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WO2025231484A1 - Procédés de préparation d'arnm coiffé avec des modifications spécifiques à un site - Google Patents

Procédés de préparation d'arnm coiffé avec des modifications spécifiques à un site

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Publication number
WO2025231484A1
WO2025231484A1 PCT/US2025/027793 US2025027793W WO2025231484A1 WO 2025231484 A1 WO2025231484 A1 WO 2025231484A1 US 2025027793 W US2025027793 W US 2025027793W WO 2025231484 A1 WO2025231484 A1 WO 2025231484A1
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Prior art keywords
rna
capped
capping
methyl
bases
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Inventor
Fengmei PI
Robert Dempcy
Khoi Ngoc Anh HA
Cheng-Hsien Wu
Dan Liu
Yu Chen
Shambhavi Shubham
Maria Andrea MAKALINAO TIU
Zongyuan Liu
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Genscript Usa Inc
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Genscript Usa Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03033—Polynucleotide 5'-phosphatase (3.1.3.33)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/0705—Nucleotidyltransferases (2.7.7) mRNA guanylyltransferase (2.7.7.50)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/01—Hydrolases acting on acid anhydrides (3.6) in phosphorus-containing anhydrides (3.6.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01003—RNA ligase (ATP) (6.5.1.3)

Definitions

  • Eukaryotic mRNA has been known to have a 5 '-capped structure, in which 7- methylguanosine forms 5 '-5' bond with the 5 '-end via a triphosphate bond.
  • the cap structure has been known to be essential for ribosome recruiting in cap dependent translation of mRNA, and has therefore been desired to be efficiently introduced into mRNA, in order to make the mRNA functional for a target protein expression.
  • Embodiments of the present disclosure may include methods of chemically capping the 5’ end of polynucleotides, such as RNA and DNA.
  • the scope of the present disclosure is not limited to chemically capping methods disclosed herein. Any chemically capping methods that are capable of capping the 5’ end of polynucleotides fall into the scope of the present disclosure.
  • embodiments of the present disclosure may include methods for producing a site-specific modified capped recombinant RNA, including: (a) obtaining a first RNAthat is monophosphorylated at the 5’ end, in which the first RNA comprises the sitespecific modification, (b) capping the 5’ monophosphorylated first RNA obtained from (a) to produce a 5’ capped first RNA, (c) obtaining a second RNAthat is monophosphorylated at the 5’ end, and (d) ligating the 3’ end of the 5’ capped first RNA obtained from (b) to the 5’ end of the second RNA obtained from (c), thereby producing the site-specific modified capped recombinant RNA.
  • embodiments of the present disclosure may also include methods for producing a site-specific modified capped recombinant RNA containing > 150 bases, including: (a) obtaining an RNAthat is monophosphorylated at the 5’ end, in which the RNA may include the site-specific modification, (b) capping the 5’ monophosphorylated RNA obtained from (a) to produce a 5’ capped RNA, thereby producing the site-specific modified capped recombinant RNA, in which the methods may be performed without ligation step.
  • embodiments of the present disclosure may include methods for producing a site-specific modified capped recombinant RNA, including: (a) obtaining a first RNAthat is monophosphorylated at the 5’ end and comprises the site-specific modification, (b) reacting an activated capping compound of Formula (I) with the first RNA of (a) in the presence of a heteroaromatic compound and/or a metal salt, and a solvent:
  • site specific modification may contain at least one modified nucleotide including a backbone modification, a sugar modification, and/or a base modification.
  • the first RNA of (a) may be obtained by a chemical synthesis.
  • the first RNA obtained from (a) may contain the site-specific modification selected from phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates, phosphotriesters, and a combination thereof.
  • the second RNA of (c) may be obtained by treating a triphosphorylated RNA produced by in vitro transcription in the presence of a RNA 5’ polyphosphatase or RNA 5’ pyrophosphohydrolase (RppH).
  • in vitro transcription may be performed in a reaction mixture containing a buffer substance in a concentration of from about 45 mM to about 55 mM, an RNase inhibitor in a concentration of from about 0.01 U/pl to about 0.03 U/pl, NTPs in a concentration of from about 3 mM to about 5 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1.5 mM to about 2.5 mM, DNA template in a concentration of from about 0.01 pg/pl to about 0.05 pg/pl, optionally, and an RNA polymerase in a concentration of from about 0.01 pg/pl to about 0.05 pg/pl.
  • in vitro transcription may be performed at from about 15°C to about 35°C or from about 35°C to about 37°C for from about 1 hour to about 12 hours.
  • in vitro transcription may be performed in the presence of Nl- Methyl-PseudoUTP
  • ligating may be performed in the presence of an RNA ligase or a DNA ligase and optionally a DNA splint that hybridizes with at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of a 3’ end region of the 5’ capped first RNA obtained from (b) and with at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of a 5’ end region of the second RNA obtained from (c).
  • methods of the present disclosure may further include (e) purifying the capped recombinant RNA.
  • activated capping compound may contain a compound represented by Formula (II), Formula (H) in which
  • metal salt may contain calcium salt.
  • the reacting may be performed at 30 to 60° C.
  • the reacting may be performed for 1 to 25 hours.
  • the solvent may contain an organic solvent that contains 0 to 20% by weight of water.
  • the concentration of the activated capping compound may be 5 to 30 mM.
  • the heteroaromatic compound may contain 1 -methylimidazole.
  • the 5’ chemically capped recombinant RNA may contain > 150 bases, > 200 bases, > 250 bases, > 300 bases, > 350 bases, > 400 bases, > 450 bases, > 500 bases, > 550 bases, > 600 bases, > 650 bases, > 700 bases, > 750 bases, > 800 bases, > 850 bases, > 900 bases, > 950 bases, > 1000 bases or > 1500 bases, which may be produced without ligation step .
  • the RNA ligase may contain T4 RNA ligase I, T4 RNA ligase II, TS2126 RNA ligase, and/or DNAzyme.
  • the DNA ligase may contain T4 DNA ligase.
  • the ligating may be performed at a ratio of the 5’ capped first RNA obtained from (b) to the second RNA obtained from (c) 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1.
  • the ligating may be performed in the presence of T4 RNA ligase I in the absence of splints.
  • base modification may contain inverted dT.
  • the second RNA may be produced by in vitro transcription performed in the presence of GMP and GTP at a ratio of GMP to GTP 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, or 20:1.
  • the in vitro transcription may be performed in the absence of a RNA 5’ polyphosphatase or RppH.
  • the purifying may be performed by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis.
  • embodiments of the present disclosure may include methods for producing a site-specific modified capped recombinant RNA, including: (a) obtaining a first RNA that is monophosphorylated at the 5’ end, in which the first RNA may contain the sitespecific modification, (b) reacting an activated capping compound of Formula (III) with the first RNA of (a) in the presence of a heteroaromatic compound and/or a metal salt, and a solvent:
  • X may be a nitrogenous base
  • R1 and/or R2 O-alkyl, halogen, a linker, hydrogen or a hydroxyl; n may be any integer from 1-9; and the capping compound may be a single stereoisomer or plurality of stereoisomers of one or more of the compounds described by Formula (III) or a salt or salts thereof, and
  • the nitrogenous base of the capping compound may be selected from the group consisting guanine, adenine, cytosine, uracil and hypoxanthine and analogs of guanine, adenine, cytosine, uracil and hypoxanthine.
  • the nitrogenous base of the capping compound may contain a modified base selected from N6-methyladenine, N1 -methyladenine, N6-2'-O-dimethyladenosine, pseudouridine, N1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2-thiouridine, 5- methyluridine, pseudoisocytosine, 5 -methoxy cytosine, 2-thiocytosine, 5 -hydroxy cytosine, N4-methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, Nl-methylguanine, 06- methylguanine, 1-methyl-guanosine, N2-methyl-guanosine, N7-methyl-guanosine, N2,N2- dimethyl-guanosine, 2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl-2'-O-methyl- guanosine, l-methyl-2'-O-methyl-guanosine,
  • the nitrogenous base of the capping compound may be attached to a sugar consisting of a ribose or a modified ribose selected from 2'- or 3'-O-alkylribose, alkoxyribose, O-alkoxyalkylribose, fluororibose, azidoribose, allylribose, deoxyribose; an arabinose or a modified arabinose; a thioribose; an 1,5 anhydrohexitol; or a threofuranose.
  • a ribose or a modified ribose selected from 2'- or 3'-O-alkylribose, alkoxyribose, O-alkoxyalkylribose, fluororibose, azidoribose, allylribose, deoxyribose; an arabinose or a modified arabinose; a thi
  • the one or more phosphates of the capping compound may contain a phosphorothioate; a phosphorodithioate; an alkyphosphonate; an arylphosphonate; a N- phosphoramidate; a boranophosphate; or a phosphonoacetate.
  • the capping compound may contain guanosine.
  • the second RNA may contain a poly A tail.
  • the leaving group L in Formula (I) and Formular (III) may contain a heteroaromatic ring compound selected from imidazole, pyrazoles, oxazoles, thiazoles, pyridines, pyrimidines, pyrazines, and triazines.
  • embodiments of the present disclosure may include methods for producing a site-specific modified recombinant RNA, including: (a) obtaining a first RNA, (b) obtaining a second RNA that is monophosphorylated at the 5’ end, in which the second RNA comprises a backbone modification, a sugar modification, and/or a base modification, and (c) ligating the 3’ end of the first RNA obtained from (a) to the 5’ end of the second RNA obtained from (b), thereby producing the site-specific modified recombinant RNA.
  • the first RNA may be capped or uncapped.
  • embodiments of the present disclosure may include methods for producing a site-specific modified capped RNA, including: (a) obtaining a RNA that is monophosphorylated at the 5’ end, and (b) reacting an activated capping compound of Formula (I) with the RNA of (a) in the presence of a heteroaromatic compound and/or a metal salt, and a solvent: Formula (I) in which
  • L a leaving group, thereby producing the site-specific modified capped RNA.
  • FIG. 1 shows a method of preparing 5’ capped RNA according to one embodiment of the present disclosure.
  • FIG. 2 shows a method of preparing 5’ capped RNA according to another embodiment of the present disclosure.
  • FIG. 3 shows protein expression of mRNAs generated by methods in accordance with some embodiments of the present disclosure.
  • FIG. 4 shows protein expression of mRNAs generated by methods in accordance with some embodiments of the present disclosure.
  • FIG. 5 shows capping reaction in accordance with some embodiments of the present disclosure.
  • FIG. 6 shows liquid chromatography-mass spectrometry (LC-MS) analysis in accordance with some embodiments of the present disclosure.
  • FIG. 7 shows strategies for generating ligation products in accordance with some embodiments of the present disclosure.
  • FIG. 8 shows effect of oligo to RNA fragment ratio on ligation reaction in accordance with some embodiments of the present disclosure.
  • FIG. 9 shows analysis of ligation products in accordance with some embodiments of the present disclosure.
  • FIG. 10 shows fluorescence-activated cell sorting (FACS) analysis in accordance with some embodiments of the present disclosure.
  • FIG. 11 A shows protein expression analysis in accordance with some embodiments of the present disclosure.
