The resulting dimers are converted into activated phosphoramidite derivatives suitable for automated oligonucleotide synthesis.

[0042] 3. Solid-phase RNA chain assembly using the dimer blocksThe process of RNA is synthesized in a direction from the 3'-end to the 5'-end of the RNA nucleotide, and the process comprises the steps of:

[0043] (a) taking a nucleoside solid support and represented by [Compound; XIV]

[0044] (b) placing a phosphoramidite represented by [Compound V; a-d] on an oligonucleotide synthesizer

[0045] (c) removing the protecting group DMT from the nucleoside solid support [compound-XV] (d) performing the process of RNA synthesis by coupling the nucleoside of solid support and the dimer phosphoramidite of in the oligonucleotide synthesizer using a mixture of ancillary regents to result in an oligonucleotide having at least one protecting group; [-compound XVII] wherein

[0046] the ancillary reagent comprises Deblock reagent (3% TCA in DCM), Activating reagent (5-ethylthio-1-H-tetrazole at 0.25 M in acetonitrile). CAP A (acetic anhydride / tetrahydrofuran / pyridine), CAP B (10% N-methylimidazole / tetrahydrofuran), Oxidation solution (0.02 M iodine / pyridine / water / tetrahydrofuran).

[0047] 4. Following synthesis:

[0048] (a) removing the cyanoethyl group from the oligonucleotide;

[0049] (b) detaching the oligonucleotide from the solid support;

[0050] (c) removing the base protecting group from the oligonucleotide;

[0051] (d) removing a silyl protecting group to result in the oligonucleotide;

[0052] (e) precipitating the oligonucleotide;

[0053] (f) purification of the crude oligonucleotide;

[0054] (g) analysing the oligonucleotide for purity determination.

[0055] There are many advantages of using this dimer amidite method of oligonucleotide synthesis. Like, it reduces the number of coupling steps required, making the process faster and more efficient.

[0056] Enhanced accuracy reduces the number of steps and minimizes errors, resulting in higher fidelity of the synthesized RNA and also advantage in improved yield by fewer steps lead to an increasedoverall yield of the desired RNA product. This method is scalable and can be adapted for both small-scale laboratory synthesis and large-scale industrial production.

[0057] The synthesized long RNA molecules can be used in various applications, including in Research: As probes, primers, and templates in molecular biology experiments. In diagnostics: the development of RNA-based diagnostic assays. In Therapeutics: the creation of RNA-based drugs, including mRNA vaccines and RNA interference (RNAi) therapies.

[0058] Brief Description of the Drawings

[0059] Coupling efficiency result with Bar graph is shown in fig. 1- fig.3

[0060] Figure No.l: Bar graph & Coupling efficiency of 5 -coupling cycles of Dimer amidites for the synthesis of 11-mer oligonucleotide.

[0061] Figure No.2: Bar graph & Coupling efficiency of 10-coupling cycles of Dimer amidites for the synthesis of 21-mer oligonucleotide.

[0062] Figure No.3: Bar graph & Coupling efficiency of 60-coupling cycles of Dimer amidites for the synthesis of 121-mer oligonucleotide.

[0063] UV analysis graph of Hetero-Dimer is shown in Fig.4- Fig.7

[0064] Figure No.4: 5’-3’-O-dihydroxy-2’-TOM-rU (N-Ac) [Compound No. Illa]

[0065] Figure No.5: 5’-O-DMT-3’-CNEt phosphoramidite 2’-T0M-rG [Compound No. Ila] Figure No.6; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate - 3’ -hydroxy-2’ -TOM-rG (N- Ac) [Compound No. Vic]

[0066] Figure No.7: 5’-O-DMT-2’-TOM-rU-3’ -5’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rG(N-Ac) [Compound No. Xc]

[0067] NMR Report [31-p-NMR analysis] of Dimer (U-G) is shown in Fig. 8-Fig. 9Figure No.8; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate -3’ -hydroxy-2 ’-TOM-rG (N-Ac) [Compound No. Vic]

[0068] Figure No.9; 5’-O-DMT-2’-TOM-rU-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rG(N-Ac)

[0069] 1-H-NMR analysis of Compound No. Vic is shown in Fig. 10

[0070] Figure No.10; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate 3 ’-hydroxy-2’ -TOM-rG (N-Ac) [Compound No. Vic]

[0071] Detailed Description of the Invention

[0072] Definitions- For the purposes of this specification:

[0073] □ “RNA oligomer” refers to an oligoribonucleotide comprising two or more ribonucleotides.

[0074] □ “Dimer block” refers to a nucleoside phosphoramidite containing two covalently linked nucleosides.

[0075] □ “Homo-dimer” refers to a dimer containing two identical nucleosides.

[0076] □ “Hetero-dimer” refers to a dimer containing two different nucleosides.

[0077] □ “TOM” refers to the 2'-O-triisopropylsilyloxymethyl protecting group.

[0078] □ “Solid support” refers to a polymeric or inorganic matrix suitable for oligonucleotide synthesis

[0079] In the following description, for the purposes of explanation, various specific details are set forth in order to provide a thorough understanding of embodiments of the present disclosure. It will be apparent, however, that embodiments of the present disclosure may be practiced without these specific details. Several features described hereafter can each be used independently of one another or with any combination of other features. An individual feature may not address all of the problems discussed above or might address only some of the problems discussed above. The ensuing description provides exemplary embodiments only and is not intended to limit the scope, applicability, or configuration of the disclosure. Rather, the ensuing description of theexemplary embodiments will provide those skilled in the art with an enabling description for implementing an exemplary embodiment. It should be understood that various changes may be made in the function and arrangement of elements without departing from the scope of the disclosure as set forth.

[0080] The word “exemplary” and / or “demonstrative” is used herein to mean serving as an example, instance, or illustration. For the avoidance of doubt, the subject matter disclosed herein is not limited by such examples. In addition, any aspect or design described herein as “exemplary” and / or “demonstrative” is not necessarily to be construed as preferred or advantageous over other aspects or designs, nor is it meant to preclude equivalent exemplary structures and techniques known to those of ordinary skill in the art. Furthermore, to the extent that the terms “includes,” “has,” “contains,” and other similar words are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising” as an open transition word without precluding any additional or other elements.

[0081] Reference throughout this specification to “one embodiment” or “an embodiment” or “an instance” or “one instance” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0082] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a”, “an”, and “the” are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.The present invention discloses an innovative method for synthesizing long RNA oligomers (>100 nucleotides) using homo- and hetero-dimer nucleoside blocks in the 3’-5’ direction. This method improves upon traditional RNA synthesis techniques by reducing the number of coupling steps, enhancing synthesis efficiency, and ensuring high fidelity in sequence assembly.

[0083] Here we are using dimers for long RNA oligonucleotide containing the 2’-O-triisopropylsilyloxymethyl (TOM) group is characterized by very high coupling efficiency along with fast, simple deprotection. High coupling efficiency is achieved because the TOM-Protec ting- Group exhibits lower steric hindrance than the 2’-O-t-butyldimethylsilyl (TBDMS) group used in current method of stepwise RNA Synthesis.

[0084] A further feature of the TOM-protecting-group is that during basic steps it cannot undergo 2’ to 3’ migration. This migration under basic conditions leads to non-biologically active 2’-5’ linkages when using the TBDMS group. These features allow the TOM-protected dimer to produce longer oligonucleotides.

[0085] The use of homo and hetero-dimer nucleotide blocks in the process is crucial to achieve the synthesis of long RNA in the range of about 100-mer to about 200-mer.

[0086] In the present, the use of TOM (triisopropylsilyl)oxy] methyl) as a 2'-OH protecting group offers significant advantages over TBDMS (t-butyldimethylsilyl), especially in terms of removal efficiency and overall synthesis time. TOM is ideally suited for removal under mild conditions, typically requiring just treatment with fluoride ions, which simplifies the deprotection process and enhances efficiency. Its smaller size reduces steric hindrance, enabling faster coupling reactions without the delays associated with bulky groups like TBDMS. These characteristics make TOM particularly advantageous for longer oligonucleotide synthesis, where rapid and efficient synthesis is essential. Moreover, TOM protecting group has milder deprotection conditions which help in preventing side reactions as well as minimizing the risk of phosphotriester bond cleavage, making it a more practical, reliable and highly promising choice for medium and long chain oligonucleotide of defined sequence.

[0087] The Inventor has chosen acetyl (Ac) as the N-protecting group, recognizing its unique advantages despite its relative simplicity. Acetyl offers mild and efficient protection, making it well-suited for oligonucleotide synthesis under standard conditions. Unlike more bulky groups such as ibu (isobutyryl) or benzoyl, acetyl is known to be easily deprotected, allowing for faster removal and minimizing difficulties in deprotection steps. While it may be more prone to hydrolysis under basic conditions, this characteristic is manageable in our optimized protocols, ensuring minimal side reactions. Moreover, acetyl offers excellent compatibility with a wide range of reagents used in oligonucleotide synthesis, making it a reliable choice for many applications. In most cases where milder conditions and speed are required, acetyl remains anideal and practical choice for effective synthesis without the need for complex deprotection strategies.

[0088] Synthesis of Long RNA Oligonucleotide using homo- or hetero-dimer nucleoside amidite blocks:

[0089] 1. Preparation of protected monomeric nucleosides

[0090] 2. Synthesis of dimer nucleoside phosphoramidite blocks

[0091] 3. Solid-phase RNA chain assembly using the dimer blocks

[0092] 4. Post-synthetic deprotection, cleavage, purification, and analysis

[0093] 1. Starting Materials; i.e. [Compound II; a-dl & [Compound III; a-dl

[0094] 2'-0-T0M (N-Ac) phosphoramidite and 2'-0-T0M protected ribonucleosides with N-acetyl (N-Ac) protection.

[0095] The ribonucleosides utilized in the invention are protected using a 2’-O-triisopropylsilyloxy methyl (2’-O-TOM) group in combination with N-acetyl (N-Ac) base protection, providing enhanced chemical stability throughout the synthesis process.

