WO2011028218A1 - Procédé pour la synthèse d'oligonucléotides triphosphates - Google Patents

Procédé pour la synthèse d'oligonucléotides triphosphates Download PDF

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Publication number
WO2011028218A1
WO2011028218A1 PCT/US2009/069201 US2009069201W WO2011028218A1 WO 2011028218 A1 WO2011028218 A1 WO 2011028218A1 US 2009069201 W US2009069201 W US 2009069201W WO 2011028218 A1 WO2011028218 A1 WO 2011028218A1
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Prior art keywords
oligonucleotide
substituted
phosphonate
heteroaryl
group
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PCT/US2009/069201
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English (en)
Inventor
Ivan Zlatev
Francois Morvan
Jean-Jacques Vasseur
Francoise Debart
Muthiah Manoharan
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Alnylam Pharmaceuticals, Inc.
Centre National De La Recherche Scientifique (Cnrs)
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Priority claimed from PCT/US2009/055775 external-priority patent/WO2010028079A2/fr
Application filed by Alnylam Pharmaceuticals, Inc., Centre National De La Recherche Scientifique (Cnrs) filed Critical Alnylam Pharmaceuticals, Inc.
Priority to US13/393,851 priority Critical patent/US9035041B2/en
Priority to US12/847,893 priority patent/US20110282044A1/en
Publication of WO2011028218A1 publication Critical patent/WO2011028218A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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

Definitions

  • the present invention describes simple, efficient, and enzyme-free methods of making oligonucleotides with 5 '-triphosphate.
  • This invention presents novel processes for synthesizing triphosphate oligonucleotides using diaryl phosphite as a reagent.
  • the process of the present invention are amenable to large-scale, economic 5 '-triphosphate oligonucleotide synthesis.
  • Oligonucleotide 5 '-triphosphates are not commercially available; still they find a great number of important biochemical applications: DNA ONTPs are used in the antisense field, in basic research or in the biotechnology industry (Brownlee, et al., Nucleic Acids Research 1995, 23, (14), 2641-2647)), as substrates for polymerases or ligases in the preparation of synthetic genes (Xiong, et al., Ferns Microbiology Reviews 2008, 32, (3), 522-540); various synthetic RNA ONTPs can be used as primers for the amplification of RNA molecules by a 5 '-pyrophosphate activated, template directed oligoribonucleotide ligation - either catalyzed by RNA ligases or non enzymatic; but also in detection of viral responses via activation of the RIG-I protein; induction of antiviral immunity (Joyce, et al., Angewandte Chemie-International
  • RNA or DNA substrate which involves the precise choice of appropriate protecting groups and overall synthetic strategy, can only be added to the existing limitations known for nucleoside triphosphate (NTP) synthesis (Burgess, et al., Chemical Reviews 2000, 100, (6), 2047- 2059.).
  • NTP nucleoside triphosphate
  • synthetic efforts are still ongoing for developing a simple, efficient and universal triphosphorylation method for nucleosides (Crauste, et al., The Journal of Organic Chemistry 2009, 74, 9165-9172; Sun, Q et al., Organic Letters 2008, 10, (9), 1703-1706; Warnecke, et al., The Journal of Organic Chemistry 2009, 74, (8), 3024-3030).
  • the present invention is directed to improved processes for making 5'- triphosphate oligonucleotides via a H-phosphonate intermediate.
  • This invention embodies a method using solid-phase oligonucleotide synthesis comprising 5' H- phosphonate intermediate of a nucleotide bound to a solid-phase support. This is preferably done in the presence of diphenyl phosphite, thereby forming a
  • FIG. 1 is a schematic of the 5 '-triphosphate synthesis FIG. 2
  • A IE-HPLC profiles of: a) dT 7 -5'-H-phosphonate; b) pppdT 7 crude; c) pppdT 7 purified.
  • B MALDI-Tof MS of: a) dT 7 -5'-H-phosphonate; b) pppdT 7 crude; c) pppdT 7 purified
  • C 31 P NMR of purified pppdT 7 (Table 1, Entry 2)
  • FIG. 3 (A) IE-HPLC profiles of: a) UUGUCUCUGGUCCUUACUUAA-5'-H- phosphonate; b) pppUUGUCUCUGGUCCUUACUUAA crude; c)
  • FIG. 4 (A) IE-HPLC profiles and (B) MALDI-Tof MS of pppAGUUGUUCCC (Table 1, Entry 18)
  • oligonucleotide 5 '-triphosphates OFTPs
  • This invention provides a new and improved process for the preparation of oligonucleotide 5 '-triphosphates (ONTPs) and for intermediates useful in this process.
  • oligonucleotide 5 '-triphosphates are prepared from a plurality of RNA and/or DNA nucleotide subunits.
  • the nucleotide subunits may be "natural” or "synthetic" moieties.
  • the term "oligonucleotide” thus effectively includes naturally occurring species or synthetic species.
  • This invention provides a highly efficient and simple method for the solid-phase synthesis of both DNA and RNA ONTPs of various length, sequence and nature.
  • a protocol was established for providing various DNA and RNA 5 '-triphosphates with high conversion, good yields and satisfactory purity of crude products, avoiding tedious chromatography purification.
  • Most, if not all, of the steps of this preparation method use inexpensive, commercially available reagents, stable upon storage, and utilize either standard automated or simple manual experimental manipulations, which make the method useful for application in both research and industrial laboratories.
  • oligonucleotide 5 '-triphosphates of the invention can be prepared by a process comprising the steps of:
  • Ri and R 2 are each independently hydrogen, haloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, subsituted cycloalkyl, heterocycle, and substituted heterocycle, acyl, phosphoryl, substituted alkyl acyl, substituted heteroalkyl acyl, substituted aryl acyl or substituted heteroaryl acyl, substituted alkyl phosphoryl, substituted heteroalkyl acyl, substituted aryl phosphoryl or substituted heteroaryl phosphoryl; followed by an aqueous base treatment;
  • step (c) activating the H-phosphonate of step (b) using a silylating agent, a
  • oligonucleotide 5 '-triphosphates of the invention can be prepared by a process comprising the steps of: (a) synthesizing an oligonucleotide using a method selected from the group consisting of solid phase phosphoramidite, solution phase phosphoramidite, solid phase H-phosphonate, solution phase H-phosphonate, hybrid phase phosphoramidite, and hybrid phase H-phosphonate-based synthetic methods;
  • R is each independently hydrogen, halogen, haloalkyl, halogen, N0 2 , CN, acyl, and sulfonyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, subsituted cycloalkyl, heterocycle, and substituted heterocycle, n is 0 to 5; followed by an aqueous base treatment;
  • step (c) activating the H-phosphonate of step (b) using a silylating agent, a
  • R comprises at least one suitable EWG (electron withdrawing groups) which include halogens, N0 2 , CN, acyl, and sulfonyl.
  • EWG electron withdrawing groups
  • oligonucleotide 5 '-triphosphates of the invention can be prepared by a process comprising the steps of:
  • step (b) converting the 5' hydroxyl moiety to 5 '-H-phosphonate with diphenyl phosphite; followed by an aqueous base treatment; (c) activating the H-phosphonate of step (b) using a silylating agent, a halogenated oxidizing agent and a nitrogen containing heteroaryl;
  • step (b) is carried out in the for at least 10 minutes, 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes.
  • the aqueous base is selected from triethyl ammonium bicarbonate, triethyl ammonium phosphate, triethyl ammonium hydrogen phosphate, triethyl ammonium sulfate, triethyl ammonium hydrogen sulftate, triethyl ammonium chloride, other ammonium aqueous buffer at pH zone 5 - 9, potassium carbonate, sodium carbonate, sodium bicarbonate, water.
  • the silylating agent used in step (c) includes ⁇ , ⁇ - bis(trimethylsilyl)-acetamide (BSA), trimethylsilyl chloride; triethylsilyl chloride, trialkylsilyl chloride, triarylsilyl chloride or mixed alkyl aryl silyl chloride,
  • BSA trimethylsilyl chloride
  • triethylsilyl chloride triethylsilyl chloride
  • trialkylsilyl chloride triarylsilyl chloride or mixed alkyl aryl silyl chloride
  • HMDS Hexamethyldisilazane
  • the halogenated oxidizing agent used in step (c) includes CC or I 2 .