  • FIG. 11B shows protein expression analysis in accordance with some embodiments of the present disclosure.
  • FIG. 12 shows analysis of ligation products in accordance with some embodiments of the present disclosure.
  • FIG. 13 A shows protein expression analysis in accordance with some embodiments of the present disclosure.
  • FIG. 13B shows protein expression analysis in accordance with some embodiments of the present disclosure.
  • FIG. 14 shows analysis of ligation products in accordance with some embodiments of the present disclosure.
  • FIG. 15 shows analysis of ligation products in accordance with some embodiments of the present disclosure.
  • mRNA Messenger RNA
  • Methods for making mRNA may include two major types: (1) an enzymatical capping method: for example, using in vitro transcription to prepare a full length RNA without 5’ cap , then using vaccinia capping enzyme to generate capO mRNA, or using vaccinia capping enzyme and 2’-O-Methyltransferase to prepare capl mRNA, and (2) the co-transcriptional capping method using cap analogues as a primer to initiate in vitro transcription.
  • Both methods may have limitations including, for example, there is no control to introduce site specific modification in the RNA sequence.
  • the NTPs used in in vitro transcription are randomly incorporated by RNA polymerase during IVT.
  • the types of capped mRNA thus generated may be highly dependent on enzymatic activity of capping enzymes or RNA polymerases with respect to 5 ’-capping.
  • types of capped mRNA such as those designed to be delivered to specific tissue in vivo, mRNA designed for latent activation, or mRNA with site specific modifications, may be needed for future mRNA therapeutic development.
  • IVT in vitro transcription
  • chemical capping refers to a reaction that is not enzymatically catalyzed, i.e., not done using a capping enzyme. Chemical capping involves a nucleophilic substitution reaction in which the leaving group of an activated nucleoside 5' mono- or polyphosphate reacts with the 5 '-phosphate of the polynucleotide, thereby releasing the leaving group and coupling the nucleoside 5' mono- or poly-phosphate to the polynucleotide via a 5' to 5' polyphosphate linkage that comprises, e.g., 2, 3, or 4 phosphates).
  • Chemical capping as described herein was used to attach a synthetic 5' cap structure (including guanosine, adenosine, cytidine, inosine and uridine caps as well as di-, tri and tetraphosphate bridges) to uncapped nucleic acids to generate polynucleotide sequencing libraries.
  • a synthetic 5' cap structure including guanosine, adenosine, cytidine, inosine and uridine caps as well as di-, tri and tetraphosphate bridges
  • Chemical capping may be carried out in the presence of activated 5 '-nucleotide precursors.
  • the synthesis of activated 5 '-nucleotides may be based on the nucleophilic substitution of a nucleoside or nucleoside 5 '-phosphate, such as a nucleoside 5'- monophosphate, NMP; a nucleoside 5 '-diphosphate, NDPs; and a nucleoside 5 '-triphosphate, NTP
  • Nucleosides and nucleoside 5 '-phosphates can be activated as a phosphorodichloridate, phosphoramidate, phosphodiester, phosphotriester, 5'-H-phosphonate, P(III) — P(V) mixed anhydride, or phosphite triester.
  • nucleoside 5'-phosphate analogues including ribonucleosides or deoxyribonucleosides, base- or sugar- modified nucleosides, as well as carbocyclic and acyclic analogues.
  • the chemistry for synthesizing nucleoside 5 '-phosphate precursors is described in more detail below.
  • P(III) and P(V) activation chemistries may be used to produce analogues containing phosphate modifications, such as thiophosphates, boranophosphates, and selenophosphates.
  • Nucleosides 5 '-phosphates, as well as their carbocyclic and acyclic analogues, activated as any of the reagents described above can be used in coupling reactions with an oligonucleotide 5'-phosphate (e.g., an oligonucleotide 5 '-monophosphate, 5 '-diphosphate, or 5 '-triphosphate) to form products comprising 5' to 5' polyphosphate linkages.
  • an oligonucleotide 5'-phosphate e.g., an oligonucleotide 5 '-monophosphate, 5 '-diphosphate, or 5 '-triphosphate
  • the coupling reaction with an oligonucleotide 5 '-phosphate may be performed in an aqueous solvent, an organic solvent, or a combination thereof; it may include an inorganic metal salt as a catalyst; and it may also include further additives such as polyethylene glycols (PEGs) and PEG derivatives.
  • PEGs polyethylene glycols
  • Phosphorodichloridates can be generated for example by the reaction of nucleosides with phosphoryl chloride in trimethylphosphate.
  • Nucleoside 5 '-phosphodiesters can be generated for example by the reaction of 2',3 '- protected nucleosides with 2-cyanoethyl phosphate in the presence of N,N'-di cyclohexyl carbodiimide (DCC), followed by in situ removal of the cyanoethyl group.
  • DCC N,N'-di cyclohexyl carbodiimide
  • Another example of a phosphorylating reagent is the 2-O-(4,4'-dimethoxytrityl)ethylsulfonylethan-2'-yl- phosphate, which forms a phosphodiester intermediate in the presence of a suitably protected nucleoside and triisopropylbenzenesulfonyl tetrazolide (TPSTAZ).
  • Nucleoside 5 '-phosphotriesters can be generated for example by a Mitsunobu-type coupling reaction between a nucleoside and a dibenzyl phosphate in the presence of triphenylphosphine and diethylazodicarboxylate; subsequent debenzylation produces the phosphotriester intermediate.
  • a phosphotriester involves the reaction of suitably protected nucleosides first with di -tert-butyl oxy N,N-diethylphosphoramidite in the presence of IH-tetrazole and then oxidation with meta-chloroperoxybenzoic acid (m-CPBA); subsequent removal of tert-butyl and acetonide groups produces the phosphotriester intermediate.
  • m-CPBA meta-chloroperoxybenzoic acid
  • a further approach to a phosphotriester makes use of salicylic alcohols to mask the phosphate group (cycloSal phosphate). CycloSal phosphotriesters can be synthesized via P(III) and P(V) chemistries.
  • cycloSal phosphotriesters via P(III) is based on the coupling of a nucleoside with saligenylchlorophosphite, followed by in situ oxidation.
  • a second approach to cycloSal phosphotriesters via P(III) involves the reaction of a nucleoside with a phosphoramidite and then the oxidation of the phosphite trimester.
  • a third approach to cycloSal phosphotriesters via P(III) involves the reaction of a nucleoside with a cyclosaligenylphosphorochloridate.
  • a fourth approach to cycloSal phosphotriesters via P(III) involves the prior synthesis of nucleoside phosphorodichloridate, which is then treated with salicylic alcohol.
  • Nucleoside 5'-H-phosphonates can be prepared for example through the transesterification of diphenyl H-phosphonate with suitably protected nucleosides in pyridine to produce phenyl H-phosphonate diesters; subsequent treatment with aqueous triethylamine produces the H-phosphonate monoester intermediate.
  • H-phosphonate monoesters may be converted into trivalent silyl phosphites, for example, by treatment with N,O- bis(trimethyl silyl) acetamide (BSA), to facilitate further oxidation to the corresponding phosphates.
  • BSA N,O- bis(trimethyl silyl) acetamide
  • Nucleosides comprising P(III) — P(V) mixed anhydrides can be generated for example by phosphitylation of nucleoside 5 '-monophosphates in the form of their tetra-N-butyl or tris- N-hexyl ammonium salts with a phosphoramidite reagent (e.g., salicylic chlorophosphite or bis-diisopropylamino chlorophosphine) followed by oxidation with an aqueous pyridine solution of iodine.
  • a phosphoramidite reagent e.g., salicylic chlorophosphite or bis-diisopropylamino chlorophosphine
  • P(III) — P(V) mixed anhydrides can be used to produce analogues containing modifications at the a-phosphate, such as 5'-(a-P-thiophosphates), 5'- (a-P-boranophosphates), and 5'-(a-P-selenophosphates).
  • Nucleoside 5 '-phosphosulfonyl reagents can be generated for example by reacting tetra n-butylammonium salts of nucleoside 5-phosphates with a sulfonylimidazolium salt in the presence of N-methylimidazole (NMI) or N,N'-diisopropylethylamine (DIPEA) as a base.
  • NMI N-methylimidazole
  • DIPEA N,N'-diisopropylethylamine
  • the sulfonylimidazolium salt can prepared by reacting phenylsulfonylimidazolide with methyl tritiate in ether.
  • Nucleoside 5'-phosphoramidates are some of the most useful precursors for the synthesis of phosphate-phosphate linkages.
  • Examples of phosphoramidates include phosphoroimidazolides, phosphoromorpholidates, phosphoropiperidates, pyrrolidinium phosphoramidates and pyridinium phosphoramidates.
  • Nucleoside 5 '-phosphoropiperidates can be generated for example by phosphitylation of carb oxybenzyl -protected nucleosides with benzyl N,N-diisopropylchlorophosphoramidite in the presence of IH-tetrazole, and then oxidative coupling with CC14/Et3N/piperidine; subsequent deprotection of carboxybenzyl and benzyl esters of the nucleosidic and phosphoramidate moieties by mild catalytic hydrogenation produces the phosphoropiperidate.
  • the coupling of 5'-phosphoropiperidates with phosphate-containing compounds to form phosphate-phosphate linkages can be promoted by 4,5-dicyanoimidazole (DCI).
  • Nucleoside 5'-phosphoromorpholidates can be generated for example by reacting a nucleoside or a nucleoside 5 '-phosphate with 2,2,2-tribromoethyl phosphoromorpholinochloridate followed by the in situ removal of the 2,2,2-tribromoethyl protecting group with Cu — Zn.
  • Another approach to 5'-phosphoromorpholidates is by coupling a nucleoside 5 '-phosphate with morpholine in the presence of DCC.
  • Nucleoside 5'-pyrrolidinium phosphoramidates can be generated for example by rearrangement of nucleoside phosphoramidate diesters derived from N-(3-chlorobutyl)-N- methylamine leading to the formation of the pyrrolidinium phosphoramidates.
  • Nucleoside 5 '-pyridinium phosphoramidates can be generated from nucleoside 5'-H- phosphonate monoesters derived either from salicylchlorophosphite or phosphorus trichloride. Silylation of the H-phosphonate monoesters with TMSC1 in pyridine, followed by oxidation with iodine result in the corresponding pyridinium phosphoramidites. Nucleoside 5'-phosphoroimidazolides are preferred reagents as they are more reactive than the corresponding phosphoromorpholidates, and more permissive when it comes to the choice of solvent.
  • Phosphoroimidazolides are also sometimes referred as phosphoroimidazolates, phosphoroimidazolidates, phosphoroimidazoles, phosphorimidazolidates, phosphorimidazolates, phosphorimidazolides, and phosphorimidazoles.
  • Nucleoside 5 '-phosphoroimidazolides can be prepared for example by treatment of nucleoside 5 '-phosphates (including nucleoside 5 '-monophosphates, NMPs; nucleoside 5'- diphosphates, NDPs; nucleoside 5 '-triphosphates, NTPs; nucleoside 5 '-tetraphosphates; nucleoside 5 '-pentaphosphates; nucleoside 5 '-hexaphosphates; and so forth) with 1,1'- carbonyldiimidazole (CDI), followed by removal of the 2', 3 '-carbonate protecting group under basic conditions.