[0096] Synthesis of 2’-TOM-3’-Phosphoramidites (Compound II; a-d)

[0097] The first nucleotide (Compound I; a-d), with a free 3'-hydroxyl group and a protected 5'-hydroxyl group, is activated by reacting it with an activating agent like tetrazole or 5-ethylthio-IH-tetrazole (ETT). This converts the phosphoramidite into a reactive phosphite intermediate. Deprotection of the 5'-Hydroxyl Group: (Compound III; a-d)

[0098] The DMT group on the first nucleotide (Compound I; a-d), is removed by treatment with an acidic solution (e.g., trichloroacetic acid in dichloromethane), exposing the 5'-hydroxyl group for coupling.

[0099] General scheme for the Phosphoramidite PreparationDMT— O

[0100] OH O-TOM

[0101]

[0102] Scheme; (1) TOM-Protected Dimers, Step-A Conventional Synthesis of 2’-TOM-3’- Phosphonamidites [Compound II; a-d]

[0103] General scheme for the Deprotection of DMT

[0104] DMT— O

[0105]

[0106] OH O-TOM OH O-TOM

[0107] [Compound; la-d] [Compound; Hla-d]

[0108] where; B {U, A(Ac>, G<Ac>} where; B {U, C(Ac), A(Ac), G(Ac)} la- U, Ib-C<Ac>, Id-G< Ac)IHa- U, nib-C(Ac), IIIc-A(Ac), ind-G(Ac)

[0109] Scheme; (2) TOM-Protected Dimers, Step-B Synthesis of 5’, 3’-dihydoxy-2’-TOM Nucleosides

[0110] In an embodiment, Other labile base-protecting groups, including but not limited to Fmoc, N-PAC, N-TBPAC, N-iPr-PAC, pivaloyl, DMF, and levulinyl, may be used without departing from the scope of the invention.

[0111] In another embodiment, the starting nucleosides may already include base modifications (e.g., 5-fluoro, 5-methyl, 7-deaza, 8-oxo, halogenated, propargylated bases).

[0112] Various modifications of nucleosides such as, alkyl, aryl, amino or acetyl, 7-deaza, 7-Iodo, 7-deaza -7-propargyl purine nucleobases, R4 is benzoyl, acetyl [ Compound; XXIV].

[0113] Various modifications of purine nucleosides such as, alkyl, aryl, amino or acetyl, 7-deaza, 7-lodo, 7-deaza -7-propargyl modification at any position of commonly known in the nucleoside chemistry would encompass in the present invention. Additionally, pyrimidine nucleosides such as alkyl, aryl, propargyl halogen modified nucleosides would further encompass the present invention. An elegant review article describing various protecting group, utilized in Oligonucleotide chemistry are incorporated in the publication; A New Amine Protecting Group, CEOC applicable in Oligonucleotide Chemistry, Manoharan et.al., 1999; 10.1080 / 07328319908044661The modified nucleosides incorporated by this method mentioned in the described art could consists of one or more of purine or pyrimidine modifications, such as but not limited to, 5-Fluoro-U, 5-Fluoro dU, 5-fluoro-dC, 5-Fluro-rC, pseudo uridine, 5-methyl-dU, 5-methyl-rU, 5-methyl-dC, 5-methyl-rC, 5-bromo-dU,5-bromo-rU,5-bromo-dC,5-bromo-rC,5-iodo-dU,5-iodo-rU,5-vinyl-dU,5-vinyl-rU,5vinylthymidine, N-3 methyl-2’-deoxyuridine, N-3 methyl-ribouridine, N-3 methyl thymidine, 4-thio uridine, 4-thio-2'-deoxyuridine,2,6-diaminopurine deoxyriboside, N-3 methyl-ribothymidine,2,6-di-aminopurineriboside,8-Oxoguanosine,8-oxoadenosine,8-bromo-2'-deoxy adenosine, 8-fluoro-2’ -deoxy guanosine and 8-fluoro-2’-deoxy adenosine, inosine, 8-bromoinosine.

[0114] Various modified nucleosides, such as 8-bromoadenosine,8-oxo-2’-deoxy-adenosine,8-oxo-riboadenos,8-oxo-2’-deoxyinosine,8-oxo-inosine,8-bromo-2’-deoxyinosine,8-bromo-ribo-inosine, N-l-methyl-adenosine, N-l methyl-2'-deoxyadenosine, N-l methyl - 2-deoxyinosine, N-1 methyl-adenosine, N-l methyldeoxyguanosine, N-l -methyl -riboguanosine, Inosine, etheno adenosine, etheno inosine, etheno 2’- deoxyinosine, purine-2'-deoxyriboside, purine -ribonucleoside, 2-aminopurine-2'-deoxyriboside,2-aminopurine-ribonucleoside.

[0115] Various modified nucleoside 2’- fluoro adenosine, 2’- fluoro cytidine, 2’- fluoro guanosine, 2’ -fluoro uridine, 2’-M0E (methoxy ethoxy) adenosine, 2’- MOE (methoxy ethoxy) cytidine, 2’-MOE (methoxy ethyl) guanosine, 2’ - MOE (methoxy ethyl) uridine.

[0116] In another embodiment, the sugar moiety of the starting nucleosides may comprise 2'-F, 2'-OMe, 2'-MOE, 2'-NH2, FANA, LNA, ANA, 4'-thio-FANA, or other sugar modifications.

[0117] The sugar modification of modified nucleosides could consist of 2'-deoxy-2'-fluoro ribonucleosides (2'-FANAs) such as A, C, G, U, Inosine and modified nucleosides containing 2'-Fluoro, in one or more positions of an RNA or DNA sequence synthesized by the method of this invention.

[0118] The sugar modification of modified nucleosides could consist of 2' deoxy-2-methoxy ribonucleosides, (2'-0Me) included, but not limited to such as A, C, G, U, Inosine and modified nucleosides containing 2'- methoxy in one or more positions of an RNA or DNA sequence synthesized by the method of this invention.

[0119] The sugar modification of modified nucleosides could consist of 2'-deoxy-2-amino ribo nucleosides (2'-NH2) such as A, C, G, U. Inosine and modified nucleosides containing 2-amino,2’ -propyl amino, in one or more positions of an RNA or DNA sequence synthesized by this method.

[0120] The sugar modification of modified nucleosides could consist of 2' -deoxy-2' -terminal amino ribo nucleosides.

[0121] The RNA synthesis may be carried out with one or more 2'-5'- linkage within the sequence, at the 3 '-end of the sequence or at the 5 '-end of the sequence.

[0122] The RNA synthesis can consist of one or more of DNA bases at the 3'-end, may be synthesized by the method of this invention containing deoxy nucleosides such as dT, dC, dG, thymidine, attached via their 3'-hydroxyl function.

[0123] The RNA having a 3 '-end may be synthesized by the method of this invention containing reverse attached ribonucleosides such as rA, rC, rG, rU and modified nucleosides and modifications, including FANA nucleosides via their 2’ or 3'-hydroxyl function.

[0124] The RNA synthesis may be achieved comprising the modified base 4'-thio-2'-deoxy-2'-fluorobeta-D arabinonucleic acid (4'-Thio-FANA).

[0125] The RNA synthesis may be achieved comprising the modified Sugar, 2'-OMethyl modification. (2'-terminal NH2), attached via spacer from 2-12 atoms nucleosides Such as A, C, G, U. Inosine and modified nucleosides containing 2'-terminal amino, in one or more positions of an RNA or DNA sequence synthesized by this method.

[0126] The sugar modification of modified nucleosides could consist of 2'-deoxy-2-methoxyethylribo nucleosides (2'-M0E), such as A, C, G, U, Inosine and modified nucleosides containing 2'-M0E, in one or more positions of an RNA or DNA sequence synthesized by this method.

[0127] The sugar modification of modified nucleosides could consist of other 2'-O-alkyl groups, such as 2'-deoxy-2'- ethoxy, propargyl, butyne ribonucleosides (2'-OEt, O-Propargyl. 2'-O-Butyne), such as A, C, G, U, Inosine and modified nucleosides containing 2'-OEt, O-Pro.

[0128] The RNA synthesis may be achieved by using Bicyclic locked nucleic acids (LNAs). such as A, C, G, T, U & 5-methyl-U.

[0129] The RNA synthesis may use the modified sugar comprising Altritol nucleic acids (ANAs).

[0130] The sugar modification of modified nucleosides could consist of 2'-deoxy-2'-fluoro arabino nucleosides (2'-FANAs) such as A, C, G, U, Inosine and modified nucleosides containing 2'-F-FANAs), in one or more positions of an RNA or DNA sequence synthesized by this method.

[0131] The sugar modification of modified nucleosides could consist of 2'-deoxy-2'-fluoro 4'-thioarabino nucleosides

[0132] 2. Dimer nucleoside amidite blocks Synthesis; i.e. [Compound V; a-dl

[0133] Monomer Activation: Activate the 3'-phosphoramidite group of the first nucleoside using an activator such as Ethyl thio tetrazole.

[0134] Coupling Reaction: Couple the activated phosphoramidite with the 5'-hydroxyl group of the second nucleoside to form a dimer.

[0135] Oxidation: Oxidize the phosphite triester linkage to a stable phosphodiester bond using iodine in water or pyridine and THF.

[0136] Homo- and hetero-dimer nucleosides are synthesized by activating a 3 ’ -phosphoramidite group of a first nucleoside, coupling it with a 5 ’-hydroxyl group of a second nucleoside, and subsequently oxidizing the resulting phosphite triester to form a stable phosphodiester linkage. The resulting dimers are converted into activated phosphoramidite derivatives suitable for automated oligonucleotide synthesis.