  • oxidizing agents for step (c) include: BSA and Et 3 N in CCl 4 /MeCN; I 2 and N,0-bis(trimethylsilyl)-acetamide in MeCN/pyridine,
  • the nitrogen containing heteroaryl is selected from the group consisting of pyridyl, substituted pyridyl, pyrimidinyl, substituted pyrimidinyl, imidazolyl, substituted imidazolyl, triazolyl, substituted triazolyl, tetrazolyl, substituted tretrazolyl, fused polyaromatic or polyheteroaromatic rings including one of the above.
  • the poly(alkylammonium)pyrophosphate is a salt of pyrophosphate and several ammonium, pyridinium or other bulky organic- solvent soluble counterions.
  • examples can be selected from tris(tri-ra-butylammonium)pyrophosphate, tetrakis(tri-ra-butylammonium)pyrophosphate, tris(tetra-w- butylammonium)pyrophosphate, tetrakis(tetra-ra-butylammonium)pyrophosphate tris(dimethylammonium)pyrophosphate, tris(tri-ethylammonium)pyrophosphate, tris(tri- isopropylammonium)pyrophosphate, tris(tri-n-propylammonium)pyrophosphate, tris(tri- i-butylammonium)pyrophosphate, tris(pyridinium)pyrophosphate, tre
  • step (c) is carried out in a solvent system selected from carbon tetrachloride, 1,1-dichloroethane, chloroform , perchloroethylene,
  • tetrachloroethylene 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, methylene chloride, trichloroethylene, methyl chloroform, 1,1,1-trichloroethane, 1,2,3-trichloropropane, ethylene dichloride, 1,2-dichloropropane, propylene dichloride, 1,2-dichloroethylene, acetonitrile, ethyl acetate, tetrahydrofuran (THF), dimethylsulfoxide(DMSO), dimethyl formamide (DMF), hexamethylphoshphoramide (HMPA), hexamethylphosphotriamide (HMPT), dimethylether (DME), pyridine, triethylamine, DIEA, dioxane, or combinations thereof.
  • THF tetrahydrofuran
  • DMSO dimethylsulfoxide
  • DMF dimethyl formamide
  • HMPA hexamethylphos
  • step (d) is carried out at room temperature for about 1 hour to about 100 hours. For example at least 10 hours, 12 hours, 15 hours, 16 hours, 17 hours, 18 hours.
  • step (d) is carried out with microwave radiation at about 30 to about 100 degrees for at least 5 minutes, 10 minutes, 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 60 minutes.
  • the oligonucleotide obtained from step (a) comprises at least one 2' protecting group selected from alkysilyl (i.e.TBMDS),
  • X and X' are independently CN, N0 2 , CF 3 , F, or OMe; Z is H or alkyl; and R 10 is aryl, substituted aryl, heteroaryl or substituted heteroaryl; and R 20 is alkyl (i.e. methyl, ethyl, propyl, butyl, i-butyl, isopropyl, isobutyl).
  • the whole process of the invention can be carried out with an automated synthesizer.
  • steps (c) and (d) can be adapted for automated synthesis.
  • the process of the invention can be adapted in one pot wherein purification is not necessary after each step.
  • the 2' protected oligonucleotide 5 '-triphosphates can be deprotected with a suitable deprotecting agent.
  • RNA is often synthesized and purified by methodologies based on: tetrazole to activate the RNA amidite, NH 4 OH to remove the exocyclic amino protecting groups, n- tetrabutylammonium fluoride (TBAF) to remove the 2'-OH alkylsilyl protecting groups, and gel purification and analysis of the deprotected RNA.
  • TBAF n- tetrabutylammonium fluoride
  • the RNA compounds may be formed either chemically or using enzymatic methods.
  • oligonucleotide synthesis One important component of oligonucleotide synthesis is the installation and removal of protecting groups. Incomplete installation or removal of a protecting group lowers the overall yield of the synthesis and introduces impurities that are often very difficult to remove from the final product. In order to obtain a reasonable yield of a large RNA molecule (i.e., about 20 to 40 nucleotide bases), the protection of the amino functions of the bases requires either amide or substituted amide protecting groups. The amide or substituted amide protecting groups must be stable enough to survive the conditions of synthesis, and yet removable at the end of the synthesis.
  • amide protecting groups benzoyl or phenoxyacetyl for adenosine, isobutyryl, acetyl, phenoxyacetyl or benzoyl for cytidine, and iso-propylphenoxyacetyl, tert-butylphenoxyacetyl or isobutyryl for guanosine.
  • the amide protecting groups are often removed at the end of the synthesis by incubating the RNA in NH 3 /EtOH or 40% aqueous MeNH 2 .
  • One aspect of the present invention relates to amino compounds with relatively low volatility capable of effecting the amide deprotection reaction.
  • the classes of compounds with the aforementioned desirable characteristics are listed below. In certain instances, preferred embodiments within each class of compounds are listed as well.
  • the polyamine compound used in the invention relates to polymers containing at least two amine functional groups, wherein the amine functional group has at least one hydrogen atom.
  • the polymer can have a wide range of molecular weights. In certain embodiment, the polyamine compound has a molecular weight of greater than about 5000 g/mol. In other embodiments, the polyamine compound compound has a molecular weight of greater than about 10,000; 20,000, or 30,000 g/mol.
  • the PEG-NH 2 compound used in the invention relates to polyethylene glycol polymers comprising amine functional groups, wherein the amine functional group has at least one hydrogen atom.
  • the polymer can have a wide range of molecular weights.
  • the PEG-NH 2 compound has a molecular weight of greater than about 5000 g/mol.
  • the PEG-NH 2 compound has a molecular weight of greater than about 10, 000; 20,000, or 30,000 g/mol.
  • the short PEG-NH 2 compounds used in the invention relate to polyethylene glycol polymers comprising amine functional groups, wherein the amine functional group has at least one hydrogen atom.
  • the polymer has a relatively low molecular weight range.
  • the cycloalkylamines used in the invention relate to cycloalkyl compounds comprising at least one amine functional group, wherein the amine functional group has at least one hydrogen atom.
  • the hydroxycycloalkyl amines used in the invention relate to cycloalkyl compounds comprising at least one amine functional group and at least one hydroxyl functional group, wherein the amine functional group has at least one hydrogen atom. Representative examples are listed below.
  • the hydroxyamines used in the invention relate to alkyl, aryl, and aralkyl compounds comprising at least one amine functional group and at least one hydroxyl functional group, wherein the amine functional group has at least one hydrogen atom.
  • Representative examples are 9-aminononanol, 4-aminophenol, and 4- hydroxybenzylamine.
  • One aspect of the present invention relates to a method of removing an amide protecting group from an oligonucleotide, comprising the steps of:
  • an oligonucleotide bearing an amide protecting group with a polyamine, PEHA, PEG-NH 2j Short PEG-NH 2 , cycloalkyl amine, hydroxycycloalkyl amine, hydroxyamine, K ⁇ COs/MeOH microwave, thioalkylamine, thiolated amine, ⁇ -amino- ethyl- sulfonic acid, or the sodium sulfate of ⁇ -amino-ethyl-sulfonic acid.
  • R 1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl
  • R is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl
  • R is aryl or heteroaryl.
  • aryl amines of the hydrofluoride salts are selected from the group consisting of (dialkyl)arylamines, (alkyl)diarylamines, (alkyl)(aralkyl)arylamines, (diaralkyl)arylamines, (dialkyl)heteroarylamines, (alkyl)diheteroarylamines,
  • the rate of the deprotection reaction can be excelerated by conducting the deprotection reaction in the presence of microwave radiation.
  • the tert-butyldimethylsilyl groups on a 10-mer or 12-mer could be removed in 2 minutes or 4 minutes, respectively, by treatment with 1 M TBAF in THF, Et 3 N-HF, or pyridine-HF/DBU in the presence of microwave radiation (300 Watts, 2450 MHz).