  • nucleoside 5 '-phosphates including nucleoside 5 '-monophosphates, NMPs; nucleoside 5'- diphosphates, NDPs; nucleoside 5 '-triphosphates, NTPs; nucleoside 5 '-tetraphosphates
  • Another strategy to prepare phosphoroimidazolides is by treatment of nucleoside 5 '-phosphates with imidazole in the presence of triphenylphosphine and 2,2' - dithiodipyridine.
  • the latter strategy can also be performed with imidazole derivatives such as N-methylimidazole, 2-methylimidazole, 4-methylimidazole, 2-aminoimidazole, 2- isopropylimidazole, 2-phenylimidazole, benzimidazole, 2-methylbenzimidazole, 2- chloroimidazole, or 2-methylaminoimidazole.
  • phosphoroimidazolides are by in situ activation of nucleoside 5 '-phosphates with trifluoroacetic anhydride (TFAA) in the presence of a tertiary amine in acetonitrile, followed by removal of excess TFAA under vacuum and treatment of the resulting mixed anhydrides with N-methylimidazole to produce the corresponding phosphoromethylimidazolides.
  • TFAA trifluoroacetic anhydride
  • Phosphoroimidazolide activation chemistry may be applied to a large number of nucleoside 5 '-phosphate analogues, including ribonucleosides or deoxyribonucleosides, base- or sugar-modified nucleosides, as well as carbocyclic and acyclic analogues.
  • the resulting nucleoside 5 '-phosphoroimidazolides can be isolated by precipitation for example by treatment with sodium or lithium perchlorate in acetone followed by filtration.
  • the nucleoside 5 '-phosphoroimidazolide can be derived from a guanosine phosphate, an adenosine phosphate, a cytidine phosphate, or an inosine phosphate where the phosphate may be a monophosphate, diphosphate, triphosphate or tetraphosphate that after chemical capping will result in a polynucleotide with a 5 '-5 ' di-, tri- or tetra- or penta-phosphate linked nucleoside cap.
  • the 2'-position of the nucleoside 5 '-phosphate may be an SH, NH2, a lower alkyl (e.g., methyl), a lower alkoxy (e.g., methoxy), a lower acyloxy (e.g., acetoxy), a lower alkylamine (e.g., methylamine), a lower acylamine (e.g., acetamide), halogenyl, allyl, propargyl, or N3.
  • a lower alkyl e.g., methyl
  • a lower alkoxy e.g., methoxy
  • a lower acyloxy e.g., acetoxy
  • a lower alkylamine e.g., methylamine
  • a lower acylamine e.g., acetamide
  • halogenyl allyl, propargyl, or N3.
  • the 3 '-position of the nucleoside 5 '-phosphate is SH, NH2, a lower alkyl (e.g., methyl), a lower alkoxy (e.g., methoxy), a lower acyloxy (e.g., acetoxy), a lower alkylamine (e.g., methylamine), a lower acylamine (e.g., acetamide), halogenyl, allyl, propargyl, or N3.
  • both 2'- and 3 '-positions of the nucleoside 5 '-phosphate are modified with the same or different groups above mentioned.
  • the nucleoside 5 '-phosphate further comprises one or more fluorescent, quencher or affinity groups attached either at the nucleobase or at the 2'- or 3 '-position.
  • nucleoside 5 '-phosphate analogues including ribonucleosides or deoxyribonucleosides, base- or sugar-modified nucleosides, as well as carbocyclic and acyclic analogues.
  • base modifications include, but are not limited to, those found in 2-aminopurine, 2,6-diaminopurine, 5-iodouracil, 5-bromouracil, 5-fluorouracil, 5- hydroxyuracil, 5-hydroxymethyluracil, 5-formyluracil, 5-proprynyluracil, 5-methylcytosine, 5-hydromethylcytosine, 5-formylcytosine, 5-carboxycytosine, 5-iodocytosine, 5- bromocytosine, 5-fluorocytosine, 5-proprynylcytosine, 4-ethylcytosine, 5-methylisocytosine, 5 -hydroxy cytosine, 4-methylthymine, thymine glycol, ferrocene thymine, pyrrolo cytosine, inosine, 1-methyl-inosine, 2-methylinosine, 5-hydroxybutynl-2'-deoxyuracil (Super T), 8- aza-7-deazagua
  • sugar modifications include but are not limited to those found in di deoxynucleotides (e.g., ddGTP, ddATP, ddTTP, and ddCTP), 2'- or 3'-O-alkyl-nucleotides (e.g., 2'-O-methyl-nucleotides and 3'-O-methyl-nucleotides), 2'- or 3'-O-methoxyethyl-nucleotides (MOE), 2'- or 3 '-fluoronucleotides, 2'- or 3'-O-allyl-nucleotides, 2'- or 3'-O-propargyl-nucleotides, 2'- or 3 '-aminenucleotides (e.g., 3'-deoxy-3'-amine-nucleotides), 2'- or 3'-O-alkylamine-nucleotides (e.g., 2'-
  • sugar modifications include those found in the monomers that comprise the backbone of synthetic nucleic acids such as 2'-O,4'-C- methylene-P-D-ribonucleic acids or locked nucleic acids (LNAs), methylene-cLNA, 2',4'-(N- methoxy)aminomethylene bridged nucleic acids (N-MeO-amino BNA), 2', d'aminooxymethylene bridged nucleic acids (N-Me-aminooxy BNA), 2'-O,4'-C- aminomethylene bridged nucleic acids (2',4'-BNA(NC)), 2'4'-C — (N- methylaminomethylene) bridged nucleic acids (2',4'-BNA(NC)[NMe]), peptide nucleic acids (PNA), triazole nucleic acids, morpholine nucleic acids, amide-linked nucleic acids, 1,5- anhydrohexitol nucleic acids (N
  • Nucleoside 5'-phosphoroimidazolides may be used to form 5 '-capped polynucleotides with phosphate modifications, such as phosphororothioates (replacement of one non-bridging oxygen atom of the phosphate group with a sulfur atom), phosphorodithioates (both nonbridging oxygen atoms of the phosphate group are replaced with sulfur), alkyphosphonates (a non-bridging oxygen atom of the phosphate group has been replaced with alkyl group, e.g. methyl), arylphosphonates (a non-bridging oxygen atom of the phosphate group has been replaced with aryl group, e.g.
  • phosphate modifications such as phosphororothioates (replacement of one non-bridging oxygen atom of the phosphate group with a sulfur atom), phosphorodithioates (both nonbridging oxygen atoms of the phosphate group are replaced with sulfur), alkyphosphonates (a non-bridging oxygen
  • N-phosphoramidates an oxygen atom is replaced with an amino group either at the 3'- or 5 '-oxygen
  • boranophosphates one non-bridging oxygen atom of the phosphate group is replaced with BH3
  • PACE phosphonoacetates
  • Nucleoside 5'-phosphoroimidazolides may be used to form 5 '-capped polynucleotides with one or more fluorescent or quencher groups, affinity groups (e.g., biotin, desthiobiotin, digoxigenin, glutathione, heparin, maltose, coenzyme A, poly-histidine, and others), haptens to an antibody (e.g., HA-tag, c-myc tag, FLAG-tag, S-tag, among many others), mono- or oligosaccharide ligands to a lectin, hormones, cytokines, toxins, and vitamins.
  • affinity groups e.g., biotin, desthiobiotin, digoxigenin, glutathione, heparin, maltose, coenzyme A, poly-histidine, and others
  • haptens to an antibody e.g., HA-tag, c-myc tag, FLAG
  • binding partners to the aforementioned affinity groups include but are not limited to avidin, streptavidin, neutravidin, maltose-binding protein, glutathione- S-transferase (GST), antibodies, lectins, nickel, cobalt, zinc, and poly-histidine.
  • Further examples include groups that form an irreversible bond with a protein tag, including benzylguanine or benzylchoropyrimidine (SNAP-Tag® (New England Biolabs, Ipswich, Mass.)); benzylcytosine (CLIP-TagTM (New England Biolabs, Ipswich, Mass.)); haloalkane (HaloTag® (Promega, Madison, Wis.)); CoA analogues (MCP-tag and ACP-tag); trimpethoprim or methotrexate (TMP-tag); FlAsH or ReAsH (Tetracysteine tag); a substrate of biotin ligase; a substrate of phosphopantetheline transferase; and a substrate of lipoic acid ligase.
  • An affinity group is used for selectively enriching samples by means of affinity purification methods, wherein the affinity binding partner is immobilized in a column, bead, microtiter plate, membrane or other solid
  • the nucleoside 5-phosphate comprises a cleavable linker between the affinity group and the site of attachment to the nucleoside 5-phosphate. This strategy allows specific elution of target of interest.
  • the cleavable linker can be cleavable, for example, by chemical, thermal or photochemical reaction.
  • Chemically cleavable linkers include disulfide bridges and azo compounds (cleaved by reducing agents such as dithiothreitol (DTT), P-mercaptoethanol or tris(2-carboxyethyl)phosphine (TCEP)); hydrazones and acylhydrazones (cleaved by transimination in a mildly acidic medium); levulinoyl esters (cleaved by aminolysis, e.g.
  • DTT dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • hydrazones and acylhydrazones cleaved by transimination in a mildly acidic medium
  • levulinoyl esters cleaved by aminolysis, e.g.
  • Further examples include, acid or base labile groups, including among others, diarylmethyl or trimethylarylmethyl groups, silyl ethers, carbamates, oxyesters, thiesters, thionoesters, and a-fluorinated amides and esters.
  • photocleavable cleavable linkers include o-nitrophenyl group, diazobenzene, phenacyl, alkoxybenzoin, benzylthioether and pivaloyl glycol derivatives.
  • Nucleoside 5'-phosphoroimidazolides may be used to form 5 '-capped polynucleotides with one or more reactive groups.
  • the analogue comprises a reactive group selected from the group consisting of a carbonyl; a carboxyl; an active ester, e.g.
  • succinimidyl ester a maleimide; an amine; a thiol; an alkyne, an azide; an alkyl halide; an isocyanate; an isothiocyanate; an iodoacetamide; a 2-thiopyridine; a 3-arylproprionitrile; a diazonium salt; an alkoxyamine; a hydrazine; a hydrazide; a phosphine; an alkene; a semicarbazone; an epoxy; a phosphonate; and a tetrazine.
  • chemoselective reactions are: between an amine reactive group and an electrophile such an alkyl halide or an N-hydroxysuccinimide ester (NHS ester); between a thiol reactive group and an iodoacetamide or a maleimide; between an azide and an alkyne (azide-alkyne cycloaddition or “Click Chemistry”).
  • an electrophile such as alkyl halide or an N-hydroxysuccinimide ester (NHS ester)
  • a thiol reactive group and an iodoacetamide or a maleimide between an azide and an alkyne (azide-alkyne cycloaddition or “Click Chemistry”.