[0137] Homo-dimer blocks consist of two same nucleosides (compound II; a-d and compound III; a-d) linked by a phosphodiester bond. Same nucleoside is chemically activated and coupled to form a homo-dimer with a phosphodiester linkage (compound IV; a-d). After that, Dimer was activated by adding the diisopropyl ammonium tetrazolium salt and then 2-cyanoethyl N, N, N, N-tetra isopropyl phosphoramidite was added under argon to synthesized the Dimer nucleoside phosphoramidite (compound V; a-d). Protective groups (e.g., triisopropylsilyl oxyethylene, dimethoxy triphenyl, N-Acetyl) are used to ensure specificity of the reaction.

[0138] General scheme for the synthesis of Homo-Dimer blocksOH O— TOM

[0139] [Compound II; a-d] [Compound III; a-d]

[0140] where; B1{U, C<Ac>, A^, G<Ac>} where; B2{U, C(AC), A<AO>, G< AC)}

[0141] Illa- U, lllb-C(Ac>, IIIC-A<AC>, llld-G(Ac>

[0142] Ila- U, llb-C<Ac>, IIC-A<A°>, lld-G<Ao>

[0143] O O-TOM

[0144] CEO— P=O

[0145] OH O-TOM

[0146] [Compound IV; a-d] [Compound V; a-d]

[0147] wherefl, and B2{U, C(Ao), A<AC>, G(AC)} wherefl, and B2{U, C< AC), A<AO>, G(AO)}

[0148] IVa-U-U, IVb-C< Ac)-C< Ac), Va-U-U, Vb-C< Ac)-C< Ac),

[0149]

[0150] IVC-A(AC)-A(AC), IVdG(Ac)-G(Ac)VC-A(AC)-A(AC), Vd-G(Ac)-G(Ao)

[0151] Scheme; (3) TOM-Protected Dimers, Step-2 Illustration of General Synthesis of TOM-Protected Homo-Dimer amidites.

[0152] Hetero-dimer blocks consist of two different nucleosides (compound II; a-d and compound III; a-d) linked by a phosphodiester bond. Similar to homo-dimers, two different nucleosides are chemically activated and coupled to form a hetero-dimer nucleoside (compound VI; a-c to compound IX; a-c). After that, Dimer was activated by adding the diisopropyl ammonium tetrazolium salt and then 2-cyanoethyl N, N, N, N-tetra isopropyl phosphoramidite was added under argon to synthesized the Dimer nucleoside phosphoramidite (compound X; a-c to compound XIII; a-c). Protective groups (e.g., triisopropylsilyl oxyethylene, dimethoxy triphenyl, N-Acetyl) are applied to ensure correct orientation and linkage.General scheme for the synthesis of Hetero dimer blocks.

[0153] [Compound II; a-d] [Compound III; a-d] whored, {U, C<Ac>, A^’, G^’} where; B2{U, C^’, A(Ac), G<Ac>}

[0154] Ila- U, llb-C(Ac>, llc-A<Ac>, lld-G(Ac> Illa- U, lllb-C<Ao>, lllc-A< Ac>, llld-G(Ac)

[0155] V? [Compound VI; a-c] [ Compound VII; a-c] [Compound VIII; a-c] [Compound IX; a-c] where; 6,-U where; B,-^’ where; BrA<Ac) where; BrG B2{, C(AC)A(AC), GAC)} B2{U, A<AC), G<AC>] B2{U, C< AO), G(AC>} B2p{U, C<Ao>, A<Ac>}

[0156] [Compound X; a-c] [Compound XI; a-c] [Compound XII; a-c] [Compound XIII; a-c] where; BrU where; B1-C(AO)where; BrAlAcl where; Bi-G

[0157]

[0158] B2P{. C^. A^. G*0)} B2P{U, A< AC), G^)} B2P{U. Cl^. G^)} B2P{U, C< AC), A<AC>} Scheme; (4) TOM-Protected Dimers, Step-2 Illustration of General Synthesis of TOM-Protected List of Hetero-Dimer amidites synthesized:

[0159] Where in Compound VI;

[0160] U coupled with of C(Ac)(Compound; VI a)

[0161] U coupled with of A'Ac)(Compound; VI b)

[0162] U coupled with of G(Ac)(Compound; VI c)

[0163] Where in Compound; VII

[0164] C(Ac)coupled with of U (Compound; VII a)

[0165] C(Ac)coupled with of A(Ac)(Compound; VII b)C(Ac)coupled with of G(Ac)(Compound; VII c)

[0166] Where in Structure; VIII

[0167] A(AC)coupled with of U (Compound; VIII a)

[0168] A(AC)coupled with of C(Ac)(Compound; VIII b)

[0169] A(AC)coupled with of G'Ac)(Compound; VIII c)

[0170] Where in Structure; IX

[0171] G(AC)coupled with of U, (Compound; IX a)

[0172] G(AC)coupled with of C(Ac)(Compound; IX b)

[0173] G(AC)coupled with of A(AC)(Compound; IX c)

[0174] In another embodiment, base-protecting groups and 5 '-hydroxyl protecting groups (DMT, MMT, TMT) may be varied during dimer synthesis.

[0175] Besides DMT at 5 ’-position other 5 ’-protecting groups, such as MMT and TMT protecting group can be utilized.

[0176] The RNA oligomers could consist of natural or modified nucleobases, gapmers, phosphodiesters, phosphorothioates, phosphonates.

[0177] Besides the specifications, broadly described in #3 above for substitution of DMT group at the 5 ’-position or 3’- position of sugar with protecting group such as MMT and TMT. Various replacement of DMT group either 5’ or 3’ position of sugar portion of nucleosides shall encompass in the present invention. Such protection of hydroxy function of nucleosides replacements were elegantly reported in the publication by E. F. Fisher & M. H. Caruthers, “Current Protocols in Nucleic Acid Chemistry (2000) 2.3.1-2.3.34 Copyright © 2000 by John Wiley & Sons, Inc.” The RNA oligomers could consist of natural or modified nucleobases, gapmers, phosphodiesters, phosphorothioates, phosphonates.

[0178] In another embodiment, each dimer may contain modified nucleobases, chromophores, biotin, fluorophores, or internal reactive handles.

[0179] A method of attachment of propargyl modifications, preferably to 2’-terminus or one or more internal base, either single or multiple propargyl modification in the short or long RNA synthesized by the method of the present invention. The propargyl modified oligonucleotidessubsequently can be conjugated with other macromolecules such as via Click chemistry preferably either at 2’position of nucleoside or, C-5 position as illustrated in formula 4 or at one or more of a pyrimidine nucleosides and by formula 4b, within the synthesized oligonucleotide by the present invention.

[0180] R could be a chromophore at internal positions of an RNA synthesized by this method is achievable with chromophores but not limited to dT-C-5-Fluoroscein, dT-C-5-Dabcyl, dT-C-5-HEX, dT-C-5-Cy-5, TAMRA, dT-C-5-Cy-3, internal carboxyl group labelled with dU-C-5-methylacrylate, biotin dT, (biotin attached via spacer to dU-C-5-biotin), C-5-dU with C-6-spacer having a terminal amine. The terminal amine shall serve as a provision for additional dyes.

[0181] 3. A process of synthesizing an RNA oligonucleotide [Compound; XVII] The process of RNA is synthesized in a direction from the 3'-end to the 5'-end of the RNA nucleotide, and the process comprises the steps of:

[0182] (a) taking a nucleoside solid support and represented by [Compound; XIV]

[0183] (b) placing a phosphoramidite represented by [Compound V; a-d] on an oligonucleotide synthesizer

[0184] (c) removing the protecting group DMT from the nucleoside solid support [compound-XV]

[0185] (d) performing the process of RNA synthesis by coupling the nucleoside of solid support and the dimer phosphoramidite of in the oligonucleotide synthesizer using a mixture of ancillary regents to result in an oligonucleotide having at least one protecting group; [-compound XVII] wherein

[0186] the ancillary reagent comprises Deblock reagent (3% TCA in DCM), Activating reagent (5-ethylthio-1-H-tetrazole at 0.25 M in acetonitrile). CAP A (acetic anhydride / tetrahydrofuran / pyridine), CAP B (10% N-methylimidazole / tetrahydrofuran), Oxidation solution (0.02 M iodine / pyridine / water / tetrahydrofuran).

[0187] RNA oligonucleotide synthesis is performed on a solid support, such as controlled pore glass (CPG), in the conventional 3’-to-5’ direction using an automated oligonucleotide synthesizer. Each synthesis cycle comprises detritylation, coupling of a dimer phosphoramidite, capping of unreacted sites, and oxidation. Sequential addition of homo- or hetero-dimer phosphoramidites enables rapid construction of long RNA chains with high sequence fidelity. Repetition of the synthesis cycle allows formation of RNA oligomers containing up to sixty dimer units or more.2’-TOM Dimer based Long RNA Synthesis Cycle.

[0188]

[0189] Scheme; (6) Illustration of RNA Synthesis cycles utilizing TOM-Protected Dimers.

[0190] Method of synthesizing long RNA oligomers comprises the following steps.

[0191] First by preparation of Solid Support: A solid support material, such as controlled pore glass (CPG) is functionalized with a protected deoxynucleoside, protected ribonucleoside, modified deoxynucleoside, modified ribonucleoside, allow the attachment of the first nucleoside block. (Compound; XIV)

[0192] General Structure of Solid support nucleoside used in example illustrated in the scheme 5.

[0193]

[0194] The Thymidine CPG was coupled with the different homo or hetero dimer nucleotides successively as shown in the scheme 5.

[0195] General scheme for the synthesis of RNA with either Dimer blocks

[0196]

[0197] Scheme; (5) Illustration of RNA Synthesis utilizing TOM-Protected Dimers.

[0198] The homo- or hetero-dimer nucleoside amidite blocks [compound; V a-d, X a-c, XI a-c, XII a-c, XIII a-c] were added in a step-wise sequential addition in 3 ’-5' direction to a Solid support [Compound; XV] leading to solid support bound trimer blocks. Each addition involves: Coupling of the 3’-phosphoramidite function of the incoming dimer block synthesized all the 16 possible dimer nucleotide followed by capping and oxidation leading to [Compound; XVI] & [ Compound; XVII] successively.