  • One aspect of the present invention relates to a method removing a silyl protecting group from a oligonucleotide, comprising the steps of:
  • an oligonucleotide bearing a silyl protecting group with pyridine-HF, DMAP-HF, Urea-HF, TSA-F, DAST, polyvinyl pyridine-HF, or an aryl amine-HF reagent of formula A:
  • R 1 is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl
  • R is alkyl, aryl, heteroaryl, aralkyl or heteroaralkyl; and R is aryl or heteroaryl.
  • the present invention relates to the aforementioned method, wherein said oligonucleotide is an oligomer of ribonucleotides.
  • the present invention relates to the aforementioned method, wherein the reaction is carried out in the presence of microwave radiation.
  • oligonucleotide 5 '-triphosphates having tens or even hundreds of individual nucleotide subunits can be prepared utilizing the processes and intermediates of this invention. Such very large oligonucleotide 5 '-triphosphates can be assembled from smaller oligonucleotide intermediates that, in turn, would be assembled from even smaller intermediates. Thus, oligonucleotide 5 '-triphosphates and
  • oligonucleotide 5 '-triphosphate intermediates of the invention contain one or more subunits.
  • the oligonucleotide 5 '-triphosphates of the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein.
  • the organism should be contacted with a oligonucleotide 5'- triphosphates having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein or is capable of specifically hybridizing with a target gene thereby modulating the gene expression. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms.
  • the preferred oligonucleotide can have all natural 2'- deoxyribo and 2'-ribonuclesides, 2'-0-methyl (2'-OMe), 2'-0-methoxyethyl (2'-MOE), 2'-deoxy-2'-ribofluoro (2'-F), 2'-deoxy-2'-arabinofluoro (2'-araF) sugar modifications and combinations there of, with and without phosphorothioate backbone at the internucleoside linkages.
  • the preferred nucleobase modifications includes 2-ThioU, 2'- amino-A, pseudouridine, inosine, 5-Me-U, 5-Me-C, chemically modified U analogues.
  • the preferred oligonucleotide can have ligands includes PK modulators such as lipophiles, Cholesterol and analogs, bile acids, steroids, circulation enhancers - PEG with different mol. wt. starting from 400 to up to 60,000 amu, small molecule protein binders (for e.g, naproxen or ibuprofen) and targeting ligands for receptor targeting, for e.g., folic acid, GalNAc and mannose.
  • PK modulators such as lipophiles, Cholesterol and analogs, bile acids, steroids, circulation enhancers - PEG with different mol. wt. starting from 400 to up to 60,000 amu
  • small molecule protein binders for e.g, naproxen or ibuprofen
  • targeting ligands for receptor targeting for e.g., folic acid, GalNAc and mannose.
  • Evaluation of the oligonucleotide can include incubating the modified strand (with or without its complement, but preferably annealed to its complement) with a biological system, e.g., a sample (e.g, a cell culture).
  • a biological system e.g., a sample (e.g, a cell culture).
  • the biological sample can be capable of expressing a component of the immune system. This allows identification of an oligonucleotide that has an effect on the component.
  • the step of evaluating whether the oligonucleotide modulates, e.g, stimulates or inhibits, an immune response includes evaluating expression of one or more growth factors, such as a cytokine or interleukin, or cell surface receptor protein, in a cell free, cell-based, or animal assay.
  • Exemplary assay methods include ELISA and Western blot analysis. Growth factors that could be evaluated include TNFa, ILla and ⁇ , IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IFNa and ⁇ , and IFNy.
  • a test includes evaluating expression of one or more of the interleukins IL- 18, IL- ⁇ , IL-10, IL-12, and IL-6.
  • Relevant cell surface receptors include the toll-like receptors, e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • a test includes evaluating expression of one or more of the toll-like receptors TL-3, TLR7, TLR8, or TLR9.
  • Ligand interaction with TLR9 stimulates expression of NFKB. Therefore, testing whether an oligonucleotide stimulates the immune response can include assaying for NFKB protein or mRNA expression.
  • the step of testing whether the modified oligonucleotide modulates, e.g., stimulates, an immune response includes assaying for an interaction between the oligonucleotide and a protein component of the immune system, e.g., a growth factor, such as a cytokine or interleukin, or a cell surface receptor protein.
  • a protein component of the immune system e.g., a growth factor, such as a cytokine or interleukin, or a cell surface receptor protein.
  • the test can include assaying for an interaction between the modified oligonucleotide and a toll-like receptor, e.g., TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9.
  • testing includes assaying for an interaction with a toll-like receptor, e.g., TLR-9.
  • exemplary assay methods include coimmunoprecipitation assays, bead-based co-isolation methods, nucleic acid footprint assays and colocalization experiments such as those facilitated by immunocytochemistry techniques.
  • the oligonucleotide includes a substitution of an adenine with a 2-substituted purine (e.g., 2-amino-adenine, ), a 6-substituted purine, a 7-deaza-alkyl-substituted purine, a 7-deaza-alkenyl-substituted purine, a 7-deaza- alkynyl-substituted purine, or a purine that is not adenine (e.g., guanine or inosine).
  • a 2-substituted purine e.g., 2-amino-adenine,
  • a 6-substituted purine e.g., 2-amino-adenine, a 6-substituted purine
  • a 7-deaza-alkyl-substituted purine e.g., a 7-deaza-alkenyl-substituted pur
  • the candidate oligonucleotide includes a substitution of a guanine with an inosine, an aminopurine, a 2-substituted guanine, a 7-deaza-alkyl-substituted guanine, a 7-deaza-alkenyl-substituted guanine, a 7-deaza-alkynyl-substituted guanine, or an O-6-alkylated guanine.
  • the candidate oligonucleotide includes a substitution of a cytosine with a 5-substituted cytosine (e.g., a 5-methyl cytosine), an N-4 substituted cytosine, a G-clamp, an analog of a G-clamp, a 2-thio- cytosine, a 4-thio-cytosine, or a uracil.
  • the candidate oligonucleotide includes a substitution of a uracil with a 5-substituted uracil, a 4-thio-uracil, a 5-methyl- 2-thio-uracil, a pseudouridine, a 1-alkylpseudouridine, a 3-alkylpseudouridine or a 2-thio- uracil.
  • the oligonucleotide includes a 2'-deoxyfluoro, 2'-0-methyl, 2'-0-methoxyethyl, 2'-0-alkyl, 2'-0-alkoxyalkyl, 2'-0-allyl, 2'-0-propyl, 2'-0-(N- methyl-acetamide (NMA), 2'-0-(N,N-dimethylaminooxyethyl), or G-clamp
  • the oligonucleotide includes an arabinose-containing nucleoside that replaces a ribonucleoside.
  • the arabinose- containing nucleoside can be a 2'-fluoroarabinose-containing nucleoside, or a T-O- methylarabinose-containing nucleoside.
  • the oligonucleotide includes a deoxynucleoside that replaces a ribonucleoside.
  • the deoxynucleoside is a 2'-fluorodeoxynucleoside, or a 2'-0-methyldeoxynucleoside.
  • an immunoselective oligonucleotide includes at least one backbone modification, e.g., a phosphorothioate, boronaphosphate, methylphosphonate or dithioate modification.
  • the oligonucleotide includes a P-alkyl modification in the linkages between one or more of the terminal nucleotides of an oligonucleotide.
  • the sense and/or antisense strand is
  • the sense and/or antisense strand is substantially free of stereogenic phosphorus atoms having an Sp configuration.
  • one or more terminal nucleotides of an oligonucleotide include a sugar modification, e.g., a 2' or 3' sugar modification.
  • the oligonucleotide includes at least two sugar 2' modifications.