  • a nucleoside 5'-phosphoroimidazolide can be used in a capping reaction with a polynucleotide 5'-phosphate (e.g., a 5 '-monophosphate, 5 '-diphosphate, 5 '-triphosphate, 5'- tetraphosphate, and so forth) to form 5' to 5' polyphosphate linkages.
  • the nucleoside 5'- phosphoroimidazolide may be used in a capping reaction in a molar excess from 2 to 1000000-fold relative to the polynucleotide 5'-phosphate, with 1000-fold (1000*) being the most preferred.
  • the capping reaction between a nucleoside 5'-phosphoroimidazolide and a polynucleotide 5 '-phosphate may be performed in an aqueous buffer, an organic solvent, or a combination thereof with a pH in the range of 4 to 7, preferably 5 to 6.
  • an aqueous buffer includes a non-nucleophilic, phosphate-free buffer, such as ADA (N-(2- Acetamido)-2-iminodiacetic acid), BES (N,N-Bis(2 -hydroxy ethyl)-2-aminoethanesulfonic acid), BICINE (N,N-Bis(2-hydroxyethyl)glycine), DIPSO (3-(N,N-Bis[2- hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), EPPS (4-(2 -Hydroxy ethyl)- 1- piperazinepropanesulfonic acid), HEPBS (N-(2 -Hydroxy ethyl)piperazine-N'-(4- butanesulfonic acid)), 4-Ethylmorpholine, MOBS (4-(N-Morpholino)butanesulfonic acid), MOPS (3-(N-Morphol
  • organic solvent examples include: alcohols, such as ethanol, 1-propanol, 2-propanol, 1-butanol, 2- butanol, t-butyl alcohol; or nitriles, such as acetonitrile or propionitrile; amides such as N,N- dimethylformamide (DMF), N,N-dimethylacetamide (DMA), N-methyl-2-pyrrolidone; sulfoxides such as dimethylsulfoxide (DMSO); ethers such as diethyl ether, diisopropyl ether, methyl t-butyl ether, tetrahydrofuran, 1,4-di oxane, 2-methoxy ethanol, anisole; or any mixtures of one or more of these solvents.
  • a preferred coupling reaction buffer includes a 10% to 40% organic solvent in aqueous buffer solution, most preferably 20%.
  • the capping reaction between a nucleoside 5'-phosphoroimidazolide and a polynucleotide 5 '-phosphate may include an inorganic metal salt as a catalyst.
  • examples include a halide, sulphate, nitrate, phosphate, hydrogen phosphate, or hydrogen sulfate salts; wherein the inorganic metal is magnesium, manganese, zinc, or cobalt.
  • the inorganic metal salt is MgCh, MnCh, ZnCh, or C0CI2.
  • the capping reaction between a nucleoside 5'-phosphoroimidazolide and a polynucleotide 5 '-phosphate may further include additives such as polyethylene glycols (PEGs) and PEG derivatives such as PEG ethers (e.g., laureths, ceteths, ceteareths, and oleths), PEG fatty acids (e.g., PEG laurates, dilaurates, stearates, and distearates), PEG amine ethers (e.g., PEG cocamines), PEG propylene glycols, or other derivates.
  • PEGs polyethylene glycols
  • PEG derivatives such as PEG ethers (e.g., laureths, ceteths, ceteareths, and oleths), PEG fatty acids (e.g., PEG laurates, dilaurates, stearates, and distearates),
  • the capping reaction between a nucleoside 5'-phosphoroimidazolide and a polynucleotide 5 '-phosphate may further include one or more surfactants.
  • surfactants include polyoxyethanyl-alpha-tocopheryl sebacate (PTS); DL-alpha-tocopherol methoxypolyethylene glycol succinate (TPGS-750-M); beta-sitosterol methoxyethylene glycol succinate (SPGS-550-M); bis(4-((2-(Methoxycarbonyl)phenyl)amino)-4-oxobutanoic acid)poly ethylene glycol 1000; and combinations thereof.
  • PTS polyoxyethanyl-alpha-tocopheryl sebacate
  • TPGS-750-M DL-alpha-tocopherol methoxypolyethylene glycol succinate
  • SPGS-550-M beta-sitosterol methoxyethylene glycol succinate
  • the capping reaction between a nucleoside 5'-phosphoroimidazolide and a polynucleotide 5'-phosphate may be performed in a range of 20° C. to 70° C.
  • the reaction times typically range from less than one hour up to 24 hours.
  • the coupling reaction temperature and time may be 50° C. and 5 hours when using 5'- phosphoroimidazolides derived from nucleoside 5 '-monophosphates (imNMPs); 37° C.
  • imNDPs 5'-phosphoroimidazolides derived from nucleoside 5 '-diphosphates
  • imNTPs room temperature and 4 hours when using 5'-phosphoroimidazolides derived from nucleoside 5 '-triphosphates
  • a suitable nucleoside 5'-phosphoroimidazolide and/or polynucleotide 5 '-phosphate salt may be formed with a suitable cation selected from, but is not limited to: inorganic cations, such as Na+, K+, Ca+, Mg+, Mn2+, Zn2+, Co2+, and Al+3; ammonium ions (i.e., NH4 +); and substituted ammonium ions (e.g., NH3R+, NH2R2 +, NHR3 +, and NR4 +), wherein the substituted ammonium ions derives from alkyl and aryl amines such as ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, hexylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, pyridine, benzylamine, and any combinations thereof.
  • the polynucleotide 5 '-phosphate may be DNA, or RNA, or a chimeric polynucleotide (chimera) consisting of RNA and DNA bases.
  • the polynucleotide 5'-phosphate may be single- stranded, double-stranded, or consist of a chimera of partially single-stranded and double-stranded segments.
  • the polynucleotide 5 '-phosphate may also be a double-stranded segment having 3' or 5' end nucleotide extensions.
  • the polynucleotide 5 '-phosphate may comprise one or more of RNA species selected from small RNAs; small nuclear RNAs (snRNAs); small nucleolar RNAs (snoRNAs); miRNAs; Piwi-interacting RNAs (piRNAs); IncRNAs; tRNAs; ribosomal RNAs (rRNAs); mRNAs; non-coding RNAs (ncRNAs); intergenic RNAs; silencing RNAs (siRNAs); small regulatory RNAs (srRNAs); or any combinations thereof.
  • small RNAs small nuclear RNAs
  • snoRNAs small nucleolar RNAs
  • miRNAs Piwi-interacting RNAs
  • piRNAs Piwi-interacting RNAs
  • IncRNAs tRNAs
  • rRNAs ribosomal RNAs
  • mRNAs non-coding RNAs
  • ncRNAs non-coding RNAs
  • the polynucleotide 5 '-phosphate may comprise samples of fragmented and/or degraded RNAs, particularly fragmented and/or degraded mRNAs and long noncoding RNAs.
  • the polynucleotide 5 '-phosphate may comprise fragmented and/or degraded DNA, such as ancient DNA, environmental DNA, forensic DNA, circulating DNA (e.g., exosomes), denatured DNA, and viral DNA.
  • fragmented and/or degraded DNA such as ancient DNA, environmental DNA, forensic DNA, circulating DNA (e.g., exosomes), denatured DNA, and viral DNA.
  • Several of these DNAs and RNAs may be used as biomarkers and in medical diagnosis applications.
  • DNAs and RNAs may be obtained from a cell or tissue extract; or from formalin-fixed, paraffin-embedded tissue (FFPE); or from a body fluid, such as saliva, blood, menstrual blood, cervicovaginal fluid, and semen.
  • FFPE formalin-fixed, paraffin-embedded tissue
  • the polynucleotide population may include polynucleotide 5 '-phosphates having one or more polynucleotides with different 5' termini.
  • the polynucleotide 5 '-phosphate will be selectively capped by the reaction with a nucleoside 5'-phosphoroimidazolide, while the other polynucleotides in the population will remain unreactive.
  • polynucleotides with no terminal phosphate at the 5' end may be pre-treated with a polynucleotide kinase to install a 5' terminal phosphate to those polynucleotides lacking a 5' phosphate.
  • a polynucleotide i.e., to remove a terminal 3' phosphate or a 2', 3 '-cyclic phosphate, to avoid unwanted formation of 3' to 5' polyphosphate linkage by reaction with a nucleoside 5'-phosphoroimidazolide; in such cases, these polynucleotides may be pre-treated with a polynucleotide kinase (e.g., T4 PNK), or a related enzyme (e.g., a phosphatase) that is able to dephosphorylated a 3' terminal nucleotide or 2', 3 '-cyclic phosphate to create a 3' OH terminus (Zhelkovsky, (2014) J Biol Chem 289: 33608-16).
  • a polynucleotide kinase e.g., T4 PNK
  • a related enzyme e.g., a phosphatase
  • a polynucleotide such as the N7-methylguanosine cap in eukaryotic mRNA; or a trimethylguanosine cap in small nuclear RNAs (snRNAs); or a y-methyl phosphate cap in snRNAs, such U6 and 7SK; or cap-like structures such as nicotinamide adenine dinucleotide (NAD+), 3 '-desphospho-coenzyme A (dpCoA), and other moi eties attached to the 5' end of RNAby an oligophosphate bridge [Warminski, et al.
  • NAD+ nicotinamide adenine dinucleotide
  • dpCoA 3 '-desphospho-coenzyme A
  • the existing cap structure is replaced by a process of decapping the 5' end so that the polynucleotides in the population have a terminal phosphate at the 5' end (e.g., a 5'- monophosphate, a 5 '-diphosphate, and/or a 5 '-triphosphate) and this terminal 5 '-phosphate then re-capped by chemical capping, (also see US 2018/0195061).
  • the decapping reaction may be mediated by an enzyme or by chemical hydrolysis (appropriate acidic or basic conditions may be selected).
  • the enzyme may be selected from a deadenylase, an apyrase, a 5'RppH, an Nudix phosphohydrolase, a tobacco acid polyphosphatase, a member of the HIT superfamily of pyrophosphatases, a DcpS, a Dcpl-Dcp2 complex, a NudC, an APTX, a member of the DXO family proteins, a APAH-like phosphatases, a Cap-Clip reagent (CELLSCRIPT, Madison, Wis.), or a combination thereof. Examples and uses of decapping enzymes are reviewed in Kramer, et al. (2019) Wiley Interdiscip Rev RNA, 10: el 511.
  • Site-specific chemically modified mRNA exists in nature and the site-specific chemically modified bases around UTR regions of mRNA might provide special functional for regulating its translational efficiency and stability. For this purpose, it is desirable to develop a method to make site-specific chemically modified mRNA in vitro for advancing the mRNA mechanistic research and for special therapeutics applications.
  • Abe et al. (“Complete Chemical Synthesis of Minimal Messenger RNA by Efficient Chemical Capping Reaction.” ACS Chem Biol.
  • RNA may have limitations including, for example, the maximum length that can be synthesized may be about 150 bases.
  • chemically synthesized short RNAs up to 150 bases were generally used as platform for noncoding RNA molecules but not coding RNAs, since the 5 ’UTR, 3’ UTR plus polyAtail structure (which is normally ⁇ 100As) requirement makes it longer than 150 bases.