[0199] Repeating the cycle of addition of any of the dimer amidite was carried out up to 60 cycles leading to a protected oligonucleotide [Compound; XVIII]

[0200] Structure of Oligonucleotide{A<AC\C<AO>, G< AO>, U}

[0201]

[0202] n-an integer ranges from [2-60]

[0203] By this approach, RNAs of various length have been successfully achieved such as 5-mer 10-dimer, 60-dimer (Compound XVIII)

[0204] In an embodiment, the synthesis may be performed on automated, semiautomated DNA / RNA or other synthesizers or manually. The synthesis can be performed at various scales from microgram to kilogram Scales.

[0205] In another embodiment, during solid-phase synthesis, 3 '-end or internal conjugation with lipophilic ligands, PEGs, fatty acids, phospholipids, PUFAs, or biologically active macromolecules may be performed.

[0206] 3'-Conjugation of RNA can be carried out with macromolecules such as Adamantane acetoyl [Compound: XVIII], Cis-eicosenoyl [Compound: XIX ], Cholesterol [Compound: XX], triethyleneglycol hexaethyleneglycol (HEG) [Compound: XXI ] and Polyethyleneglycol (PEG) [Compound: XXII ] and 2-O-butyne[Compound: XXIII ]other fatty acid which are being used in sgRNA utilizing automated RNA synthesizer. As example a general structure of either short or long RNA having 3’- conjugation with ligand, L as shown in [Compound: XXIV] and [Compound: XXV]

[0207] The RNA synthesis may comprise the step of conjugation of lipophilic or hydrophobic groups atthe 3'-end of the RNA either through an amidite function on the hydrophobic moiety or through an amino linker at the 3'-end or at an internal position of the synthesized RNA of a synthesized oligonucleotide having a terminal amino group. The later synthesis involves a coupling step between amino at the 3 '-terminal of oligonucleotide and carboxylic function on the lipophilic moiety. The lipophilic moieties consist of various glycol, Such as, various lipids, propargyl. 2'-O-Butyne, in one or more positions of an RNA or DNA sequence synthesized by this method.

[0208] 1 Cell membrane phospholipids or PUFAs (Polyunsaturated fatty acid), such as prostaglandins, thromboxanes, and leukotrienes, as well as the more recently discovered hydroxy-, hydroperoxy-, epoxy-, and oxo-eicosanoids and the specialised pro-resolving (lipid) mediators, lipoxygenases, or cytochrome P450 can be incorporated as a ligand in to defined sequence RNA as a carrier of such biologically significant macromolecules in to defined cellular system. The control of blood and vascular inflammation is greatly aided by these eicosanoids.

[0209] 2 Various class of molecules, which have shown high promise are short and long chain polyethyene glycols (PEG); Bonora, G. M., Burcovich, B. Veronese, F. M., Plyasunova, O., Pokrovsky, A. and Zary tova, V., “XII International Round Table Conference, Nucleosides, Nucleotides and their Biological Applications, La Jolla, Calif., September 15-19, PPI 89, 1996.” For efficient delivery of synthetic RNA, and DNA molecules with PEG attachment to various oligonucleotides have shown to possess very favourable properties. PEG-oligomers and other unsaturated fatty acid such as Cis-eicosanoid have shown nice enzymatic stability by preventing fast digestion. The thermal melting behaviour was shown not to be affected, thereby still retaining properties of double strand formation.

[0210] Macular degeneration is a progressive bilateral ocular condition that affects the macula of the eye. The most prevalent type of this disorder is age-related macular degeneration (AMD), which stands as the primary cause of irreversible blindness among individuals over the age of 50 in developed nations. Autosomal dominant Stargardt disease-3 (STGD3) is a hereditary macular dystrophy, a form of dry AMD. This condition arises from a mutation in the elongation of very-long-chain fatty acids-like 4 (ELOVL4) gene, which plays a crucial role in encoding the elongase enzyme; “Aihua Liu, Yanhua Lin et.al, Role of long-chain and very-long-chain polyunsaturated fatty acids in macular degenerations and dystrophies.2011;6(5):593— 613.”

[0211] Such fatty acids can be attached as ligand (Li or L2) to carry in to a within the human or animal cell in to localized organ as targeted delivery of the conjugates of oligonucleotide and macromolecules.[Compound: XVIII] [Compound: XIX]

[0212] 2,3-Di hexadecyl-rac-glycerol [Compound: XX] [Compound: XXI]

[0213]

[0214] CH3-C=C — CH3PEG 2’- O-butyne

[0215] [Compound: XXII] [Compound: XXIII]

[0216] Where; Z is a protecting group selected from the group consisting

[0217] of DMT, MMT and TMT. or as illustrated by the labile sugar protecting groups as describe; E. F. Fisher & M. H. Caruthers.

[0218] R4 is propargyl | CH2-C≡CH |

[0219] R4 is propyl amino | -CH2-CH2-CH2-NHB I, alkyl amino where amino is protected with a transient protecting group

[0220] B could be; TFA, Fmoc

[0221] L is Adamantane acetoyl, Cis-eicosenoyl, Cholesterol,

[0222]

[0223] 2'-O-Butyne, triethylene glycol, hexaethyleneglycol (HEG),

[0224] Polyethyleneglycol(PEG),

[0225] [Compound: XXIV]O 0-R4

[0226] ⊖-P=O

[0227] I

[0228] o

[0229] O 0-R4

[0230] O 0-R4

[0231] Where; Z is a protecting group selected from the group consisting

[0232] of DMT, MMT and TMT. or as illustrated by the labile sugar

[0233] protecting groups as describe; E. F. Fisher & M. H. Caruthers.

[0234] R4 is propargyl CH2-C≡CH at one or more position.

[0235] R4 is propyl amino | -CH2-CH2-CH2-NHB I, alkyl amino

[0236] where amino is protected with a transient protecting group

[0237] L[ is described in the paragraph 6.0, 6.1, 6.2

[0238]

[0239] n is any number from 2-60

[0240] [Compound: XXV]

[0241] In another embodiment, the RNA synthesis can consist of one or more of DNA bases at the 3'-end, may be synthesized by the method of this invention containing deoxy nucleosides such as dT, dC, dG, thymidine, attached via their 3'-hydroxyl function.In another embodiment, the RNA having a 3'-end may be synthesized by the method of this invention containing reverse attached ribonucleosides such as rA, rC, rG, rU and modified nucleosides and modifications, including FANA nucleosides via their 2’ or 3'-hydroxyl function.

[0242] 4. Following synthesis:

[0243] (a) removing the cyanoethyl group from the oligonucleotide;

[0244] (b) detaching the oligonucleotide from the solid support;

[0245] (c) removing the base protecting group from the oligonucleotide;

[0246] (d) removing a silyl protecting group to result in the oligonucleotide;

[0247] (e) precipitating the oligonucleotide;

[0248] (f) purification of the crude oligonucleotide;

[0249] (g) analysing the oligonucleotide for purity determination.

[0250] Following chain assembly, standard post-synthesis processing steps are performed, including removal of cyanoethyl groups, cleavage from the solid support, deprotection of nucleobases and 2’-0-T0M groups, precipitation, purification, and analytical quality assessment of the RNA product.

[0251] In another embodiment, the terminal protecting group Z may be selectively converted to 5'-mono-, di-, or triphosphates.

[0252] Subsequently, Z can be replaced selectively to 5’- monophosphate, 5’- diphosphate and 5’-triphosphtate in the oligonucleotides.

[0253] There are many advantages of using this dimer amidite method of oligonucleotide synthesis. Like, it reduces the number of coupling steps required, making the process faster and more efficient.

[0254] Enhanced accuracy reduces the number of steps and minimizes errors, resulting in higher fidelity of the synthesized RNA and also advantage in improved yield by fewer steps lead to an increasedoverall yield of the desired RNA product. This method is scalable and can be adapted for both small-scale laboratory synthesis and large-scale industrial production.

[0255] The synthesized long RNA molecules can be used in various applications, including in Research: As probes, primers, and templates in molecular biology experiments. In diagnostics: the development of RNA-based diagnostic assays. In Therapeutics: the creation of RNA-based drugs, including mRNA vaccines and RNA interference (RNAi) therapies.

[0256] The RNA synthesis may comprise the step of conjugation of peptides, such as cell penetrating peptides (CPPs) or membrane permeant peptide (MPPs) utilizing either the free amine function of such peptides and a 3'-terminal carboxylic function of the synthesized RNA. The CPPs and MPPs having an appropriate carboxyl function can be coupled to the free terminal amino function of a 3'-end of the reverse synthesized RNA (4'-S-FANAs) such as A, C, G, U, Inosine and modified nucleosides containing 4'-S-FANAs in one or more positions of an RNA or DNA sequence synthesized by this method. We anticipate synthesis of RNA

[0257] Z B

[0258] Z O O-R4

[0259] ⊖-P=O

[0260] I

[0261] O 0-R4

[0262] ⊖-P=O

[0263] O 0-R4 1

[0264] ⊖-P=O

[0265] O 0-R4

[0266] ⊖-P=O

[0267] L1— n2

[0268] O 0-R4

[0269] ⊖-P=O

[0270] o 1

[0271] L2

[0272] Where; Z is a protecting group selected from the group

[0273] consisting of DMT, MMT and TMT. or as illustrated by the labile sugar protecting groups as describe; E. F. Fisher & M. H. Caruthers.

[0274] R4 is propargyl; CH2— C≡CH at one or more position;

[0275] R4 could also be a chromophore at one or more R4 position

[0276] as defined in paragraph 11.