  • Exemplary sugar modifications include, for example, a 2'-fluoro nucleotide, a 2'-0-alkyl nucleotide, a 2'- O-alkoxyalkyl nucleotide, a 2'-0-allyl nucleotide, a 2' O-propyl nucleotide, a T-O- methylated nucleotide (2'-0-Me), a 2'-deoxy nucleotide, a 2'-deoxyfluoro nucleotide, a 2'-0-methoxyethyl nucleotide (2'-0-MOE), a 2'-0-N-MeAcetamide nucleotide ( -O- NMA), a 2'-0-dimethylaminoethyloxyethyl nucleotide (2'-0- DMAEOE), a 2'- aminopropyl, a 2'-hydroxy, a 2'-ara-fluoro, or 3'-
  • the oligonucleotide includes a methylphosphonate.
  • the oligonucleotide includes a difluorotoluyl (DFT) modification, e.g., 2,4-difluorotoluyl uracil, or a guanidine to inosine substitution.
  • DFT difluorotoluyl
  • the oligonucleotide includes a 5 '-uridine- adenine- 3' (5'-UA-
  • the uridine is a 2' -modified nucleotide, or a 5 '-uridine-guanines' (5'-UG-3') dinucleotide, wherein the 5 '-uridine is a 2' -modified nucleotide, or a 5'- cytidine-adenine-3' (5'-CA-3') dinucleotide, wherein the 5'-cytidine is a 2' -modified nucleotide, or a 5'-uridine-uridine-3' (5' -UU-3') dinucleotide, wherein the 5 '-uridine is a 2' -modified nucleotide, or a 5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the 5'-cytidine is a 2' -modified nucleotide, or a 5'-cytidine-cytidine-3' (5
  • the chemically modified nucleotide in the oligonucleotide may be a 2'-0-methylated nucleotide.
  • the modified nucleotide can be a 2' -deoxy nucleotide, a 2'-deoxyfluoro nucleotide, a 2'-O-methoxyethyl nucleotide, a 2'-O-NMA, a 2'- DMAEOE, a 2'-aminopropyl, 2'-hydroxy, or a 2'-ara-fluoro, or 3'-amidate (3'-NH in place of 3'-0), a locked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA).
  • LNA locked nucleic acid
  • ENA extended nucleic acid
  • HNA hexose nucleic acid
  • CeNA cyclohexene nucleic acid
  • the oligonucleotide has a single overhang, e.g., one end of the oligonucleotide has a 3' or 5' overhang and the other end of the oligonucleotide is a blunt end.
  • the oligonucleotide has a double overhang, e.g., both ends of the oligonucleotide have a 3' or 5' overhang, such as a dinucleotide overhang.
  • both ends of the oligonucleotide have blunt ends.
  • the oligonucleotide includes a sense RNA strand and an antisense RNA strand, and the antisense RNA strand is 18 - 30 nucleotides in length.
  • the oligonucleotide includes a nucleotide overhang having 1 to 4 unpaired nucleotides, which may be at the 3 '-end of the antisense RNA strand, and the nucleotide overhang may have the nucleotide sequence 5'-GC-3' or 5'-CGC-3' .
  • the unpaired nucleotides may have at least one phosphorothioate dinucleotide linkage, and at least one of the unpaired nucleotides may be chemically modified in the 2' -position.
  • the double strand region of the candidate oligonucleotide includes phosphorothioate linkages on one or both of the sense and antisense strands.
  • the candidate oligonucleotide includes phosphorothioate linkages between nucleotides 1 through 5 of the 5' or 3' end of the sense or antisense agent.
  • the antisense RNA strand and the sense RNA strand are connected with a linker.
  • the chemical linker may be a hexaethylene glycol linker, a poly-(oxyphosphinico-oxy-l,3-propandiol) linker, an allyl linker, or a polyethylene glycol linker.
  • the immuno selective oligonucleotide can include at least two modifications.
  • the modifications can differ from one another, and may be applied to different RNA strands of a double- stranded oligonucleotide.
  • the sense strand can include at least one modification
  • the antisense strand can include a modification that differs from the modification or modifications on the sense strand.
  • the sense strand can include at least two different modifications
  • the antisense strand can include at least one modification that differs from the two different modifications on the sense strand. Accordingly, the sense strand can include multiple different modifications, and the antisense strand can include further multiple
  • a double strand oligonucleotide comprises two triphosphate moieties at the 5' end of each strand.
  • 5'-phosphoramidites it would be possible to introduce the triphosphate at the 3'-end.
  • the invention features a method of evaluating an oligonucleotide that includes providing a candidate single stranded oligonucleotide having at least one ribonucleotide modification; contacting the candidate single stranded oligonucleotide to a cell-free system, cell, or animal; and evaluating the immune response in the cell-free system, cell, or animal as compared to an immune response in a cell-free system, cell, or animal that is contacted with an unmodified single stranded oligonucleotide.
  • the candidate single stranded oligonucleotide stimulates an immune response to a lesser or greater extent than a reference.
  • an unmodified oligonucleotide is determined to be an oligonucleotide that modulates an immune system response.
  • the candidate single- stranded oligonucleotide is 15-2000 nucleotides in length (e.g., 17, 19, 21, 23, 25, 27, 28, 29, 30, 100, 500, 1000, or 1500 nucleotides in length).
  • linker or "spacer” generally refers to any moiety that can be attached to an oligoribonucleotide by way of covalent or non-covalent bonding through a sugar, a base, or the backbone.
  • the linker/spacer can be used to attach two or more nucleosides or can be attached to the 5' and/or 3' terminal nucleotide in the oligoribonucleotide.
  • Such linker can be either a non-nucleotidic linker or a nucleotidic linker.
  • non-nucleotidic linker generally refers to a chemical moiety other than a nucleotidic linkage that can be attached to an oligoribonucleotide by way of covalent or non-covalent bonding.
  • non-nucleotidic linker is from about 2 angstroms to about 200 angstroms in length, and may be either in a cis or trans orientation, (e.g. d(T) n ; wherein n is 1-10) or non-nucleotidic (for example a linker described herein, e.g. optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl or heteroaryl).
  • nucleotidic linkage generally refers to a chemical linkage to join two nucleosides through their sugars (e.g. 3'-3', 2'-3',2'-5', 3'-5') consisting of a phosphate, non-phosphate, charged, or neutral group (e.g., phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate (e.g. methylphosphonate), amide, ester, disulfide, thioether, oxime and hydrazone linkage between adjacent nucleosides.
  • sugars e.g. 3'-3', 2'-3',2'-5', 3'-5'
  • neutral group e.g., phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate (e.g. methylphosphonate)
  • the linker/spacer between the two oligonucleotides comprises a cleavable linking group, for example a group that is potentially biodegradable by enzymes present in the organism such as nucleases and proteases or cleavable at acidic pH or under reductive conditions, such as by glutathione present at high levels intraceluUarly.
  • cleavable linking groups include, but are not limited to, disulfides, amides, esters, peptide linkages and phosphodiesters.
  • the cleavable linking group can be internal to the spacer or may be present at one or both terminal ends of the spacer. In one embodiment, the cleavable linking group is between one of the oligonucleotides and the spacer. In one embodiment, the cleavable linking group is present on both ends of the spacer. In one embodiment, the cleavable linking group is internal to the spacer.
  • halo refers to any radical of fluorine, chlorine, bromine or iodine.
  • aliphatic refers to non- aromatic moiety that may contain any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contain one or more units of unsaturation, e.g., double and/or triple bonds.
  • An aliphatic group may be straight chained, branched or cyclic and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms.
  • aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted.
  • alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms.
  • CrC 12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it.
  • haloalkyl refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl).
  • Alkyl and haloalkyl groups may be optionally inserted with O, N, or S.
  • aralkyl refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group.
  • Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.
  • alkenyl refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3- octenyl groups.
  • alkynyl refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and
  • the sp and sp carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.
  • alkylamino and dialkylamino refer to -NH(alkyl) and -N (alkyl) 2 radicals respectively.
  • aralkylamino refers to a -NH(aralkyl) radical.
  • alkoxy refers to an -O-alkyl radical
  • cycloalkoxy and “aralkoxy” refer to an -O-cycloalkyl and O-aralkyl radicals respectively.
  • sioxy refers to a R 3 SiO- radical.
  • mercapto refers to an SH radical.
  • thioalkoxy refers to an -S-alkyl radical.