  • the purely chemical synthesis without using any enzymes for preparing capped mRNA could not be translated for RNA therapeutics development.
  • Chemical synthesis of RNA are generally used for siRNA, miRNA, sgRNA, etc., at non-coding RNA field.
  • embodiments of the present disclosure may include (1) methods of preparing site specific modified mRNAby ligating short capped RNAs up to 150 bases, which may be produced by chemical synthesis followed by chemical capping at the 5’ end without using any enzymes, to another RNA produced by in vitro transcription; and (2) methods of chemically capping RNAs with more than 150 bases with or without chemical modification produced by in vitro transcription.
  • FIG. 1 shows a method of producing a 5’ capped recombinant RNA according to one embodiment of the present disclosure.
  • a first RNA (RNA1) with, e.g., up to 150 bases, which is monophosphorylated at the 5’ end and may or may not contain sitespecific modified nucleotides may be obtained by chemical RNA synthesis using automated processes in conjunction with the phosphoramidite methods.
  • the 5’ monophosphorylated RNA1 may be reacted with an activated capping compound of Formula (I) (see below), e.g., 7-methylguanosine 5-diphosphate imidazolide (Im-m 7 GDP), with RNA1 in the presence of an appropriate heteroaromatic compound and/or a metal salt, and a solvent, thereby producing a 5’ capped RNA1.
  • an activated capping compound of Formula (I) see below
  • RNA1 7-methylguanosine 5-diphosphate imidazolide (Im-m 7 GDP)
  • RNA2 A second 5’ monophosphorylated RNA (RNA2) may be produced by in vitro transcription reaction using T7 polymerase in the absence of cap analogs to generate a 5’ triphosphorylated RNA , which will be further dephosphorylated using enzymes, e.g., RNA 5' polyphosphatase or RNA 5’ pyrophosphohydrolase (RppH), to produce 5’ monophosphorylated RNA2; or the 5’ monophosphorylated RNA2 can be prepared by in vitro transcription with including a certain ratio of GMP nucleoside to force GMP to initiate IVT.
  • enzymes e.g., RNA 5' polyphosphatase or RNA 5’ pyrophosphohydrolase (RppH)
  • a ratio of GMP to GTP may be 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10:1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, or 20: 1.
  • the 3’ end of 5’ capped RNA1 and the 5’ end of 5’ monophosphorylated RNA2 may be ligated in the presence of RNA ligases and splint DNA that hybridizes the 3’ end of 5’ capped RNA1 and the 5’ end of 5’ monophosphorylated RNA2, thereby producing 5’ capped recombinant RNA1 and RNA2 (RNA1/2).
  • RNA1 and RNA2 may be ensured by splint DNA, which can hybridize the ends of RNA1 and RNA2, directing DNA or RNA ligase (e.g. RNA ligase II) activity in joining 5’ and 3’ ends.
  • DNA splint may hybridize with at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of a 3’ end region of the 5’ capped RNA1 and may hybridize with at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of a 5’ end region of the 5’ monophosphorylated RNA2.
  • FIG. 2 shows a method of producing a 5’ capped RNA according to another embodiment of the present disclosure.
  • a 5’ triphosphorylated RNA with, e.g., more than 150 bases may be produced by in vitro transcription reaction using T7 polymerase in the absence of cap analogs.
  • 5’ triphosphorylated RNA may be then treated with enzymes, e.g., RNA 5' polyphosphatase or RNA 5’ pyrophosphohydrolase (RppH), to produce 5’ monophosphorylated RNA, which may then be reacted with an activated capping compound of Formula (I), e.g., Im-m 7 GDP, in the presence of an appropriate heteroaromatic compound and/or a metal salt, and a solvent, thereby producing a 5’ capped RNA.
  • enzymes e.g., RNA 5' polyphosphatase or RNA 5’ pyrophosphohydrolase (RppH)
  • RppH RNA 5' polyphosphatase or RNA 5’ pyrophosphohydrolase
  • 5’ monophosphorylated RNAs with up to 150 bases may be generated directly by chemical synthesis.
  • 5’ triphosphorylated RNAs generated by in vitro transcription may be treated with (1) RNA 5' polyphosphatase, which can remove the y and P phosphates from 5'- triphosphorylated and the P phosphates from diphosphorylated RNA, but may not dephosphorylate monophosphorylated or 5 '-capped RNA; or (2) RNA 5’ pyrophosphohydrolase (RppH), which can remove pyrophosphate from the 5' end of triphosphorylated RNA.
  • RppH RNA 5’ pyrophosphohydrolase
  • the nitrogenous base of the 5' nucleoside cap of Formula (III) may be selected from the group consisting guanine, adenine, cytosine, uracil and hypoxanthine and analogs of guanine, adenine, cytosine, uracil and hypoxanthine or modifications thereof.
  • a modified nitrogenous base of the 5' nucleoside cap may comprise a modified base selected from N6-methyladenine, N1 -methyladenine, N6-2'-O- dimethyladenosine, pseudouridine, N1 -methylpseudouridine, 5-iodouridine, 4-thiouridine, 2- thiouridine, 5 -methyluridine, pseudoisocytosine, 5 -methoxy cytosine, 2-thiocytosine, 5- hydroxy cytosine, N4-methylcytosine, 5-hydroxymethylcytosine, hypoxanthine, Nl- methylguanine, 06-methylguanine, 1-methyl-guanosine, N2-methyl-guanosine, N7-methyl- guanosine, N2,N2-dimethyl-guanosine, 2-methyl-2'-O-methyl-guanosine, N2,N2-dimethyl- 2'-O-methyl-guanosine, l-methyl-2'-O-methyl-methyl-
  • the nitrogenous base of the 5' nucleoside cap may be attached to a sugar selected from a ribose or a modified ribose selected from 2'- or 3'-O-alkylribose, alkoxyribose, O-alkoxyalkylribose, fluororibose, azidoribose, allylribose, deoxyribose; an arabinose or a modified arabinose; a thioribose; an 1,5 anhydrohexitol; or a threofuranose.
  • a sugar selected from a ribose or a modified ribose selected from 2'- or 3'-O-alkylribose, alkoxyribose, O-alkoxyalkylribose, fluororibose, azidoribose, allylribose, deoxyribose; an arabinose or a modified
  • the one or more phosphates of the 5' nucleoside cap may consist of a phosphorothioate; a phosphorodithioate; an alkyphosphonate; an arylphosphonate; a N-phosphoramidate; a boranophosphate; or a phosphonoacetate.
  • the 5' nucleoside cap includes guanosine.
  • the leaving group L of Formula (I) and (III), besides the imidazole group, may be exemplified by heteroaromatic ring compounds, such as pyrazoles, oxazoles, thiazoles, pyridines, pyrimidines, pyrazines, and triazines.
  • heteroaromatic ring compounds such as pyrazoles, oxazoles, thiazoles, pyridines, pyrimidines, pyrazines, and triazines.
  • Activated capping compound represented by Formula (II) may be synthesized by a method of diphosphorylation of guanosine, followed by dehydration condensation with imidazole.
  • Activated capping compound represented by Formula (II) may be synthesized by a scheme that includes, for example, phosphorylation of the 5 '-position of a ribose of guanosine to synthesize guanosine monophosphate (guanosine-5 '-phosphate), to which imidazole may be then reacted, thereby bonding imidazole to the phosphate group followed by reacting the product typically with triethylammonium phosphate, in the presence of a divalent metal salt, to synthesize guanosine diphosphate, and further typically with iodomethane or dimethyl sulfate to methylate the 7-position of the base followed by reacting the product with imidazole to bond the imidazole to the phosphate group.
  • the monophosphate RNA having the 5 '-end monophosphorylated may be a target compound to which the activated capping compound can bind.
  • the 5'- monophosphate RNA may be synthesized typically by a method of removing pyrophosphate from 5 '-triphosphate RNA with use of enzymes, e.g., RNA 5' polyphosphatase or RNA 5'- pyrophosphohydrolase (RppH), or by a chemical solid phase synthesis method.
  • a counter salt of monophosphate RNA may be exemplified by tetraalkylammonium salt, trialkyl acetate salt, and sodium acetate salt. In particular, use of an organic salt as a counter cation of phosphoric acid can improve the reactivity.
  • the activated capping compound and the monophosphate RNA may be reacted in the presence of the heteroaromatic compound and/or at least one metal salt selected from the group consisting of calcium salt, zinc salt, manganese salt, magnesium salt, nickel salt, and copper salt; and the solvent.
  • the heteroaromatic compound may be preferably an imidazole compound having an imidazole group.
  • the imidazole compound may be exemplified by N- alkylimidazole, in which an alkyl group may be bound to nitrogen of imidazole, wherein the alkyl group may be particularly exemplified by those having the alkyl group with 1 to 5 carbon atoms.
  • the N-alkylimidazole may be exemplified by 1 -methylimidazole, 1- ethylimidazole, 1 -propylimidazole, 1 -methyl- lH-imidazole-2-carboxylate, 1- methylimidazole-4-carboxylate, 5-chloro-l-methyl-4-nitroimidazole, and 2-hydroxymethyl-
  • 1 -methylimidazole 1 -methylimidazole.
  • N-alkylimidazoles preferred may be 1 -methylimidazole for its high activity of cap introduction.
  • the imidazole compound may be also exemplified by imidazoles other than N-alkylimidazole, which may include l-(2-hydroxyethyl)imidazole and
  • 2-nitroimidazole preferred may be 2-methylimidazole for its high activity of cap introduction.
  • the metal salt may be selected from the group consisting of calcium salt, zinc salt, magnesium salt, manganese salt, nickel salt, and copper salt, or may be mixture of these salts (calcium and zinc salts, for example).
  • the calcium salt may be exemplified by calcium chloride (CaCh) and calcium hydroxide (Ca(0H)2).
  • the zinc salt may be exemplified by zinc chloride (ZnCh).
  • the manganese salt may be exemplified by manganese chloride. Among them, preferred may be CaCh for its high activity of cap introduction.
  • the solvent may be exemplified by water and an organic solvent.
  • the organic solvent may be exemplified by dimethyl sulfoxide (DMSO), or N,N-dimethylformamide (DMF). Among them, preferred may be dimethyl sulfoxide (DMSO) for its high ability of solubilizing organic salts.
  • DMSO dimethyl sulfoxide
  • the solvent may be preferably organic solvent that contains 0 to 20 wt % of water, and more preferably contains 1 to 10 wt % of water, in consideration of high activity of cap introduction.
  • Concentration of the activated capping compound in the reaction liquid preferably may fall within the range from 5 to 30 mM.
  • Concentration of the heteroaromatic compound in the reaction liquid may be preferably in the range from 0.5 to 20 mM, more preferably from 5 to 15 mM, and particularly preferably 10 mM.
  • Concentration of the metal salt in the reaction liquid may be preferably in the range of 0.5 to 10 mM.
  • the reaction conditions may be suitably set, typically with the reaction temperature adjusted within the range from 30 to 60° C., preferably from 35 to 40° C., and particularly preferably at 37° C.