[0277] L2is a Cell penetrating peptide

[0278]

[0279] n2is any number from 2-60[Compound: XXVI]

[0280] B could be a modified nucleoside base radical selected from the group consisting of 1-(N-benzoyl-5-methylcytosinyl), l-((N. N-dimethylformamidinyl)-5-methylcytosinyl), l-(N-acetyl-5methylcytosinyl), l-(5-methyl-uracilyl), l-(5-fluoro-uracilyl), l-(N-benzoyl-5-fluorocytosinyl)-,9-(N-benzoyl-7-deazaadeninyl), 9-(N-(N, N-dimethylformamidinyl)-7-deazaadeninyl), 9-(N-(N, N-dibutylformamidinyl)-7-deazaadeninyl), 9-(N -isobutyryl-7-deazaguaninyl), and 9-(N ’ (N. N dimethylformamidinyl)-7-deazaguaninyl), 9-(N6(N. N dibutylformamidinyl)-7-deazaguaninyl)

[0281]

[0282] [Compound: XXVII]

[0283] In Compound: XXVII, Z is a protecting group selected from the group consisting of dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl (MMT) and trimethoxytriphenyl methyl (TMT).

[0284] Besides the specifications, broadly described in #3 above for substitution of DMT group at the 5 ’-position or 3’- position of sugar 5 ’-protections such as MMT, & TMT protecting group. Various replacement of DMT group from either 5’ or 3’ position of sugar portion of nucleosides shall encompass to the present invention. Such protection of hydroxy function of nucleosides replacements were elegantly reported by the publication by E. F. Fisher & M. H. Caruthers; “Current Protocols in Nucleic Acid Chemistry (2000) 2.3.1-2.3.34 Copyright © 2000 by John Wiley & Sons, Inc.” The RNA oligomers could consist of natural or modified nucleobases, gapmers, phosphodiesters, phosphorothioates, phosphonates.

[0285] Subsequently, Z can be replaced selectively to 5’- monophosphate, 5’- diphosphate and 5’-triphosphtate oligonucleotides.

[0286] Ri is an alkyl or aryl radical;

[0287] R2 is an alkyl or aryl radical; and,

[0288] R3 is a cyanoethyl radical, alkyl radical or aryl radical.

[0289] Y is an oxygen atom or a Sulfur atom;

[0290] W is selected from the group consisting of an oxygen diradical, a N — H diradical and a fluorine radical, and R is selected so that, if W is an oxygen diradical, then R is (triisopropylsilyloxymethylene), TOM, TBDMS, PIVOM;

[0291] if W is a N-H diradical, then R is of the form R.5Xis selected from the group consisting of fluorenyl ethyloxycarbonyl (Fmoc), trifluoroacetyl, acetyl, alkanoyl, and aroyl; and if W is a fluorine radical, then R4 is not present.Z is a protecting group selected from the group consisting

[0292] of dimethoxytriphenylmethyl (DMT),

[0293] monomethoxytriphenylmethyl (MMT) and

[0294] trimethoxytriphenylmethyl (TMT).

[0295] W is represented by; O

[0296] R4 is represented by; TOM, TBDMS, PIVOM

[0297] M is represented by; Succinyl Solid support

[0298] [Compound: XXVIII]

[0299] Z is a protecting group selected from the group consisting of dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl

[0300] (MMT) and trimethoxytriphenylmethyl (TMT).

[0301] W is represented by; NH

[0302] R4 is represented by; Fmoc, Trifluoroacetyl, Acetyl, Alkanoyl, Aroyl

[0303] M is represented by; Succinyl Solid support

[0304] M is a hydrogen radical or if M is a linker, then it is represented by

[0305]

[0306] the formula K -C(O) and optionally connected to a solid Support

[0307] which is CPG, CPSG, Polystyrine, Nittophase.

[0308] [Compound: XXIX]

[0309] In Compound: XXVII, M is a hydrogen radical or a linker,

[0310] if M is a linker (LCAA), then it is represented by the formula K — C(O) and, optionally, connected to a solid Support which is CPG, CPSG, Polystyrine, Nittophase suitable for oligonucleotide synthesis,

[0311] Wherein; K is a chain of between 2 and 20 carbons, selected from the group consisting of alkyl, alkenyl, cycloalkyl, aryl, and aralkyl, in a hydrocarbyldiradical moiety, optionally comprising intervening — O —, — S, — S(O), — C(O) —, and — NR, where R is a hydrogen radical, or a Substituted C to Co alkyl or a substituted aralkyl;

[0312] In Compound: XXVIII,

[0313] W is selected from the group consisting of an oxygen diradical, a N — H diradical and a fluorine radical, and R4 is selected so that, if W is an oxygen diradical, then R4 is triisopropylsilyloxymethylene (TOM), TBDMS, PIVOM.

[0314] In Compound: XXIX,

[0315] if W is a N-H diradical, then R4 is of the form R5xis selected from the group consisting of fluorenyl ethyloxycarbonyl (Fmoc), trifluoroacetyl, acetyl, alkanoyl, and aroyl;

[0316] if W is a fluorine radical, then R4 is not present:

[0317] B is selected from the group of nucleoside base radicals consisting of 9-(N6-benzoyladeninyl), 9-(N-acetyl adeninyl), 9-(N6-tert butyl phenoxyacetyladeninyl)- 9-(N6-phenoxyacetyladeninyl), 9-(N6-isopropyl phe noxyacetyladeninyl), 1-(N4-benzoylcytosinyl), 1-(N4-acetylcytosinyl), 1-(N4(N. N-dimethylformamidi nyl)cytosinyl), l-(N4-phenoxyacetylcytosinyl), 1-(N4-tertbutylphenoxyacetylcytosinyl)-1-(N4-isopropyl phenoxyacetylcytosinyl), 9-(N2-isobutyrylguaninyl), 9-(N2-tertbutylphenoxyacetylguaninyl), 9-(N2-isopropylphenoxyacetylguaninyl)-N4-phenoxyacetylcytosinyl)- 1-(N4-tertbutylphenoxyacetylcytosinyl), 1-(N4-isopropyl phenoxyacetylcytosinyl), and 1-uracilyl or Bis a modified nucleoside base radical selected from the group consisting of 1-(N4-benzoyl-5-methylcytosinyl)- 1-(N4(N. N-dimethylformamidinyl)-5-methyl cytosinyl)- 1 -(N4-acetyl-5-methylcytosinyl)-l-(5-methyl-uracilyl), (5-fluoro-uracilyl)-l-(N4-benzoyl-5- fluorocytosinyl), 9-(N6-benzoyl-7-deazaadeninyl)- 9-(N2-(N, N-dimethylformamidinyl)-7-deazaadeninyl), 9-(N2-isobutyryl-7-deazaguaninyl), and 9-(N2(N. N dimethylformamidinyl)-7-deazaguaninyl).

[0318] LNA nucleoside, 2’-0Me nucleoside, 2’ -Fluoro nucleoside, 2’ -MOE nucleoside, 3’-0Me nucleoside, arabino nucleoside, 3 ’-Fluoro nucleoside, 3 ’-MOE nucleoside, 3 ’-PEG, ligand, chromophore, abasic sugar

[0319] Z is a protecting group selected from the group consisting of dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl (MMT) and trimethoxytriphenylmethyl (TMT).

[0320] Besides the specifications, broadly described in the #3 above for substitution of DMT group at the 5 ’-position or 3’- position of sugar 5 ’-protections such as MMT & TMT protecting group. Various replacement of DMT group from either 5’ or 3’ position of sugar portion of nucleosides shall encompass to the present invention. Such protection of hydroxy function of nucleosides replacements were elegantly reported by the publication by E. F. Fisher & M. H. Caruthers, Current Protocols in Nucleic Acid Chemistry (2000) 2.3.1-2.3.34 Copyright © 2000 by John Wiley & Sons, Inc. The RNA oligomers could consist of natural or modified nucleobases, gapmers, phosphodiesters, phosphorothioates, phosphonates.

[0321] 30. A process of synthesizing RNA oligomers represented by Compound XXX & XXXI wherein oligonucleotide is synthesized in the 3 '-to 5 '-direction,

[0322] No.

[0323] No.

[0324]

[0325] In Compound XXX & XXXI it should be noted as follows;

[0326] a) W is selected from the group consisting of an oxygen diradical, a N — H diradical and a fluorine radical, and R4 is selected so that,

[0327] if W is an oxygen diradical, then R4 is TOM (triisopropylsilyloxymethylene), TBDMS, PIVOM; if W is a N-H diradical, then R4 is of the form R5X, where x is selected from the group consisting of fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, acetyl, alkanoyl, and aroyl;

[0328] if W is a fluorine radical, then R4 is not present;

[0329] b) B is selected from the group of nucleoside base radicals consisting of 9-(N6-benzoyladeninyl), 9-(N6-acetyladeninyl), 9-(N6-tert butyl phenoxyacetyladeninyl)- 9-(N6-phenoxyacetyladeninyl), 9-(N6-isopropyl phenoxyacetyladeninyl)- 1-(N4-benzoylcytosinyl)-1-(N4acetylcytosinyl), 1-(N4(N. N- dimethylformamidi nyl)cytosinyl), l-(N4-phenoxyacetylcytosinyl), 1-(N4-tertbutylphenoxyacetylcytosinyl), 1-(N4-isopropyl phenoxyacetylcytosinyl), 9-(N2-isobutyrylguaninyl)-, 9-(N2-tertbutylphenoxyacetylguaninyl), 9-(N2-isopropylphenoxyacetylguaninyl)-N4-phenoxyacetylcytosinyl), 1-(N4-tertbutylphenoxyacetylcytosinyl), 1-(N4isopropyl phenoxyacetylcytosinyl), and 1 -uracilyl or B is a modified nucleoside base radical selected from the group consisting of 1-(N4-benzoyl-5-methylcytosinyl), 1-(N4-(N, N-dimethylformamidinyl)-5-methylcytosinyl), 1-(N4-acetyl-5-methylcytosinyl)- 1-(5-methyl-uracilyl), -(5-fluoro-uracilyl)-, 1-(N4- benzoyl-5-fluorocytosinyl), 9-(N6-benzoyl-7-dea Zaadeninyl), 9-(N6-(N, N-dimethylformamidinyl)- 7-deazaadeninyl), 9-(N6- 2

[0330] isobutyryl-7- deazaguaninyl) and 9-(N — (N. N- dimethylformamidinyl)-7-deazaguaninyl).