  • alkylene refers to a divalent alkyl (i.e., -R-), e.g., -CH 2 -, -CH 2 CH 2 -, and -CH 2 CH 2 CH 2 -.
  • alkylenedioxo refers to a divalent species of the structure -0-R-0-, in which R represents an alkylene.
  • aryl refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom can be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.
  • cycloalkyl as employed herein includes saturated cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom can be substituted.
  • the cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.
  • heterocyclyl refers to a nonaromatic 3-10 membered monocyclic, 8- 12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted.
  • the heterocyclyl groups herein described may also contain fused rings.
  • Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings).
  • heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted.
  • the heteroaryl groups herein described may also contain fused rings that share a common carbon-carbon bond.
  • oxo refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
  • acyl refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.
  • substituted refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group.
  • Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, azide, amino, SO 3 H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(0) n alkyl (where n is 0-2), S(0) n aryl (where n is 0-2), S(0) n heteroaryl (where n is 0-2), S(0) n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-,
  • a “protected” moiety refers to a reactive functional group, e.g., a hydroxyl group or an amino group, or a class of molecules, e.g., sugars, having one or more functional groups, in which the reactivity of the functional group is temporarily blocked by the presence of an attached protecting group.
  • Protecting groups useful for the monomers and methods described herein can be found, e.g., in Greene, T.W., Protective Groups in Organic Synthesis (John Wiley and Sons: New York), 1981, which is hereby
  • the oligonucleotides of the invention is an oligonucleotide.
  • An "iRNA agent,” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, stimulate or inhibit an immune response, or have no effect on an immune response.
  • An oligonucleotide may also down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA.
  • an oligonucleotide that down regulates expression of a target gene may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA (sometimes referred to in the art as RNAi), or pre-transcriptional or pre- translational mechanisms.
  • RNAi post-transcriptional cleavage of a target mRNA
  • An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded oligonucleotide. If the oligonucleotide is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
  • oligonucleotides used in accordance with this invention may be with solid phase synthesis, see for example “Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2'-0— Methyloligoribonucleotide- s: synthesis and applications, Chapter 4,
  • oligoribonucleotides is described in U.S. Pat. No. 5,508,270.
  • the preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863.
  • the preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No.
  • oligoribonucleotides is described in U.S. Pat. No. 5,023,243.
  • the preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198.
  • the preparation of 3'-Deoxy-3'-amino phosphor amidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925.
  • 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.
  • Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Cosstick et al. Tetrahedron Lett. 1989, 30, 4693.
  • oligonucleotide having increased stability in yet another aspect, relates to methods for identifying oligonucleotide having increased stability in biological tissues and fluids such as serum, oligonucleotide having increased stability have enhanced resistance to degradation, e.g., by chemicals or nucleases (particularly endonucleases) which normally degrade RNA molecules.
  • Methods for detecting increases in nucleic acid stability are well known in the art. Any assay capable of measuring or detecting differences between a test oligonucleotide and a control oligonucleotide in any measurable physical parameter may be suitable for use in the methods of the present invention.
  • the inhibitory effect of an oligonucleotide on a target gene activity or expression requires that the molecule remain intact, the stability of a particular oligonucleotide can be evaluated indirectly by observing or measuring a property associated with the expression of the gene.
  • the relative stability of an oligonucleotide can be determined by observing or detecting (1) an absence or observable decrease in the level of the protein encoded by the target gene, (2) an absence or observable decrease in the level of mRNA product from the target gene, and (3) a change or loss in phenotype associated with expression of the target gene.
  • the stability of an oligonucleotide may be evaluated based on the degree of the inhibition of expression or function of the target gene, which in turn may be assessed based on a change in the disease condition of the patient, such as reduction in symptoms, remission, or a change in disease state.
  • the method includes preparing an oligonucleotide as described above (e.g., through chemical synthesis), incubating the oligonucleotide with a biological sample, then analyzing and identifying those oligonucleotide that exhibit an increased stability as compared to a control oligonucleotide.
  • oligonucleotide is produced in vitro by mixing/annealing complementary single-stranded RNA strands, preferably in a molar ratio of at least about 3:7, more preferably in a molar ratio of about 4:6, and most preferably in essentially equal molar amounts (e.g., a molar ratio of about 5:5).
  • the single-stranded RNA strands are denatured prior to mixing/annealing, and the buffer in which the mixing/annealing reaction takes place contains a salt, preferably potassium chloride.
  • Single- stranded RNA strands may be synthesized by solid phase synthesis using, for example, an Expedite 8909 synthesizer (Applied Biosystems, Applera Kunststoff GmbH, Darmstadt, Germany), as described above.
  • Oligonucleotide are incubated with a biological sample under the conditions sufficient or optimal for enzymatic function. After incubating with a biological sample, the stability of the oligonucleotide is analyzed by means conventional in the art, for example using RNA gel electrophoresis as exemplified herein.
  • the oligonucleotide may be incubated at a concentration of 1-10 ⁇ , preferably 2-8 ⁇ , more preferably 3-6 ⁇ , and most preferably 4-5 ⁇ .
  • the incubation temperature is preferably between 25°C and 45°C, more preferably between 35°C and 40°C, and most preferably about 37°C.
  • the biological sample used in the incubation step may be derived from tissues, cells, biological fluids or isolates thereof.
  • the biological sample may be isolated from a subject, such as a whole organism or a subset of its tissues or cells.
  • the biological sample may also be a component part of the subject, such as a body fluid, including but not limited to blood, serum, plasma, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen.
  • the biological sample is a serum derived from a blood sample of a subject.
  • the subject is preferably a mammal, more preferably a human or a mouse.
  • the method includes selecting an oligonucleotide having increased stability by measuring the mRNA and/or protein expression levels of a target gene in a cell following introduction of the oligonucleotide.
  • an oligonucleotide of the invention inhibits expression of a target gene in a cell, and thus the method includes selecting an oligonucleotide that induces a measurable reduction in expression of a target gene as compared to a control oligonucleotide.
  • Assays that measure gene expression by monitoring RNA and/or protein levels can be performed within about 24 hours following uptake of the oligonucleotide by the cell.
  • RNA levels can be measured by Northern blot techniques, RNAse Protection Assays, or Quality Control-PCR (QC-PCR) (including quantitative reverse transcription coupled PCR (RT- PCR)) and analogous methods known in the art.
  • Protein levels can be assayed, for example, by Western blot techniques, flow cytometry, or reporter gene expression (e.g., expression of a fluorescent reporter protein, such as green fluorescent protein (GFP)).
  • RNA and/or protein levels resulting from target gene expression can be measured at regular time intervals following introduction of the test oligonucleotide, and the levels are compared to those following introduction of a control oligonucleotide into cells.
  • a control oligonucleotide can be a nonsensical oligonucleotide (i.e., an oligonucleotide having a scrambled sequence that does not target any nucleotide sequence in the subject), an oligonucleotide that can target a gene not present in the subject (e.g., a luciferase gene, when the oligonucleotide is tested in human cells), or an oligonucleotide otherwise previously shown to be ineffective at silencing the target gene.
  • the mRNA and protein levels of the test sample and the control sample can be compared.
  • oligonucleotide is selected as having increased stability when there is a measurable reduction in expression levels following absorption of the test oligonucleotide as compared to the control oligonucleotide.
  • mRNA and protein measurements can be made using any art-recognized technique (see, e.g., Chiang, M.Y., et al., J. Biol Chem. (1991) 266:18162-71; Fisher, T, et al, Nucl. Acids Res. (1993) 21:3857; and Chen et al, J. Biol. Chem. (1996) 271:28259).
  • oligonucleotide composition of the invention can be measured using a variety of techniques known in the art. For example, Northern blot analysis can be used to measure the presence of RNA encoding a target protein. The level of the specific mRNA produced by the target gene can be measured, e.g., using RT- PCR. Because oligonucleotide directs the sequence- specific degradation of endogenous mRNA through RNAi, the selection methods of the invention encompass any technique that is capable of detecting a measurable reduction in the target RNA. In yet another example, Western blots can be used to measure the amount of target protein present.