  • the reaction time may be within the range from 1 to 25 hours, preferably from 5 to 15 hours, and particularly preferably 9 hours. Cap introduction activity may be kept high within these conditions, enabling efficient introduction of the cap structure into mRNA.
  • RNA in vitro transcription may relate to a process wherein RNA is synthesized in a cell-free system (in vitro).
  • DNA particularly plasmid DNA
  • RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the present disclosure, may be preferably a linearized plasmid DNA template.
  • the promoter for controlling in vitro transcription can be any promoter for any DNA- dependent RNA polymerase.
  • DNA-dependent RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • a DNA template for in vitro RNA transcription may be obtained, for example, by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, for example, into plasmid DNA.
  • DNA template may be linearized with a suitable restriction enzyme before it is transcribed in vitro.
  • the cDNA may be obtained by reverse transcription of mRNA or chemical synthesis.
  • the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis.
  • reagents used in in vitro transcription may include:
  • RNA polymerase such as bacteriophage-encoded RNA polymerases
  • NTPs ribonucleoside triphosphates
  • RNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SP6 RNA polymerase);
  • RNase ribonuclease
  • pyrophosphatase or RNA 5’ polyphosphatase or RNA 5’ pyrophosphohydrolase (RppH)
  • RppH pyrophosphohydrolase
  • MgCh and/or magnesium acetate (Mg(C 214302)2) (MgOAc), which supplies Mg 2+ ions as a co-factor for the polymerase;
  • a buffer to maintain a suitable pH value which can also contain antioxidants (e.g., DTT), amines, such as, betaine and/or polyamines, such as, spermidine at optimal concentrations.
  • antioxidants e.g., DTT
  • amines such as, betaine and/or polyamines, such as, spermidine at optimal concentrations.
  • mRNAs ranging from about 100 bases (100b) to about 20Kb, > 100b, > 150b, > 200b, > 250b, > 300b, > 350b, > 400b, > 450b, > 500b, > 650b, > 700b, > 750b, > 800b, > 900b, > 1Kb, > 2Kb, > 3Kb, > 4Kb, > 5Kb, > 6Kb, > 7Kb, > 8Kb, > 9Kb, > 10Kb, from about 150b to about 20Kb, from about 150b to about 19Kb, from about 150b to about 18Kb, from about 150b to about 17Kb, from about 150b to about 16Kb, from about 150b to about 17Kb, from about 150b to about 16Kb, from about 150b to about 15Kb, from about 150
  • Common buffer systems used in RNA in vitro transcription may include 4-(2- hydroxy-ethyl)-l -piperazineethanesulfonic acid (HEPES) and tri s(hydroxymethyl)aminom ethane (Tris).
  • HEPES 4-(2- hydroxy-ethyl)-l -piperazineethanesulfonic acid
  • Tris tri s(hydroxymethyl)aminom ethane
  • the pH value of the buffer may be commonly adjusted to a pH value of 6 to 8.5.
  • Some commonly used transcription buffers may contain 80 mM HEPES/KOH, pH 7.5 and 40 mM Tris/HCl, pH 7.5.
  • the transcription buffer may also contain a magnesium salt, such as MgCh and/or MgOAc commonly in a range between 5-50 mM.
  • MgCh magnesium salt
  • MgOAc magnesium salt
  • Mg 2+ may be an essential component in an RNA in vitro transcription buffer system because free Mg 2+ may function as cofactor in the catalytic center of the RNA polymerase and may be critical for the RNA polymerization reaction. In diffuse binding, fully hydrated Mg ions may also interact with the RNA product via nonspecific long-range electrostatic interactions.
  • in vitro transcription may be performed for from about 10 minutes to about 12 hours, e.g., 20-180 minutes and about 1 hour to about 12 hours.
  • in vitro transcription may be performed at a temperature of 15-55° C., 15-45° C., 15-37° C., 15-35° C., 15-25° C., 15-20° C., 25-55° C., 25-45° C., 25-37° C., 25- 35° C., 25-30° C., 35-55° C., or 35-37° C.
  • IVT reaction may be performed at a temperature of about 25° C.
  • in vitro transcription may be performed at a temperature of about 45° C. or higher, including, e.g., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C. or higher.
  • RNA in vitro transcription reactions may be performed as batch reactions in which all components are combined and then incubated to allow the synthesis of RNA molecules until the reaction terminates.
  • fed-batch reactions were developed to increase the efficiency of the RNA in vitro transcription reaction (Kern et al. (1997) Biotechnol. Prog. 13: 747-756; Kern et al. (1999) Biotechnol. Prog. 15: 174-184).
  • all components are combined, but then additional amounts of some of the reagents are added over time (e.g., NTPs, MgCh and/or MgOAc) to maintain constant reaction conditions.
  • co-transcription refers to mRNA prepared as with a cap structure through one step in vitro transcription reaction using RNA polymerase, e.g., T7 RNA polymerase.
  • RNA polymerase e.g., T7 RNA polymerase.
  • one or more traditional post transcriptional capping method may need to prepare an uncapped RNA through in vitro transcription (IVT) with RNA polymerase, and then add Cap using capping enzyme, e.g., vaccinia capping enzyme, with the aid of 2’-O-Methyltransferase to add methylation at the +1 base of the mRNA.
  • IVTT in vitro transcription
  • capping enzyme e.g., vaccinia capping enzyme
  • RNA synthesis of RNA can allow for site-selective incorporation of modified nucleotides.
  • the phosphoramidite approach may be the most widely used. This method may involve four repeating steps that occur on a solid support (typically CPG or polystyrene beads) following removal of the 5 '-hydroxyl protecting group on the 3' nucleotide: i) coupling at the 5' site with a protected phosphoramidite, ii) capping of the unreacted 5 '-hydroxyl groups, iii) oxidation of the newly formed phosphite linkage, and iv) removal of the 5 '-protecting group on the newly added nucleotide.
  • the synthetic RNA may be cleaved from the solid support, the remaining protecting groups are removed, and the final RNA product is purified (typically by high-performance liquid chromatography (HPLC) or polyacrylamide gel electrophoresis).
  • RNAs with single or multiple modifications can be generated in order to study the individual effects of the natural modified nucleotides, ii) large quantities of RNA can be generated that may be 100% modified at the desired locations, and iii) the roles of specific functional groups in RNA can be studied by using synthetic analogs with only minor changes in the chemical composition.
  • these approaches have been used to generate small, modified RNAs in high quantities for biophysical studies.
  • unnatural modifications have allowed mechanistic studies to be carried out on RNAs such as ribozymes.
  • the modified nucleotides may be incorporated into RNA site-specifically using T7 RNA polymerase and in vitro transcription. Site-specific modifications, however, may be made to RNA by using unnatural base pair systems.
  • the unique complementarity of an unnatural base pair may allow one modified nucleotide to direct the incorporation of another into an RNA strand by using RNA polymerase.
  • a site-specific isoC within a DNA template could direct the incorporation of the modified nucleotide isoG into an RNA transcript.
  • the unnatural nucleotide 2-amino-6-(2-thienyl)purine can direct the site-specific incorporation of 2-oxopyridine into RNA.
  • Hydrophobic base pairs may be used for the site-specific incorporation of nucleotide analogs, cross-linking analogs, biotinylated nucleotides, and fluorescent analogs into RNA. The potential applications of these systems may be numerous.
  • RNAs that are representative of the natural RNAs, which are typically longer than is routinely achievable by direct chemical synthesis.
  • These RNAs can be generated with internal modifications using a semisynthesis approach.
  • the RNA may be generated in two or more segments, either chemically or enzymatically, in which one or more segment contains the modification(s) of interest.
  • One RNA segment can be modified at the 3' end using a bisphosphate analog of the modified nucleotide and T4 RNA ligase.
  • the advantage of this approach is that any size RNA can be modified and the reaction may be generally not limited by sequence or nucleoside composition.
  • the modified RNA segment can be generated using available phosphoramidites and chemical synthesis.
  • T4 RNA ligase can be used to ligate RNA fragments with a 5' phosphate on the donor strand and a 3' hydroxyl on the acceptor strand.
  • One major drawback, however, may be the possibility of circularization of the individual RNA fragments. Therefore, the ends of the RNAs to be joined need to be appropriately modified with phosphate, hydroxyl, or 2', 3 '-cyclic phosphate groups.
  • RNA ligase, modified RNA fragments, and DNA splints may be used to achieve highly efficient and rapid ligation of synthetic RNA fragments.
  • RNA fragments may be single-stranded (i.e., not paired with the DNA splint) at the ligation site in order to accommodate T4 RNA ligase, which prefers singlestranded substrates.
  • long splints may be used to increase the ligation efficiencies with T4 DNA ligase. The improved hybridization of longer splints may reduce the amount of RNA secondary structure formation, which could affect the ligation efficiency.
  • modified nucleoside triphosphate refers to chemical modifications comprising backbone modifications as well as sugar modifications, and/or base modifications. These modified nucleoside triphosphates are also termed herein as (nucleotide) analogues.
  • the modified nucleoside triphosphates as defined herein are nucleotide analogues/modifications, e.g., backbone modifications, sugar modifications, and/or base modifications.
  • a backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides are chemically modified.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides.
  • a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides.
  • nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
  • modified nucleosides and nucleotides which may be used in the context of the present invention, can be modified in the sugar moiety.
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
  • alkoxy or aryloxy — OR, e.g
  • “Deoxy” modifications include hydrogen, amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleotide can include nucleotides containing, for instance, arabinose as the sugar.
  • the phosphate backbone may further be modified in the modified nucleosides and nucleotides.
  • the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).
  • the modified nucleosides and nucleotides which may be used in the present disclosure, can further be modified in the nucleobase moiety.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.
  • nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • the nucleotide analogues/modifications may be selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5 '-triphosphate, 2-Aminopurine-riboside-5'- triphosphate; 2-aminoadenosine-5 '-triphosphate, 2'-Amino-2'-deoxycytidine-triphosphate, 2- thiocytidine-5 '-triphosphate, 2-thiouridine-5 '-triphosphate, 2'-Fluorothymidine-5 triphosphate, 2'-O-Methyl inosine-5 '-triphosphate 4-thiouridine-5 '-triphosphate, 5- aminoallylcytidine-5 '-triphosphate, 5-aminoallyluridine-5 '-triphosphate, 5-bromocytidine-5'- triphosphate, 5-bromouridine-5 '-triphosphate, 5-
  • nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5 '-triphosphate, 7-deazaguanosine-5 '-triphosphate, 5- bromocytidine-5 '-triphosphate, and pseudouridine-5 '-triphosphate.
  • modified nucleosides may include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1- carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudo
  • modified nucleosides may include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5 -formylcytidine, N4- methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-ze
  • modified nucleosides may include 2-aminopurine, 2, 6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8- aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1- methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine,
  • modified nucleosides may include inosine, 1 -methylinosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.
  • modified nucleosides may include inverted deoxythymidine (dT).
  • dT inverted deoxythymidine
  • the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.
  • a modified nucleoside may be 5'-O-(l- Thiophosphate)- Adenosine, 5'-O-(l-Thiophosphate)-Cytidine, 5'-O-(l-Thiophosphate)- Guanosine, 5'-O-(l-Thiophosphate)-Uridine or 5'-O-(l-Thiophosphate)-Pseudouridine.