[0331] C) Z is a protecting group selected from the group consisting of dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl (MMT) and trimethoxytriphenylmethyl (TMT) Besides the specifications, broadly described in the #3 above for substitution of DMT group at the 5 ’-position or 3’- position of sugar 5 ’-protections such as MMT, & TMT protecting group. Various replacement of DMT group from either 5’ or 3’ position of sugar portion of nucleosides shall encompass to the present invention. Such protection of hydroxy function of nucleosides replacements were elegantly reported by the publication by E. F. Fisher & M. H. Caruthers, Current Protocols in Nucleic Acid Chemistry (2000) 2.3.1-2.3.34 Copyright © 2000 by John Wiley & Sons, Inc. The RNA oligomers could consist of natural or modified nucleobases, gapmers, phosphodiesters, phosphorothioates, phosphonates.

[0332] Subsequently, Z can be replaced selectively to 5’- monophosphate, 5’- diphosphate and 5’-triphosphtate oligonucleotide;O W-R4

[0333] O^-P=O _

[0334] I

[0335] O W-R;

[0336] O^-P=O

[0337]

[0338] M-O W-R4

[0339] Where; P can be monophosphate, diphosphate and triphosphate

[0340] W is represented by; O

[0341] R4 is represented by; TOM, TBDMS, PIVOM

[0342] M is represented by; Succinyl Solid support.

[0343] n1is mono nucleotide

[0344] n2is dimer nucleotide

[0345] [Compound: XXXII]

[0346] Standard literature having the procedure for Phosphate addition.

[0347] n is an integer between 2 and 120; and wherein, the process of RNA synthesis in the 3'->5' direction com prises the steps of:

[0348] (a) providing a Support attached nucleoside represented by Compound XXVIII & XXIX;

[0349] (b) placing one or more appropriate phosphoramidite represented by Compound XXVII in an oligonucleotide synthesize;

[0350] (c) performing oligonucleotide synthesis and coupling on the synthesize;

[0351] (d) detaching synthesized oligonucleotide from the solid Support;

[0352] (e) removing the base protecting group or groups, if present, from the oligonucleotide;

[0353] (f) removing the Sugar silyl protecting group or groups;

[0354] (g) precipitating crude oligonucleotide;

[0355] (h) optionally, analysing the crude oligonucleotide for purity;

[0356] Here we are using dimers for long RNA oligonucleotide containing the 2’-O-triisopropylsilyloxymethyl (TOM) group is characterized by very high coupling efficiency along with fast, simple deprotection. High coupling efficiency is achieved because the TOM-Protecting- Group exhibits lower steric hindrance than the 2’-O-t-butyldimethylsilyl (TBDMS) group used in current method of stepwise RNA Synthesis.

[0357] A further feature of the TOM-protecting-group is that during basic steps it cannot undergo 2’ to 3’ migration. This migration under basic conditions leads to non-biologically active 2’-5’ linkages when using the TBDMS group. These features allow the TOM-protected dimer to produce longer oligonucleotides.

[0358] The use of homo and hetero-dimer nucleotide blocks in the process is crucial to achieve the synthesis of long RNA in the range of about 100-mer to about 200-mer.

[0359] Examples:

[0360] The following examples are provided to illustrate the invention and are not intended to limit the scope of the claims. Unless otherwise indicated, standard reagents and procedures known in the field of oligonucleotide chemistry were employed.

[0361] The Inventor used all 16 combination of dimer nucleoside amidite a block to achieve the synthesis of Long RNA. We prepared Four Homo-dimer nucleosides amidite blocks and Twelve Hetero-Dimer blocks. Each Dimer was linked by a phosphodiester bond. The synthesis of dimer nucleosides amidite blocks involves several key steps. Suitable nucleosides (e.g., Adenine (A), Cytosine (C), Guanine (G), and Uracil (U.) are selected based on the desired RNA sequence. Then, each nucleotide is chemically activated by Ethyl thio tetrazole to facilitate coupling with another nucleoside under controlled conditions to form the homo-dimer nucleoside. The coupling reaction results in the formation of a stable phosphodiester linkage between the two nucleosides, ensuring integrity during RNA synthesis. Oxidize the phosphite triester linkage to a stable phosphodiester bond using iodine in water or pyridine.

[0362] Purify the synthesized dimer block using chromatography techniques to ensure high purity.

[0363] Synthesis of Dimer nucleoside phosphoramidite: Dimer was activated by adding the diisopropyl ammonium tetrazolium salt 1g (1.2 eq.) and then 2-cyanoethyl N, N, N, N-tetra isopropyl phosphoramidite 2.1ml (146mmol) was added under argon. The product was purified by column chromatography, using fuji fine silica gel.

[0364] Example 1: Synthesis of Dimer [U-G]-OP

[0365] Synthesis of 5’ -O-dimethoxy trityl- P-cy anoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) uridyl-3’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl guanosineScheme for the synthesis of Dimer [U-G]-OP

[0366] Ila Hid

[0367] Compound na;5'-O-DMT-3'-CNEt Compound Did; 5'-3'-dihydroxy- phosphoramidite 2'-TOM-rU 2'-TOM-rG (N-Ac)

[0368]

[0369] Compound VIc;5'-O-DMT-2'-TOM-rU- Compound Xc; 5’-O-DMT-2'-TOM-rU- 3'-5’-CNEt phosphate -3'-hydroxy- 3' -5’-CNEt phosphate -3'-CNEt

[0370] 2'-TOM-rG (N-Ac) phosphoramidite-2'-TOM-rG(N-Ac)

[0371] Scheme; (7) Synthesis of Dimer [U-G]-OP (Compound Xc)

[0372] Step-A: Synthesis of 5’-O-dimethoxytrityl-uridine-2’-O-triisopropylsilyloxymethyl-3’-[(2-cyanoethyl)- (N, N-diisopropyl)] phosphoramidite (Compound II a)

[0373] Compound la; 5 ’ -O-dimethoxytrityl-2’ -O-triisopropylsilyloxymethyl-3 ’ -hydroxy-uridine, 50g (136.43mmol) was dissolved in 500ml acetonitrile. The activation solution (Ethyl Thio Tetrazole)17.75g (272.72mmol) / 2eq. was added in above solution under argon atmosphere. Then subsequently DIPEA 24ml (413 mmol) and 1-methyl imidazole 4.9ml (272mmol), followed by 2-cyanoethyl N, N, N, N-tetra isopropyl phosphoramidite(92ml), (545mmol) was added under argon atmosphere. The reaction was stirrer for 90 minutes between 20-27°C. The progress of reaction was monitored through TLC. After completion of reaction 500ml saturated solution of aq. sodium bicarbonate was added, the aq. layer was removed, followed by washing with saturated solution of aq. sodium chloride (500ml). The organic layer was dried by passing over anhydrous sodium sulfate, followed by concentration of the organic layer. The product was purified by column chromatography, using silica gel (230-400 mesh size), and solvent system hexane: ethylacetate: triethyl amine;50:40:10. The yield was 58 g, (yield;84%). The Rf of crude compound II d; 0.7, UV (λmax-250nm,0.239), (εmax-13497.2)

[0374] Step-B; Synthesis of 5’-3’-O-dihydroxy-2’-O- triisopropylsilyloxymethyl guanosine (Compound III d)Compound Id, 5’-O-dimethoxytrityl -2’-O-triisopropylsilyloxymethyl-3’- hydroxy-N-acetyl-guanosine, 100g (136mmol) was dissolved in 1000ml of DCM. Trifluroacetic acid 15ml (316.66 mmol) in above solution was added at 20-27°C. The reaction mixture was stirrer for 90 minutes between 20-27°C. The progress of reaction was monitored through TLC. After completion of the reaction, 1000ml saturated solution of aq. sodium bicarbonate was added, the aq. layer was removed and followed by washing with saturated solution of aq. sodium chloride (1000ml). The organic layer was dried with passed over anhydrous sodium sulfate, followed by concentration of the organic layer. The product was purified by column chromatography, using silica gel (230-400 mesh size), and solvent system consisting of Chloroform: Hexane: Acetone + Methanol;50:30:20+5%. The yield was 10 g. The Percent Yield was 16%. The Rf of crude compound III d; 0.4, UV (λmax- 255nm,0.719), (εmax- 17658.06)

[0375] Step-C; Synthesis of 5’-O-dimethoxytrityl-2’-O-(triisopropylsilyloxymethyl) uridyl -3’-O-[cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’-hydroxy guanosine (Compound VI c)

[0376] Compound II a, 5 ’-dimethoxytrityl -uridine -2’-O-triisopropylsilyloxymethyl-3’-[(2-cyanoethyl)-(N, N-diisopropyl)] phosphoramidite,10g (102.64mmole) was dissolved in acetonitrile (100ml). The activator solution (ethyl thio tetrazole), 2.78g (250mmole) / 2eq. was added in above solution under argon atmosphere, followed by addition of Compound IHd; 5’-3’-O-dihydroxy-2’-O-triisopropylsilyloxymethyl N-acetyl-guanosine, 8.22g (200mmole) / 1.5eq. under argon atmosphere. The reaction was stirred for 30minutes between 20-27°c. The progress of reaction was monitored through TLC. Then the iodine oxidation solution (4.56g; 0.2mole) was added. The progress of reaction was checked after 2 minutes by TLC. After completion of 2 minutes of oxidation 100ml saturated sodium bisulphite solution was added to neutralize the excess iodine. After completion of reaction, a 100ml saturated solution of aq. sodium chloride. The organic layer was dried with passed over anhydrous sodium sulfate, followed by concentration of the organic layer. The product was purified by column chromatography, using silica gel (230-400 mesh size), and a solvent system consisting of chloroform: acetone: hexane;50:40:10. The yield was 5g (yield; 33%) The Rf of crude compound VI a; 0.35. UV (λmax- 250nm, 0.335), (εmax-25304.03),32P-NMR (CDCl3), δ ppm-(-3.095, -3.145)