  • a phenotype influenced by the amount of the protein can be detected.
  • Techniques for performing Western blots are well known in the art (see, e.g., Chen, et al, J. Biol. Chem. (1996) 271:28259).
  • the target gene When the target gene is to be silenced by an oligonucleotide that targets a promoter sequence of the target gene, the target gene can be fused to a reporter gene, and reporter gene expression (e.g., transcription and/or translation) can be monitored.
  • reporter gene expression e.g., transcription and/or translation
  • a portion of the target gene (e.g., a portion including the target sequence) can be fused with a reporter gene so that the reporter gene is transcribed.
  • a portion of the target gene e.g., a portion including the target sequence
  • By monitoring a change in the expression of the reporter gene in the presence of the oligonucleotide it is possible to determine the effectiveness of the oligonucleotide in inhibiting the expression of the reporter gene.
  • the expression levels of the reporter gene in the presence of the test oligonucleotide versus a control oligonucleotide are then compared.
  • the test oligonucleotide is selected as having increased stability when there is a measurable reduction in expression levels of the reporter gene as compared to the control oligonucleotide.
  • reporter genes useful for use in the present invention include, without limitation, those coding for luciferase, GFP, chloramphenicol acetyl transferase (CAT), ⁇ -galactosidase, and alkaline phosphatase. Suitable reporter genes are described, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, New York (Ausubel, F.A., et al., eds., 1989); Gould, S. J., and S. Subramani, Anal. Biochem.
  • a two hour treatment with saturated aqueous ammonia at room temperature allowed removing of the oligomers from the solid support and provided the
  • FIG. 2 A the 5 '-triphosphate oligonucleotide purity from the crude mixture was above 80% (A.b) as determined by ion exchange HPLC; and minor contamination by the non
  • RNA 5 '-triphosphate sequence was chosen since it is particularly important as a precursor for the 5 '-cap structure of flaviviruses and is particularly difficult of access.
  • the 10-mer RNA ONTP was successfully prepared providing high yield and good purity of the crude product (Table 1, Entry 18 and FIG. 4). The synthesis of longer RNA 5 '-triphosphates using the PivOM approach are currently in progress.
  • Oligonucleotides la-c were synthesized on CPG solid support (Glen Research) using standard automated oligonucleotide synthesis on an ABI394 (Applied Biosystems) synthesizer on the 40 umol scale. Standard synthesis cycle was used for detritylation, phosphor amidite coupling, oxidation (sulfurization) and capping steps. 3'-di- isopropylphosphoramidites bearing fast labile nucleobase protecting groups (i.e. Ac for C, Pac for A, iPrPac for G) were commercially available (Chem Genes), and used as a 0.2 M solution in anhydrous acetonitrile (Glen).
  • a reagent was a Pac 2 0 solution.
  • a Trityl Off synthesis was performed as the last DMTr group was removed at the end of the synthesis.
  • the oligonucleotide was washed with anhydrous acetonitrile and reverse flushed with argon.
  • the solid supported oligonucleotide was then stocked at -20 °C.
  • Solid supported H-Phosphonates 2 (0.25 to 2 umol) were placed in an empty Twist synthesis column. 5 to 6 beads of activated 4 A molecular sieves were introduced inside the column. The column was closed and flushed with argon. The oxidation solution was then prepared as follows: 150 mg (2 mmol) of imidazole (Aldrich) were coevaporated twice with anhydrous acetonitrile and then dried under vaccum over P 2 O 5 .
  • the dried solid support CPG carrying the oligonucleotide triphosphates 2" was transferred from the Twist column to an empty screw cap plastic vial (10 mL). 2 mL of 30% NH 4 OH (JTBaker)/ethanol - 3:1 (v/v) were added and left to react for 2 hours at room temperature. The solution was decanted, evaporated and then lyophilized from water, affording partly or fully deprotected oligonucleotide triphosphates 3-1. Further deprotection of the 2' silyl groups was performed as follows: lyophilized 3-1 was placed in plastic vial and dissolved in 0.5 mL of 1 M TBAF (Aldrich, THF solution). The solution was left to react for 24 hours at room temperature. It was then diluted with 2 mL of water and applied on an equilibrated illustra NAP-25 des salting column (GE
  • oligonucleotides were analyzed by ion exchange HPLC using a gradient of 0 to 0.5 M NaCl (10 mM TRIZMA) in 40 min on a Dionex BioLC DNA Pac PA100 column, installed on a Waters apparatus. Samples were injected on a 25 OD per mL concentration, injecting 10 uL. Data were processed using the Empower 2 software. Molecular weight of the appropriate compounds was determined after an LC/MS analysis on a RP LC - Q-Tof mass spectrometer (Applied Biosystems). Ion deconvolution was applied for determination of the molecular weight.
  • Oligonucleotide Triphosphate and Diphosphate Synthesis Diphosphates. Annealing and production of dsRNAi TPs 1. Synthesis of PTEN 21 mer oligonucleotides RNAs (antisense strand) on solid
  • Oligonucleotides ld-h are synthesized on CPG solid support (Glen Research) using
  • a Trityl Off synthesis is performed as the last DMTr group is removed at the end of the synthesis.
  • the oligonucleotide is ished with anhydrous acetonitrile and reverse flushed with argon.
  • the solid supported oligonucleotide is then stocked at -20 °C.
  • Oligonucleotide synthesis of Id is in progress, pending delivery of custom synthesized 2'Fluoro 3' phosphoramidites of G and A, bearing the fast labile protecting groups (iPrPac and Pac, respectively). It is synthesized in a similar fashion as previously described for oligonucleotides la-c and le-f.
  • the oxidation solution is then prepared as follows: 150 mg (2 mmol) of imidazole (Aldrich) are coevaporated twice with anhydrous acetonitrile and then dried under vaccum over P 2 O 5 . The residue is then redissolved in anhydrous acetonitrile (0.8 mL), anhydrous CC (Aldrich, 0.8 mL), anhydrous triethylamine (Sigma, 0.1 mL) and ⁇ , ⁇ - bis-trimethylsilyl acetamide (Aldrich, 0.4 mL).
  • the resulting solution is dried over activated 4 A molecular sieves for 10 min, and then degassed with Argon for 30 seconds; it is then pushed gently through the column for 5 hours at room temperature.
  • the column is emptied and ished quickly twice with methanol, then reverse flushed with argon.
  • the dried solid support CPG carrying the oligonucleotide tri or di phosphates 2" is transferred from the Twist column to an empty screw cap plastic vial (10 mL). 2 mL of 30% NH 4 OH (JTBaker)/ethanol - 3:1 (v/v) are added and left to react for 2 hours at room temperature. The solution is decanted, evaporated and then lyophilized from water, affording partly or fully deprotected oligonucleotide tri or di phosphates 3-1. Further deprotection of the 2' silyl groups is performed as follows: lyophilized 3-1 is placed in plastic vial and dissolved in 0.5 mL of 1 M TBAF (Aldrich, THF solution).
  • the solution is left to react for 24 hours at room temperature. It is then diluted with 2 mL of water and applied on an equilibrated illustra NAP-25 desalting column (GE Healthcare). It is then eluted with 3.5 mL of water. The solution is collected and lyophilized, affording fully deprotected oligonucleotide tri or di phosphate 3-2.
  • RNA TP is washed with cold ethanol, then decanted, dried under vacuum and stored at -20 °C.
  • IE-HPLC semi prep column: Dionex DNA; gradient (buffer A: 25 mM TRIZMA HC1 - Aldrich; buffer B: 25 mM TRIZMA HC1 1 M NH 4 C1 - Aldrich; 0 to 0.7 M NH 4 C1 in 5 CV; flow 10 mL/min).
  • RP-HPLC semi prep column: CI 8; gradient (buffer A: 25 mM TEAB - Aldrich; buffer B: Acetonitrile - E. Merck; 0 to 50% acetonitrile in 5 CV; flow 10 mL/min). Collected desalted fraction was freezed and lyophilized, then stored at -20 °C.