  • the modified nucleotides may include nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uri dine, Nl-methyl-pseudouri dine, 5,6- dihydrouridine, a-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl- guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, Nl-methyl-adenosine, 2- amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cy
  • a 5' cap may be typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an RNA molecule.
  • 5' cap may be added using a 5 '-5 '-triphosphate linkage.
  • a 5' cap may be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5 '-end of an RNA.
  • the naturally occurring 5' cap may include m7GpppN.
  • 5' cap structures may include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3 ',4'- seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety
  • site-specific modification refers to selectively modify nucleotide in one RNA chain at certain positions as designed. This is in contrast to using traditional enzymatic in vitro transcription technology, in which modified nucleotides may be included in the IVT reaction mixture at a defined ratio so that enzymes may randomly incorporate modified nucleotides into synthesized RNA chain.
  • site specific modification of the present disclosure one can define at which position adenine (A) should be modified with m6A instead of all the As in the RNA chain are randomly modified.
  • Enhanced green fluorescent protein (eGFP) mRNA were prepared using a combined chemical synthesis and in vitro transcription method as shown in FIG. 1.
  • a 5’-Capl eGFP mRNA was made by first synthesizing a RNA 14-mer oligonucleotide containing a 5 ’-phosphate group (sequence: mAGGAAAUAAGAGAG; mA is 2’-O-methyladenosine) (SEQ ID NO: 4) (RNA1) using well established solid phase oligonucleotide chemistry.
  • this oligonucleotide was chemically (non-enzymatically) capped (5’ m7Gppp-) (5’ capped RNA1) using organic synthetic process as described in the present disclosure.
  • the synthetic oligonucleotide was incubated with 7-methyl GDP imidazolide reagent in DMSO using 1 -methylimidazole as a catalyst in an overnight reaction at room temperature.
  • HPLC analysis (Cl 8) revealed that the capping reaction was of great efficiency and the 5’ capped oligonucleotide product was purified by HPLC using a Cl 8 column to generate a purified 5’ Capped 14-mer RNA sequence.
  • RNA2 The remaining later sequence of the eGFP mRNA fragment (RNA2) was prepared by an IVT approach. Briefly, the template was prepared by cleaving the eGFP mRNA plasmid with restriction enzyme, e.g., EARI, to cut after 14-mer of the 5’end of eGFP, then using PCR to add T7 promoter sequence using forward primer: TAATACGACTCACTATAGGAAAAGAAGAGTAAGAAG (SEQ ID NO: 1) and to add poly A tail using reverse primer: ttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttttt
  • the 5’ capped oligonucleotide product (14-mer) (i.e., the 5’-end of the eGFP mRNA fragment) (5’ capped RNA1) was then ligated to 5 ’-monophosphate RNA (964nt) (i.e., the 3’-end of the eGFP mRNA fragment) (RNA2) in ligation reaction in the presence of T4 RNA ligasel and DNA splint CTTACTCTTCTTTTCTCTCTTATTTC (SEQ ID NO: 3).
  • the ligated RNA was further purified by lithium chloride precipitation method to generate full-length recombinant eGFP mRNA (5’ capped recombinant RNA1/2).
  • eGFP mRNA The function of the full-length recombinant eGFP mRNA (978nt) was verified from cell expression assay. [00159] Briefly, A549 cells plated on 96 well plate was transfected with 100 ng of eGFP mRNA prepared by the chemical capping coupled with ligation method as disclosed here using lipofectamine MessengerMax, and the eGFP expression was measured using plate reader.
  • FIG. 3 shows eGFP expression in A549s using plate reader.
  • the term “eGFP - 14” indicates the truncated eGFP mRNA without the ligation of the 14-mer capped oligonucleotide (i.e., the 5’-end of the eGFP mRNA fragment).
  • eGFP +14 indicates the full-length recombinant eGFP mRNA, which was the ligation product of 5 ’-end 14-mer capped oligonucleotide (i.e., the 5’-end of the eGFP mRNA fragment) and truncated 3 ’-end 964 bases of monophosphate eGFP mRNA sequence prepared by IVT with GMP (i.e., the 3 ’-end of the eGFP mRNA fragment).
  • eGFP-RppH +14 indicates a version of full length recombinant eGFP mRNA prepared by ligating 14-bases capped oligo with a 964- bases monophosphate 3 ’end of eGFP mRNA sequence prepared by IVT using GMP followed up with a dephosphorylation reaction.
  • RNA containing a 5 ’-monophosphate group was prepared as shown in FIG. 2.
  • RNA was prepared using IVT, under 100% Nl- Methyl-PseudoUTP substitution in the absence of Cap analogs. This process generated modified, uncapped RNA with 5 ’-triphosphates.
  • the RNA product was further treated with 5 ’-RNA polyphosphatase to generate 5 ’-monophosphate groups to undergo chemical capping reactions.
  • Sample 3 chemically capped capO-eGFP mRNA (978 bases) prepared by directly chemical capping reaction on an in vitro transcribed full length monophosphate eGFP RNA is functional and has positive expression of eGFP protein, although less efficient than the positive control of enzymatically prepared capO-eGFP mRNA (Sample 4).
  • These results show technology breakthrough of direct chemical reaction on a full length eGFP mRNAs (978 bases) produced by in vitro transcription., That is, mRNA with more than 150 bases can be chemically capped and are functional as compared with that produced by co-transcriptional capping and enzymatic capping.
  • Pyridine was also evaluated as an alternative nucleophilic catalyst in the chemical capping incubation mixture. While expression was observed, the level of expression was lower as compared with that using 1 -methylimidazole as the catalyst.
  • FIG. 5 shows the absence of the starting oligonucleotide peaks in HPLC chromatograms, indicating that capping reaction of the present disclosure could reach completion within 24 hours. These results are consistent with subsequent LC-MS analysis of chemically capped RNA oligonucleotides.
  • FIG. 6 and Table 1 show the LC-MS results of a chemically capped 41-mer RNA oligonucleotide. If successfully capped, the capped 41-mer should have a theoretical mass of 14052 Da. If uncapped, the 41-mer should have a theoretical mass of 13595 Da.
  • T4 RNA ligase I T4 Rnll
  • T4 RNA ligase II T4 Rnl2
  • T4 DNAL T4 DNA ligase
  • T4 Rnll can catalyze covalent joining of single- stranded 5’ phosphoryl termini to single-stranded 3’ hydroxyl termini of DNA or RNA in an ATP-dependent manner.
  • T4 Rnl2 in comparison can preferentially join nicks on doublestranded RNA (dsRNA) rather than joining single-stranded RNA (ssRNA) termini.
  • single-stranded DNA (ssDNA) splints may be generally used to facilitate ligation of single-stranded RNA.
  • Intermolecular RNA ligation reactions using any of T4 Rnll, T4 Rnl2, and T4 DNAL may be complicated, however, by low efficiency and reaction byproducts, such as unwanted circularized and/or concatemer RNAs.
  • the annealed mixtures were then incubated at 37°C for 1 hour with either 10 units of T4 Rnl2 in the presence of lx buffer and 1 mM ATP, or 12 units of T4 DNAL in the presence of lx buffer and 20 units of RNase inhibitor.
  • the reaction mixtures were subsequently treated with RNase-free DNase I. As control, reactions without DNA splint were also included.
  • the reaction products were analyzed on 4% EX-gels. Successful ligation should generate a 353 nt (258 nt + 95 nt) RNA.
  • small ultra-red fluorescent protein developed from an allophycocyanin a-subunit (Nature Methods volume 13, pages763-769 (2016)) was used.
  • smURFP small ultra-red fluorescent protein
  • BV biliverdin
  • BVMe2 A synthetic derivative of biliverdin derivative biliverdin dimethyl ester
  • a synthetic uncapped 41-mer corresponding to smURFP mRNA 5’ sequences was used as the acceptor, and a truncated smURFP mRNA missing the first 41 nucleotides (A41 smURFP) (624 nt in length) served as the 5 ’-phosphate donor.
  • Capped full-length mRNA is 666 nt in length.
  • the 5’ truncated mRNA missing the first 41 nt (A41 smURFP) is 624 nt in length and served as the 5’-phosphate donor.
  • a reaction mixture containing A41 smURFP and a chemically capped 41-mer RNA oligo (i.e., 42-mer containing a cap analog) that corresponds to the 5’ of smURFP mRNA (at a ratio of 1 :30) were incubated with T4 Rnll in the presence of lx buffer, 1 mM ATP, and 25% PEG8000 at 37°C for 1 hour. Reaction products were examined on 4% EX-gels.
  • FIG. 9 shows a shift in migration of ligation product (666 nt) (lane 3) as compared with the truncated mRNA (624 nt) (lane 1), suggesting efficient ligation of the capped 41-mer RNA oligo (42-mer) with the truncated mRNA (624 nt).
  • the ligation mixture was purified on an Agilent 1260 Infinity II Preparative HPLC System using a DNAPac RP HPLC column (10x150 mm 4 um), with 0.1 M triethylammonium acetate (TEAA) in H2O as mobile phase A and 0. IM TEAA in 25% acetonitrile as mobile phase B.
  • TEAA triethylammonium acetate
  • IM TEAA in 25% acetonitrile
  • HPLC-collected fractions from above were transfected into CHO-K1 cells using Lipofectamine MessengerMax in the presence of 5 pM BVMe2 and fluorescence was analyzed by FACS after 24 hours. As controls, ligation mixtures without HPLC purification were also included. FACS analysis revealed that further RP-HPLC purification further enhanced smURFP mRNA expression. For example, FIGS. 10 and 11 A show that, following HPLC purification, the percentage of smURFP-positive cells increased by 14% (from 42.2% to 56.3%.), while mean smURFP fluorescence intensity increased by almost two fold (FIG. 11B). These data indicate successful generation of full-length functional smURFP mRNAs through ligation of chemically capped oligos and RNA fragment.
  • mRNA messenger RNA
  • One strategy to enhance mRNA stability may involve modifying poly(A) tail with chemically resistant nucleotides.
  • inverted deoxythymidine (dT)-containing poly(A) sequence may be appended to the 3' end of the mRNA to create a 3 ’-3’ linkage that may protect oligonucleotides from 3’ exonuclease cleavage.
  • incorporation of inverted dT residues may confer resistance against exonuclease-mediated degradation.
  • Firefly luciferase Flue
  • mRNA (1914 nt) SEQ ID NO: 6
  • Mobile phase A 0.1 M TEAA in H2O
  • mobile phase B 0.1M TEAA in 25% acetonitrile.
  • HPLC data show that samples eluted at around 50% to 60% mobile phase B. The gradient on semi-prep HPLC for purification was then extended between 50% B to 60% B for better separation.
  • FIG. 12 top panel, shows two peaks on HPLC chromatogram. Comparing with control samples, the second peak is indicated as the ligated product (1924 nt).