[0377] Step-D; Synthesis of 5’-O-dimethoxytrityl- P-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) uridyl-3’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl guanosine (Compound Xc)

[0378] Compound VI c; 5’-O-dimethoxytrityl-2’-O-(triisopropylsilyloxymethyl) uridyl -3’-O-[cyanoethoxyphosphino] (3 ’ -5 ’)-N-acetyl-2’ -O-triisopropylsilyloxymethyl-3 ’ -hydroxy guanosine, 5g (was dissolved in 50ml ACN. The activator solution (ethyl thio tetrazole), 0.957g 0.25M and DIPEA 1.60 ml (2.5eq) was added in to the compound. Followed by addition of 2-cyanoethyl N, N, N, N-tetra isopropyl phosphoramidite 2.21ml (3.10M) was added under argon.The reaction was stirred for 45 minutes between 20-27°c. The progress of reaction was checked by TLC. After completion of the reaction, a saturated solution of aq. sodium bicarbonate (50 ml) was added, the aq. layer was removed and followed by washing with saturated solution of aq. sodium chloride (50ml). The organic layer was dried by passing over anhydrous sodium sulfate, followed by concentration of the organic layer. The product was purified by column chromatography, using silica gel (Fuji-fine), and solvent system consisting of Chloroform: Acetone;(50:50) The yield was 3g, (yield;52.26%). The Rf of crude Compound V d; 0.5. UV (λmax- 250nm, 0.265), (εmax-23768.38)

[0379] 32P-NMR (CDCl3), δ ppm-(152.108, 151.511,150.165,149.879, -2.716, -3.414, -3.566, -3.801, -3.944)

[0380] QC data (HPLC, Mass & UV)

[0381] Table No. 1

[0382]

[0383] Table No. 2The reaction was stirred for 45 minutes between 20-27°c. The progress of reaction was checked by TLC. After completion of the reaction, a saturated solution of aq. sodium bicarbonate (50 ml) was added, the aq. layer was removed and followed by washing with saturated solution of aq. sodium chloride (50ml), The organic layer was dried by passing over anhydrous sodium sulfate, followed by concentration of the organic layer. The product was purified by column chromatography, using silica gel (Fuji-fine), and solvent system consisting of Chloroform: Acetone;(50:50) The yield was 3g, (yield;52.26%). The Rf of crude Compound V d; 0.5. UV (λmax- 250nm, 0.265), (εmax-23768.38)

[0384] 32P-NMR (CDCl3), δ ppm-(152.108, 151.511,150.165,149.879, -2.716, -3.414, -3.566, -3.801, -3.944)

[0385] QC data (HPLC, Mass & U V )

[0386] Dimer [U-U]-OP

[0387]

[0388]

[0389] Table No. 2Table No. 3

[0390] Dimer [A-A]-OP HPLC UV (methanol)

[0391] Table No. 5 MF‘l£ Mass UV (methanol)

[0392]

[0393] Table No. 6

[0394]

[0395]

[0396] Dimer [U-G]-OP

[0397] HPLC Mass UV (methanol):

[0398]

[0399] TableNo.8

[0400] HPLC Mass UV (methanol)

[0401]

[0402]

[0403]

[0404] Table No.9

[0405] Table No.10

[0406]

[0407] Table No.11

[0408] Dimer[A-C]-OP

[0409] UV (methanol):

[0410] Peaks observed

[0411]

[0412] Table No.12

[0413] Dimer [A-G]-OP HPLC Mass UV (methanol)

[0414] Table No.13 Dimer [G-U]-OP

[0415] Table No.14 Dimer [G-C]-OP HPLC Mass UV (methanol)

[0416] Product Name

[0417]

[0418] Table No.15

[0419]

[0420] Example 2: Solid-Phase Synthesis of RNA Oligonucleotides

[0421] Solid Support:

[0422] Functionalization: Use controlled pore glass (CPG) of different size depend upon length of oligo, like 1000 A or 2000A as the solid support, functionalized with a cleavable linker.

[0423] 5’ DMT-dt-CPSG-1000 A, Trityl value-69umol

[0424] 5’DMT-dt-CPSG-2000 A, Trityl value-25, umol

[0425] Oligonucleotides seq. (10-mer, 20-mer, 120-mer) were synthesized using 3 ’-5’ directed in lumole scale. The synthesis was performed on Expedite 8909 Synthesizer using TOM RNA lumole cycle and Coupling time of dimer with solid support 1.5 minute.

[0426] First take the dimer amidite in 30 ml expedite bottle and dissolve in dry acetonitrile to make the solution 0.06M. After that, attach the dimer bottle to the synthesizer on their port.

[0427] Example 3: Oligonucleotide synthesis

[0428] Attachment of first dimer amidite block: Attach the first 2-O-TOM (N-Ac) dimer block to die solid support.

[0429] Deprotection of DMT group from first nucleoside containing CPG at the 5'-end of the using 3% trichloroacetic acid (TCA) in dichloromethane (DCM).and then Activation of dimer blocks by 5- ethylthio-1-H-tetrazole, 0.25 M and coupled with nucleoside attach CPG. Continue the activation of the 3‘-phosphoraimdite group of the next dimer block and couple it to the growing RN A chain on the solid support. Then acetylate any unreacted 5'-OH groups using acetic anhydride and N- methylirnidazole to prevent deletion sequences with the use of CAP A (acetic anhydride / tetrahydroluran / pyridine), CAP B (10% N-methylimidazole / tetrahydrofuran). Oxidize the phosphite triester linkage to a stable phosphodiester bond using oxidation solution (0.02 M iodine / pyridine / water / tetrahydro furan).

[0430] Repeat the deprotection, coupling, capping, and oxidation steps for each additional dimer block until the desired RNA sequence is synthesized.The invention is applicable to large-scale synthesis of RNA oligonucleotides for research, diagnostics, and therapeutic use, including mRNA vaccines, siRNA, sgRNA, prime-editing guide RNA (pegRNA) antisense oligonucleotides, and RNA conjugates.

[0431] Common name and Chemical name

[0432] [Compound -Via]

[0433] Common name; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate -3’ -hydroxy-2’ -TOM-rC (N-Ac) Chemical name;5’-O-dimethoxytrityl-2’-O-(triisopropylsilyloxymethyl) uridyl -3’-O-[cyanoethoxyphosphino] (3’ -5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy-cytidine [Compound -Xa]

[0434] Common name; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rC(N-Ac)

[0435] Chemical name;5’-O-dimethoxytrityl- P-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) uridyl-3 ’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl cytidine

[0436] 6. Dimer (U-A)

[0437] Common name and Chemical name

[0438] [Compound -VIb]

[0439] Common name; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate -3’ -hydroxy-2’ -TOM-rA (N-Ac) Chemical name;5’-O-dimethoxytrityl-2’-O-(triisopropylsilyloxymethyl) uridyl -3’-O-[cyanoethoxyphosphino] (3’ -5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy adenosine [Compound -Xb]

[0440] Common name; 5’-O-DMT-2’-TOM-rU-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rA(N-Ac)

[0441] Chemical name; 5’-O-dimethoxytrityl- P-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) uridyl-3 ’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl

[0442] 7. Dimer (U-G)

[0443] Common name and Chemical name

[0444] [Compound -Vic]

[0445] Common name; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate -3’ -hydroxy-2’ -TOM-rG (N-Ac)Chemical name; 5’-O-dimethoxytrityl-2’-O-(triisopropylsilyloxymethyl) uridyl -3’-0-[cyanoethoxyphosphino] (3’ -5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy guanosine [Compound -Xc]

[0446] Common name; 5’-O-DMT-2’-TOM-rU-3’-5’-CNEt phosphate-3’-CNEt phosphoramidite-2 ’-TOM-rG(N-Ac)

[0447] Chemical name; 5’-O-dimethoxytrityl- P-cyanoethylphosphoryl -2’-0-( triisopropylsilyloxymethyl) uridyl-3 ’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl guanosine

[0448] 8. Dimer (C-U)

[0449] Common name and Chemical name

[0450] [Compound -Vila]

[0451] Common name; 5’-O-DMT-2’-TOM-rC(N-Ac)-3’-5’-CNEt phosphate -3’ -hydroxy-2 ’-TOM-rU Chemical name; 5’-O-dimethoxytrityl-N-acetyl— 2’-O-(triisopropylsilyloxymethyl) cytidyl -3’-0- [cyanoethoxyphosphino] (3 ’-5’)- 2 ’-O-triisopropylsilyloxymethyl-3’ -hydroxy-uridine [Compound -Xia]

[0452] Common name; 5’-O-DMT-2’-TOM- rC(N-Ac)-3 ’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2 ’ -TOM-rU

[0453] Chemical name; 5’-O-dimethoxytrityl- N-Acetyl -p-cyanoethylphosphoryl -2’-0-( triisopropylsilyloxymethyl) cytidyl -3’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-2’-O-triisopropylsilyloxymethyl uridine

[0454] 9. Dimer [C-A]

[0455] Common name and Chemical name

[0456] [Compound -Vllb]

[0457] Common name; 5’-O-DMT-2’-TOM-rC(N-Ac)-3’-5’-CNEt phosphate-3’ -hydroxy-2’ -TOM-rA(N-Ac)

[0458] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) cytidyl-3’-O-[cyanoethoxyphosphino] (3 ’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy-adenosine

[0459] [Compound -Xlb]

[0460] Common name; 5’-O-DMT-2’-TOM-rC (N-Ac)-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2 ’-TOM-r A (N-Ac)Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-0-( triisopropylsilyloxymethyl) cytidyl -3’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl adenosine

[0461] 10. Dimer [C-G]

[0462] Common name and Chemical name

[0463] [Compound -Vile]

[0464] Common name; 5’-O-DMT-2’-TOM-rC(N-Ac)-3’-5’-CNEt phosphate-3 ’-hydroxy-2’ -TOM-rG(N-Ac)