  • the dried solid support CPG carrying the oligonucleotide tri or di phosphates 2" is transferred from the Twist column to an empty screw cap plastic vial (10 mL). 10 mL of 30% NH 4 OH (JTBaker)/ethanol - 3:1 (v/v) are added and left to react for 2 hours at room temperature. The solution is decanted, evaporated and then lyophilized from water, affording partly or fully deprotected oligonucleotide tri or di phosphates 3-1. Further deprotection of the 2' silyl groups is performed as follows: lyophilized 3-1 is placed in plastic vial and dissolved in 5 mL of 1 M TBAF (Aldrich, THF solution).
  • the solution is left to react for 24 hours at room temperature. It is then diluted with water and desalted by RP CI 8 semi preparative HPLC an AKTA purifying unit. The fractions are collected and lyophilized, affording fully deprotected oligonucleotide tri or di phosphate 3-2.
  • RNA TP is washed with cold ethanol, then decanted, dried under vacuum and stored at -20 °C. 3.
  • the complementary sequence strands of all target oligonucleotides 3 are synthesized on solid support CPG using standard oligoribonucleotide synthesis employing the commercially available 2'0-TBDMS 3' phosphor amidite building blocks. After deblocking with ammonia and removal of the 2'OTBDMS groups using the 3HF-NEt 3 complex, the oligonucleotides are purified by ion exchange HPLC then desalted on reverse phase HPLC and lyophilized from water.
  • dsRNAi are generated by annealing an equimolar amounts of complementary sense and antisense strands.
  • RNA is the ligand for retinoic acid-inducible protein I (RIG-I), a key sensor of viral infections.
  • RIG-I retinoic acid-inducible protein I
  • Purified monocytes are stimulated with single- stranded or double- stranded synthetic or in vitro-transcribed RNA oligonucleotide 5' triphosphates as described by Scheele et al. (Immunity, 2009, 31, 25).
  • Human PBMCs are isolated from whole human blood of healthy, voluntary donors by Ficoll-Hypaque density gradient centrifugation.
  • Plasmacytoid dendritic cells are positively depleted with magnetically labeled anti-CD304 antibody (Miltenyi Biotec). Untouched monocytes are obtained by negative depletion from PBMCs according to the manufacturer's instructions (Human Monocyte Isolation Kit II, Miltenyi Biotec). Cells are kept in RPMI 1640 containing 10% FCS, 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 ⁇ streptomycin. All compounds are tested for endotoxin contamination prior to use.
  • Mouse embryonic fibroblasts (MEFs) from MDA-5 "7” , RIG- ⁇ 7" , and IPS- 7" mice are prepared as described (Kato et al., 2006, Nature, AA ⁇ , 101).
  • 0.2 ug nucleic acid and 0.5 ul Lipofectamine (Invitrogen) are mixed in 50 ul Optimem
  • the amount of IFN-a production is determined with the IFN-a module set from Bender MedSystems.
  • the ELISA assay is performed according to the manufacturer's protocol.
  • the concentration of cytokines is determined by standard curve obtained using known amounts of recombinant cytokines.
  • Anti-FLAG beads are ished subsequently with lysis buffer and high-salt ish buffer (300 mM NaCl, 50 mM Tris/HCl [pH 7.4], 5 mM MgCl 2 , 1 mM DTT, and 0.1% CHAPS).
  • RIG-I-FLAG is eluted by an addtion of FLAG-peptide (300 ug/ml) solution to the beads. Purity of recombinant RIG-I is determined by SDS-PAGE separation and subsequent Coomassie blue staining.
  • RNA for (His 6 )FLAG-tagged RIG-I (HF-RIG-I) is determined as described ([Haas et al., 2008, Immunity, 28, 315] and [Latz et al., 2007, Nat. Immun., 8, 772]) by an amplified luminescent proximity homogenous assay (AlphaScreen;
  • biotinylated RNA for 1 hr at 37°C in buffer (50 mM Tris [pH 7.4], 100 mM NaCl, 0.01% Tween20, and 0.1% BSA) and subsequently incubated for 30 min at 25 °C with HF-RIG-Tbinding Nickel Chelate Acceptor Beads (PerkinElmer) and biotin- RNA-binding Streptavidine donor beads (PerkinElmer).
  • the donor bead contains the photosensitizer phtalocyanine, which converts ambient oxygen into a "singlet" oxygen after illumination with a 680 nm laser light.
  • the "singlet" oxygen can diffuse up to 200 nm and activate a thioxene derivative on the acceptor bead that is brought into proximity by interaction of the test molecules bound to the beads.
  • the resulting chemiluminescence with subsequent activation of a fluorochrome (contained within the same bead) emitting in the range of 520-620 nm correlates with the number and proximity of associated beads that is inversely correlated with the dissociation constant of donor (biotin-RNA) and acceptor (HF-RIG-I).
  • the assay is performed in wells of 384-well plates (Proxiplate; PerkinElmer). Plates are analyzed for emitted fluorescence with a multilabel reader (Envision; PerkinElmer).
  • oligonucleotide with 5' triphosphate ends can successfully synergize the RIG-I mediated imuune response triggering, with the oligonucleotide mediated silencing of targeted mRNAs.
  • the Bcl2 mRNA can be targeted by the appropriate oligonucleotide, inducing silencing of the Bcl2 protein which, along with RIG-I mediated immune response activation, provokes massive apoptosis of tumor cells in lung metastases in vivo (Nat. Med., 2007, 14, 1256).
  • RNAs 1 mg ml-1
  • Lipofectamine 2000 or Lipofectamine RNAiMAX both from Invitrogen
  • Dendritic cells are transfected as well as enriched lymphocyte subsets with 200 ng of nucleic acid with 0.5 ml of Lipofectamine in a volume of 200 ml.
  • mice Female C57BL/6 and BALB/c mice are used. Mice are 6-12 weeks of age at the beginning of the experiments. For tumor treatment, Tlr7- or Ifnarl -deficient mice are used that are crossed into the C57BL/6 genetic background for at least ten generations. RNAs are intravenously injected after complexation with in vivo-jetPEI (201-50,
  • CDl lc-DTR transgenic mice are injected intraperitoneally with 100 ng of diphteria toxin in PBS (Sigma D-0564).
  • Lung metastasis is experimentally induced by injection of 4 _ 105 B16 melanoma cells into the tail vein.
  • 50 mg of RNA complexed with jetPEI is administered in a volume of 200 ml on days 3, 6 and 9 after tumor challenge by retro- orbital or tail vein injection.
  • the number of macroscopically visible melanoma metastases is counted on the surface of the lungs.
  • western blot analyses are performed with lysed tumor cells and tumor tissue, flow cytometric analyses with single- cell suspensions, and quantitative RT-PCR and 5c-RACE analyses with extracted total RNA.
  • B16 melanoma cells are transfected stably with a mutated Bcl2 cDNA specifically designed to disrupt the target cleavage site of the Bcl2-specific oligonucleotide 2.2 without affecting the amino acid sequence of the Bcl-2 protein.
  • the production of cytokines in culture supernatants is measured by ELISA, and then the activation of dendritic cells and NK cells is assessed by flow cytometry and the stimulation of NK cell lytic activity against tumor cells is determined with a standard 51Cr release assay.
  • a luciferase- based IFN-b reporter gene assay is used.
  • the induction of apoptosis in cells cultured in vitro or freshly isolated ex vivo is measured by staining for annexin the cell surface and by using flow cytometry.
  • viable cells are quantified with a fluorimetric assay (CellTiter-Blue Cell Viability Assay, Promega) in vitro. Apoptosis is further verified in vivo by
  • GpppX-capped RNAs In order to get GpppX-capped RNAs, several approaches can be taken that differ widely in their efficiency. They can be synthesized chemically starting from mono- or diphosphate RNA. A 7Me GpppA cap can also be added to di- or triphosphate RNA using vaccinia virus capping enzyme that contains RNA triphosphatase, guanylyltransferase and N7MTase activities (Peyrane et al., Nucl. Acid Res., 2007, 35, e26; Brownlee et al., Nucl. Acid. Res., 1995, 23, 2641; Shuman, J. Biol. Chem., 264, 9690).