  • FIG. 12 shows that samples ligated with capped mRNA (1914 nt) (SEQ ID NO: 6) and inverted dT oligo (10 nt) (SEQ ID NO: 5) exhibited two distinct peaks, e.g., capped mRNA (1914 nt) and ligated product (1924 nt).
  • HEK293T and HepG2 cells were seeded at a density of 10,000 cells per well in a 96-well plate. Transfections were performed using 100 ng of capped Flue mRNA (1914 nt) or 100 ng of ligated capped Flue mRNA (1924 nt) with Messenger Max reagent, according to the manufacturer’s protocol. Following a 24-hour incubation period, luciferase activity was assessed by Luciferase Assay System (Promega) using SpectraMax ID3 instrument.
  • RNA ligases derived from T4 bacteriophage may be well-characterized and widely-used enzymes used for covalent linking of two single-stranded RNA fragments.
  • Other RNA ligase 1 enzymes from different bacteriophages have been characterized, but may be less commonly used.
  • One of these enzymes may be a moderately-thermostable RNA ligase 1 from TS2126 bacteriophage.
  • this enzyme may be functional at temperatures up to 65°C (Blondal et al., “Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties. Nucleic Acids Res. 2005 Jan 7;33(1): 135-42; the content of which is hereby incorporated by reference in its entirety).
  • TS2126 RNA ligase 1 has also been used to successfully perform both intra- and inter-molecular ligations.
  • a chemically-capped, synthetic 42-mer containing the first 41 nucleotides of a 666-nt long smURFP mRNA (i.e., 42-mer containing a cap analog) was used as phosphate acceptor.
  • a truncated smURFP mRNA lacking the first 41 nucleotides (A41 smURFP) (624 nt) was used as phosphate donor.
  • a mixture containing nuclease-free water, chemically- capped, synthetic 41-mer (42-mer) and A41 smURFP (624 nt) was made.
  • RNALigase 1 Chemically-capped, synthetic 41-mer (42-mer) and A41 smURFP (624 nt) were added in a ratio of 30: 1 as previously determined. This RNA was denatured by an incubation at 70°C for two minutes, followed by incubation at 4°C for three minutes. After denaturation step, the following reagents were added (the concentrations listed are the final concentrations of the reagents in the reaction mixture): 50 mM MOPS pH 7.5, 10 mM KC1, 5 mM MgCh, 1 mM DTT, 0.05 mM ATP, 2.5 mM MnCh, and 0.4 pg of TS2126 RNALigase 1.
  • TS2126 RNA ligase 1 has been shown to have relatively high intra-molecular ligation efficiency. The possibility of products arising from intra-molecular ligation instead of, or in addition to, inter-molecular ligation was of particular concern with this enzyme.
  • a control reaction mixture containing all of the components listed above except for the 41-mer was made was run.
  • An additional control containing all of the components listed in the initial reaction except TS2126 RNA Ligase 1 was also made. These reaction mixtures were incubated at 37°C for one hour. At the conclusion of this incubation period, reaction products were analyzed on a 4% agarose eGel- EX.
  • FIG. 14 shows a higher-molecular-weight band the same size as full-length smURFP was observed in the reaction containing chemically-capped, synthetic 41-mer (41- mer), A41 smURFP (624 nt) (A41), and TS2126 RNA ligase 1.
  • the molecular weight of this band also does not correspond to any of the intra-molecular ligation products seen in the reaction containing A41 smURFP without 41-mer.
  • RNA mixture was denatured by heating at 70°C for two minutes, followed by incubation at 4°C for three minutes.
  • the following reagents were added to each mixture (the listed concentrations are the final concentrations of reagents in the reaction mixture): 50 mM MOPS pH 7.5, 10 mM KC1, 5 mM MgCh, 1 mM DTT, 0.05 mM ATP, 2.5 mM MnCh, and 0.4 pg of TS2126 RNALigase 1.
  • TS2126 Ligation A41 Alone a control containing the aforementioned reagents with the exception of the 38- mer was made (referred to as TS2126 Ligation A41 Alone). Additional controls containing all of the listed reagents except TS2126 RNALigase 1 were also made. All reactions were incubated at 37°C for one hour. After incubation, each reaction was analyzed on a 4% agarose eGel -Ex.
  • FIG. 15 shows higher-molecular-weight bands, i.e., full-length smURFP, in the + TS2126 RNALigase 1 reaction mixtures containing PS-modified Oligo 1 and A41 smURFP or PS-modified Oligo 2 and A41 smURFP.
  • This full-length smURFP band does not correspond to any of the intra-molecular ligation products observed in the reaction mixture containing A41 smURFP without 38-mer.
  • Advantage of the present disclosure may include, for example, essentially, any modified capped RNA oligonucleotides up to 150 bases or > 150 bases can be produced by the methods of the present disclosure, which may allow for the use of a large plethora of commercially available modified nucleoside phosphorami dite reagents to produce modified mRNAthat could otherwise not be prepared by enzymatic capping processes due to T7 polymerase incompatibility.
  • Modified 5’-cap analogs can be incorporated into mRNAto allow for mRNA properties related to removal groups useful for mRNA purification and/or mRNA tissue specific activation (i.e. “Caged mRNA”).
  • methods of the present disclosure may allow generation of functional, 5’ capped, site-specific modification of long RNAs, e.g., > 150 bases, for protein expression in in vitro and in vivo applications.
  • Additional advantages may include (1) site-specific chemical capping: the use of activated capping compound (N 7 -methyl-GDP-imidazolide) may allow for direct, sitespecific installation of a methylated guanosine cap onto the 5 ’-monophosphate of the RNA oligonucleotide, ensuring the cap structure is uniform and correct and avoiding mixed populations; (2) catalyst-driven and enzyme-free: the heteroaromatic compound (1- methylimidazole) may promote efficient coupling under mild conditions, enabling cap formation without enzymatic assistance, reducing cost, and simplifying storage and handling.
  • RNAs containing non-natural nucleotides or modifications that can inhibit enzymatic reactions may also make the capping effective for RNAs containing non-natural nucleotides or modifications that can inhibit enzymatic reactions; (3) customizable cap chemistry: the activated GDP analog may allow incorporation of specific cap structures (e.g., N 7 -methyl) or other derivatives; and (4) scalability and cost-effectiveness: reagents may be synthetically accessible and suitable for batch processing, offering a potential cost advantage at scale.
  • the present invention may be defined by the following aspects:
  • a method for producing a site-specific modified capped recombinant RNA comprising: (a) obtaining a first RNA that is monophosphorylated at the 5’ end and comprises the site-specific modification, (b) reacting an activated capping compound of Formula (I) with the first RNA of (a) in the presence of a heteroaromatic compound and/or a metal salt, and a solvent:
  • the site specific modification comprises at least one modified nucleotide comprising a backbone modification, a sugar modification, and/or a base modification.
  • RNA polymerase in a concentration of from about 0.01 pg/pl to about 0.05 pg/pl.
  • the activated capping compound comprises a compound represented by Formula (II), Formula ⁇ I I) wherein
  • a method for producing a site-specific modified capped RNA comprising:
  • L a leaving group, thereby producing the site-specific modified capped RNA.
  • RNA of (a) comprises at least one modified nucleotide comprising a backbone modification, a sugar modification, and/or a base modification.
  • RNA that is monophosphorylated at the 5’ end of (a) is obtained by treating a triphosphorylated RNA produced by in vitro transcription in the presence or in the absence of a RNA 5’ polyphosphatase or a RNA 5’ pyrophosphohydrolase (RppH).
  • the in vitro transcription is performed in a reaction mixture comprising a buffer substance in a concentration of from about 45 mM to about 55 mM, an RNase inhibitor in a concentration of from about 0.01 U/pl to about 0.03 U/pl, NTPs in a concentration of from about 3 mM to about 5 mM, the cap analogue in a concentration of from about 6 mM to about 8 mM, one or more magnesium salts in a concentration of from about 20 mM to about 30 mM, a polyamine in a concentration of from about 1.5 mM to about 2.5 mM, the DNA template in a concentration of from about 0.01 pg/pl to about 0.05 pg/pl, optionally, the RNA 5’ polyphosphatase or the RppH in a concentration of from about
  • RNA polymerase in a concentration of from about 0.01 pg/pl to about 0.05 pg/pl.

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Abstract

L'invention concerne un procédé de production d'un ARN recombinant coiffé modifié spécifique à un site qui comprend (a) l'obtention d'un premier ARN qui est monophosphorylé à l'extrémité 5' et contient la modification spécifique à un site, (b) la réaction d'un composé de coiffage activé de formule (I), de formule (II) ou de formule (III) avec le premier ARN monophosphorylé en 5' obtenu à partir de (a) en présence d'un composé hétéroaromatique, d'un sel métallique et d'un solvant, produisant ainsi un premier ARN coiffé en 5', (c) l'obtention d'un second ARN qui est monophosphorylé à l'extrémité 5' et (d) la ligature de l'extrémité 3' du premier ARN coiffé en 5' obtenu à partir de (b) à l'extrémité 5' du second ARN obtenu à partir de (c), ce qui permet de produire l'ARN recombinant coiffé modifié spécifique à un site.
PCT/US2025/027793 2024-05-03 2025-05-05 Procédés de préparation d'arnm coiffé avec des modifications spécifiques à un site Pending WO2025231484A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130029326A1 (en) * 2007-08-17 2013-01-31 Epicentre Technologies Corporation Selective 5' Ligation Tagging of RNA
US20230250127A1 (en) * 2021-11-24 2023-08-10 Nanjing GeneLeap Biotechnology Co., Ltd. MODIFIED mRNA 5'-CAP ANALOGS
WO2023183825A2 (fr) * 2022-03-21 2023-09-28 Ptc Therapeutics Gt, Inc. Thérapie génique de maladies associées à ush2a
WO2024044741A2 (fr) * 2022-08-26 2024-02-29 Trilink Biotechnologies, Llc Procédé efficace de production d'oligonucléotides coiffés en 5' hautement purifiés
WO2025043082A2 (fr) * 2023-08-23 2025-02-27 The Broad Institute, Inc. Compositions et procédés d'amélioration des propriétés d'arn à l'aide d'une base, d'une liaison phosphodiester, d'un squelette de sucre et de modifications de coiffe

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130029326A1 (en) * 2007-08-17 2013-01-31 Epicentre Technologies Corporation Selective 5' Ligation Tagging of RNA
US20230250127A1 (en) * 2021-11-24 2023-08-10 Nanjing GeneLeap Biotechnology Co., Ltd. MODIFIED mRNA 5'-CAP ANALOGS
WO2023183825A2 (fr) * 2022-03-21 2023-09-28 Ptc Therapeutics Gt, Inc. Thérapie génique de maladies associées à ush2a
WO2024044741A2 (fr) * 2022-08-26 2024-02-29 Trilink Biotechnologies, Llc Procédé efficace de production d'oligonucléotides coiffés en 5' hautement purifiés
WO2025043082A2 (fr) * 2023-08-23 2025-02-27 The Broad Institute, Inc. Compositions et procédés d'amélioration des propriétés d'arn à l'aide d'une base, d'une liaison phosphodiester, d'un squelette de sucre et de modifications de coiffe

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