[0465] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) cytidyl-3’-O-[cyanoethoxyphosphino] (3 ’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy-guanosine

[0466] [Compound -XIc]

[0467] Common name; 5’-O-DMT-2’-TOM-rC (N-Ac)-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rG (N-Ac)

[0468] Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) cytidyl -3’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl guanosine

[0469] 11. Dimer (A-U)

[0470] Common name and Chemical name

[0471] [Compound -Villa]

[0472] Common name; 5’-O-DMT-2’-TOM-rA (N-Ac)-3’ -5 ’-CNEt phosphate -3 ’-hydroxy-2 ’-TOM-rU

[0473] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) adenyl -3’-O-[cyanoethoxyphosphino] (3 ’ -5 ’)-2’ -O-triisopropylsilyloxymethyl-3 ’ -hydroxy-uridine [Compound -XIIa]

[0474] Common name;5-O-DMT-2’-TOM-rA(N-Ac)-3’-5’-CNEt phosphate-3 ’-CNEt phosphoramidite-2’-TOM-rU

[0475] Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) adenyl -3’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-2’-O-triisopropylsilyloxymethyl uridine

[0476] 12. Dimer (A-C)

[0477] Common name and Chemical name

[0478] [Compound -VIIIb]Common name; 5’-O-DMT-2’-TOM-rA(N-Ac)-3’-5’-CNEt phosphate-3 ’-hydroxy-2’ -TOM-rC(N-Ac)

[0479] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) adenyl -3’-O-[cyanoethoxyphosphino] (3’ -5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy-cytidine [Compound -XIIb]

[0480] Common name; 5’-O-DMT-2’-TOM-rA (N-Ac)-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rC (N-Ac)

[0481] Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) adenyl -3’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl cytidine

[0482] 13. Dimer (A-G)

[0483] Common name and Chemical name

[0484] [Compound -VIIIc]

[0485] Common name; 5 ’ -O-DMT-2’ -TOM-rA(N-Ac)-3 ’ -5 ’ -CNEt phosphate-3 ’ -hydroxy-2’ -TOM-rG(N-Ac)

[0486] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) adenyl-3’-O-[cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl -3 ’-hydroxy-guanosine

[0487] [Compound -XIIc]

[0488] Common name; 5’-O-DMT-2’-TOM-rA (N-Ac)-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rG (N-Ac)

[0489] Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-O-( triisopropylsilyloxymethyl) adenyl-3’-O- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethylene guanosine

[0490] 14. Dimer (G-U)

[0491] Common name and Chemical name

[0492] [Compound -IXa]

[0493] Common name; 5’-O-DMT-2’-TOM-rG (N-Ac)-3’-5’-CNEt phosphate-3’ -hydroxy-2’ -TOM-rU Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) guanosyl -3’-O- [cyanoethoxyphosphino] (3 ’ -5’)-2’ -O-triisopropylsilyloxymethyl-3 ’ -hydroxy-uridine [Compound -XIIIa]

[0494] Common name; 5’-O-DMT-2’-TOM-rG (N-Ac)-3’-5’-CNEt phosphate-3 ’-CNEt phosphoramidite-2 ’ -TOM-rUChemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-0-( triisopropylsilyloxymethyl) guanosyl -3’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-2’-O-triisopropylsilyloxymethyl uridine

[0495] 15. Dimer (G-C)

[0496] Common name and Chemical name

[0497] [Compound -IXb]

[0498] Common name;5’-O-DMT-2’-TOM-rG(N-Ac)-3’-5’-CNEt phosphate -3 ’-hydroxy-2 ’-TOM-rC(N-Ac)

[0499] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) guanosyl -3’-0- [cyanoethoxyphosphino] (3 ’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’ -hydroxy-cytidine

[0500] [Compound -XIIIb]

[0501] Common name; 5’-O-DMT-2’-TOM-rG (N-Ac)-3’ -5’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rC (N-Ac)

[0502] Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-0-( triisopropylsilyloxymethyl) guanosyl -3’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3 ’ -5 ’ )-N- acetyl-2 ’ -0 -triisopropylsilyloxymethyl cytidine

[0503] 16. Dimer (G-A)

[0504] Common name and Chemical name

[0505] [Compound -IXc]

[0506] Common name; 5 ’ -O-DMT-2’ -T0M-rG(N-Ac)-3 ’ -5 ’ -CNEt phosphate-3 ’ -hydroxy-2’ -TOM-rA(N-Ac)

[0507] Chemical name; 5’-O-dimethoxytrityl-N-acetyl-2’-O-(triisopropylsilyloxymethyl) guanosyl -3’-0- [cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl-3’-hydroxy-adinosine

[0508] [Compound -XIIIc]

[0509] Common name; 5’-O-DMT-2’-TOM-rG (N-Ac)-3’ -5 ’-CNEt phosphate -3’-CNEt phosphoramidite-2’-TOM-rA (N-Ac)

[0510] Chemical name; 5’-O-dimethoxytrityl-N-Acetyl- p-cyanoethylphosphoryl -2’-0-( triisopropylsilyloxymethyl) guanosyl -3’-0- [(N, N-diisopropylamino) cyanoethoxyphosphino] (3’-5’)-N-acetyl-2’-O-triisopropylsilyloxymethyl adenosine

Claims

We claim:

1. A method for synthesizing an RNA oligonucleotide, comprising:(a) providing a solid support having a first ribonucleoside attached thereto;(b) sequentially coupling to the solid support pre-formed dimer ribonucleoside phosphoramidite blocks, each dimer block comprising two ribonucleosides joined by an internucleotide phosphate linkage;(c) wherein each ribonucleoside comprises a 2'-O-triisopropylsilyloxymethyl (2'-O- TOM) protecting group;(d) oxidizing or sulfurizing the internucleotide linkage after each coupling step; and (e) deprotecting the assembled RNA to yield an RNA oligonucleotide having a length of from 16 nucleotides to at least 220 nucleotides;the said RNA oligonucleotide comprise oligomers having lengths selected from 5-mer to 220-mer;the ribonucleosides further comprising:(a) 2'-0-T0M protecting groups on the ribose moiety; and(b) N-acetyl base-protecting groups or a labile base protecting group, which together allow high coupling efficiency and mild deprotection conditions and the base-protecting group is selected from acetyl, fluorenylmethyloxycarbonyl (Fmoc), phenoxyacetyl, levulinyl, pivaloyl, or combinations thereof.

2. The method of claim 1, wherein the dimer ribonucleoside phosphoramidite blocks are selected from:(a) homo-dimer blocks comprising two identical ribonucleosides; or(b) hetero-dimer blocks comprising two different ribonucleosides.

3. The method of claim 1, wherein the RNA oligonucleotide has a length of greater than 100 nucleotides, preferably from 150 nucleotidesto 220 nucleotides.

4. The method of claim 1, wherein the solid support is selected from controlled pore glass (CPG), controlled pore silica gel (CPSG), polystyrene, nittophase supports, or other supports suitable for solid-phase oligonucleotide synthesis.

5. The method of claim 1, wherein each synthesis cycle comprises steps as illustrated in Scheme 6..

6. A dimer ribonucleoside phosphoramidite block suitable for RNA synthesis, comprising: (a) two ribonucleosides linked by a phosphodiester or phosphorothioate bond;(b) ribose moieties protected with 2'-O-triisopropylsilyloxymethyl groups; and(c) a terminal phosphoramidite group enabling solid-phase coupling.

7. The dimer ribonucleoside phosphoramidite block of claim 6, wherein ribonucleoside comprises a modified sugar or nucleobase selected from 2'-fluoro, 2'-O-methyl, 2'-M0E, LNA, FANA, ANA, 4'-thio, halogenated, alkylated, propargylated, or methylated nucleosides.

8. A synthetic RNA oligonucleotide produced by the method of claim 1, wherein the RNA comprises phosphodiester or phosphorothioate linkages, a 5 '-mono-, di-, or triphosphate group and has a defined nucleotide sequence.

9. The synthetic RNA oligonucleotide of claim 8, wherein the RNA is conjugated at a 3 '-end, or internal position to a ligand selected from lipids, fatty acids, polyethylene glycol (PEG), peptides, fluorophores, chromophores, antibodies, drugs, or targeting moieties.

10. The synthetic RNA oligonucleotide of claim 8, wherein the RNA comprises a 5' cap analog selected from a di -phosphate or tri-phosphate cap structure linked via a 5 '-5' phosphodiester bond.

11. The synthetic RNA oligonucleotide of claim 10, wherein the cap analog is selected from: (a) m7GppG;(b) m7GpppG; or(c) m7GpppNm (Cap-1 structure).

12. The synthetic RNA oligonucleotide of claim 8, wherein the RNA is an uncapped 5'-triphosphate RNA (pppRNA) capable of inducing an innate immune response.

13. The synthetic RNA oligonucleotide of claim 8, wherein the RNA is bioconjugated via an alkyne-based or click chemistry linkage at the 3 '-end, 5 '-end, or internal position to dyes, PEGs, drugs, polymers, lipids, or targeting ligands.

14. Use of the synthetic RNA oligonucleotide of claim 8 as a guide RNA (gRNA), singleguide RNA (sgRNA), or prime-editing guide RNA (pegRNA) in a CRISPR-Cas genomeediting system.

15. Use of the synthetic RNA oligonucleotide of claim 8 for therapeutic or prophylactic applications, including mRNA therapy, RNA interference, vaccination, gene regulation, or gene therapy.

16. Use of the synthetic RNA oligonucleotide of claim 8, wherein the RNA is a long noncoding RNA (IncRNA) of greater than 200 nucleotides, lacking protein-coding function, for regulating chromatin remodeling, transcription, cell differentiation, inflammation, or disease mechanisms.

17. Use of the synthetic RNA oligonucleotide of claim 8, wherein the RNA is conjugated to one or more antibodies to form bar-coded or multiplexed RNA- antibody constructs for diagnostic or therapeutic applications.

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