  • Guanylyltransferase Reaction mixtures (20 uL) containing 50 mM Tris-HCl, pH 7.5, 5 mM DTT, 1.25 mM MgCI 2 , 25 uM [cc- 32 P] GTP (9900 cpm/pmol), 39 pmol (of 5'ends) triphosphate-terminated RNA oligonucleotide, and 2 uL of enzyme are incubated for 30 min at 37 °C. Reactions are halted by the addition of 5% trichloroacetic acid, and acid- insoluble material is collected by filtration. The filters are counted in liquid scintillation fluid.
  • Capping and 32P-labeiling of phosphorylated oligonucleotides Capping of the RNA 5' triphosphates using 1 U guanylyl transferase (Gibco BRL) and 1 uM [a - 32 P]GTP (3000 Ci/mmol; Amersham) in 0.05 M Tris-HCl, pH 7.8, 1.25 mM MgC12, 6mM KCl, 2.5 mM dithiothreitol, 20 U human placental ribonuclease inhibitor (Promega), 0.1 mM S- adenosylmethionine in a 5 ,1 reaction volume for 1 h at 37 °C.
  • bovine serum albumin (0.4 ug) is added.
  • the reaction products are analysed, or in preparative experiments purified, by electrophoresis on 20% polyacrylamide-7 M urea gels.
  • the major radioactive band are detected by autoradiography and eluted in 0.25 M ammonium acetate, as above.
  • the eluate is centrifuged to remove gel pieces and the RNA precipitated from the supernatant with 3 vol ethanol in the presence of 2 M ammonium acetate and 20 ug yeast carrier RNA.
  • Modulation of the immune system can be measured for example by (i) measurement of either the mRNA or protein expression levels of a component ⁇ e.g., a growth factor, cytokine, or interleukin) of the immune system, e.g., in a cell or in an animal, (ii) measurement of the mRNA or protein levels of a protein factor activated by a component of the immune system (for example, NFKB), e.g., in a cell or in an animal, (iii) measurement of cell proliferation, e.g., in a tissue explant or a tissue of an animal.
  • a component e.g., a growth factor, cytokine, or interleukin
  • a component of the immune system for example, NFKB
  • cell proliferation e.g., in a tissue explant or a tissue of an animal.
  • Evaluation of the oligonucleotide can include incubating the modified strand (with or without its complement, but preferably annealed to its complement) with a biological system, e.g., a sample (e.g, a cell culture).
  • a biological system e.g., a sample (e.g, a cell culture).
  • the biological sample can be capable of expressing a component of the immune system. This allows identification of an oligonucleotide that has an effect on the component.
  • the step of evaluating whether the oligonucleotide modulates, e.g, stimulates or inhibits, an immune response includes evaluating expression of one or more growth factors, such as a cytokine or interleukin, or cell surface receptor protein, in a cell free, cell-based, or animal assay.
  • Protein levels can be assayed, for example, by Western blot techniques, flow cytometry, or reporter gene expression (e.g., expression of a fluorescent reporter protein, such as green fluorescent protein (GFP)).
  • reporter gene expression e.g., expression of a fluorescent reporter protein, such as green fluorescent protein (GFP)
  • the levels of mRNA of the protein of interest can be measured by Northern blot techniques, RNAse Protection Assays, or Quality Control-PCR (QC-PCR) (including quantitative reverse transcription coupled PCR (RT-PCR)) and analogous methods known in the art.
  • RNA and/or protein levels resulting from target gene expression can be measured at regular time intervals following introduction of the test oligonucleotide, and the levels are compared to those following introduction of a control oligonucleotide into cells.
  • the step of testing whether the modified oligonucleotide modulates, e.g., stimulates, an immune response includes assaying for an interaction between the oligonucleotide and a protein component of the immune system, e.g., a growth factor, such as a cytokine or interleukin, or a cell surface receptor protein.
  • a protein component of the immune system e.g., a growth factor, such as a cytokine or interleukin, or a cell surface receptor protein.
  • Exemplary assay methods include coimmunoprecipitation assays, bead-based co-isolation methods, nucleic acid footprint assays and colocalization experiments such as those facilitated by immunocytochemistry techniques.
  • Cell proliferaton can be monitored by following the uptake of [ H] thymidine or of a fluorescent dye.
  • Cells are plated in a 96-well tissue culture plate and then incubated with the oligonucleotide.
  • [ H]thymidine is added and incubation is continued.
  • the cells are subsequently processed on a multichannel automated cell harvester (Cambridge Technology, Cambridge, MA) and counted in a liquid scintillation beta counter (Beckman Coulter).
  • a commercially available assay like the LIVE/DEAD Viability/Cytotoxicity assay from Molecular Probes can be used.
  • the kit identifies live versus dead cells on the basis of membrane integrity and esterase activity. This kit can be used in microscopy, flow cytometry or microplate assays.

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Abstract

La présente invention décrit un procédé simple, efficace et sans enzyme pour la fabrication d'oligonucléotides avec le 5'-triphosphate. Cette invention présente un nouveau procédé pour la synthèse des oligonucléotides triphosphates à l'aide d'un phosphonate de diaryle comme réactif. Le procédé de la présente invention se prête à la synthèse d'oligonucléotides 5'-triphosphates, de façon économique et à une grande échelle.
PCT/US2009/069201 2008-09-02 2009-12-22 Procédé pour la synthèse d'oligonucléotides triphosphates WO2011028218A1 (fr)

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WO2012130886A1 (fr) * 2011-03-28 2012-10-04 Rheinische Friedrich-Wilhelms-Universität Bonn Purification d'oligonucléotides triphosphorylés en utilisant des marquages de capture
CN103193843A (zh) * 2013-04-15 2013-07-10 江西科技师范大学 由全保护核苷磷酰胺中间体通过酸催化合成核苷三磷酸和核苷二磷酸的方法
EP2712870A1 (fr) * 2012-09-27 2014-04-02 Rheinische Friedrich-Wilhelms-Universität Bonn Nouveaux ligands de RIG-I et procédés pour les produire
US9035041B2 (en) 2008-09-02 2015-05-19 Alnylam Pharmaceuticals, Inc. Process for triphosphate oligonucleotide synthesis
US9381208B2 (en) 2006-08-08 2016-07-05 Rheinische Friedrich-Wilhelms-Universität Structure and use of 5′ phosphate oligonucleotides
US9738680B2 (en) 2008-05-21 2017-08-22 Rheinische Friedrich-Wilhelms-Universität Bonn 5′ triphosphate oligonucleotide with blunt end and uses thereof
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WO2017221929A1 (fr) * 2016-06-21 2017-12-28 株式会社ジーンデザイン Procédé de synthèse de monomère de h-phosphonate d'acide ribonucléique, et procédé de synthèse d'oligonucléotide mettant en œuvre ce monomère
US11174279B2 (en) 2016-06-21 2021-11-16 National Institute Of Advanced Industrial Science And Technology Method for synthesizing ribonucleic acid H-phosphonate monomer, and oligonucleotide synthesis in which said monomer is used
JP7045669B2 (ja) 2016-06-21 2022-04-01 株式会社ジーンデザイン リボ核酸ｈ－ホスホネートモノマーの合成方法および本モノマーを用いたオリゴヌクレオチド合成
CN109641931B (zh) * 2016-06-21 2022-10-18 基因设计有限公司 核糖核酸h-磷酸酯单体的合成方法和使用了该单体的寡核苷酸合成
WO2019053609A1 (fr) * 2017-09-12 2019-03-21 Vilnius University Nucléotides de cytidine modifiés en position n 4 et leur utilisation
LT6615B (lt) * 2017-09-12 2019-04-25 Vilniaus Universitetas N4-modifikuoti citidino nukleotidai ir jų panaudojimas
US11584772B2 (en) 2017-09-12 2023-02-21 Vilnius University N4-modified cytidine nucleotides and their use
WO2022212442A1 (fr) * 2021-03-31 2022-10-06 Modernatx, Inc. Synthèse de coiffes trinucléotidiques et tétranucléotidiques pour la production d'arnm

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