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US 6,649,751 B2Granted
Synthesis, deprotection, analysis and purification of RNA and ribozymes
AssigneeSirna Therapeutics, Inc.
InventorsNassim Usman, Francine Wincott, David Sweedler, Leonid Beigelman, Lech W. Dudycz, Susan Grimm, Anthony DiRenzo, Danuta Tracz
Life of this patent
Estimating the term…
Abstract
Provided is a method for purification and synthesis of RNA molecules that have at least one chemical modification.
Claims184
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
- (a) deprotection, wherein said deprotection comprises removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with an alkylamine or a mixture of ammonium hydroxide and alkylamine at a temperature of about 60-70 degrees centigrade for about 5-15 minutes, under conditions suitable for the removal of said exocyclic protecting groups, wherein said alkylamine is selected from the group consisting of ethylamine, propylamine and butylamine;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to reverse phase high-performance liquid chromatography (HPLC) column, wherein said RNA comprises a 5′-protecting group;
- (ii) eluting said RNA by passing a suitable buffer through said reverse phase column;
- (iii) removing the 5′-protecting group from said RNA;
- (iv) loading said RNA lacking said 5′-protecting group on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (v) eluting said RNA by passing a suitable buffer through said anion exchange column;
- and (vi) collecting the eluate from said anion exchange column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
6 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
- (a) deprotection, wherein said deprotection comprises removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to reverse phase high-performance liquid chromatography (HPLC) column, wherein said RNA comprises a 5′-protecting group;
- (ii) eluting said RNA by passing a suitable buffer through said reverse phase column;
- (iii) removing the 5′-protecting group from said RNA;
- (iv) loading said RNA lacking said 5′-protecting group on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (v) eluting said RNA by passing a suitable buffer through said anion exchange column;
- and (vi) collecting the eluate from said anion exchange column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
6 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
- (a) deprotection, wherein said deprotection comprises:
- (i) removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with an alkylamine or a mixture of ammonium hydroxide and alkylamine at a temperature of about 60-70 degrees centigrade for about 5-15 minutes, under conditions suitable for the removal of said exocyclic protecting groups, wherein said alkylamine is selected from the group consisting of ethylamine, propylamine and butylamine;
- and (ii) removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to reverse phase high-performance liquid chromatography (HPLC) column, wherein said RNA comprises a 5′-protecting group;
- (ii) eluting said RNA by passing a suitable buffer through said reverse phase column;
- (iii) removing the 5′-protecting group from said RNA;
- (iv) loading said RNA lacking said 5′-protecting group on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (v) eluting said RNA by passing a suitable buffer through said anion exchange column;
- and (vi) collecting the eluate from said anion exchange column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
8 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising the steps of:
- a) deprotection, wherein said deprotection is a one pot deprotection process comprising:
- (i) removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with anydrous alkylamine at a temperature of about 60-70 degrees centigrade for at least 5 minutes under conditions suitable for the removal of said exocyclic protecting groups;
- and (ii) removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to reverse phase high-performance liquid chromatography (HPLC) column, wherein said RNA comprises a 5′-protecting group;
- (ii) eluting said RNA by passing a suitable buffer through said reverse phase column;
- (iii) removing the 5′-protecting group from said RNA;
- (iv) loading said RNA lacking said 5′-protecting group on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (v) eluting said RNA by passing a suitable buffer through said anion exchange column;
- and (vi) collecting the eluate from said anion exchange column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
8 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
- (a) deprotection, wherein said deprotection comprises removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with an alkylamine or a mixture of ammonium hydroxide and alkylamine at a temperature of about 60-70 degrees centigrade for about 5-15 minutes, under conditions suitable for the removal of said exocyclic protecting groups, wherein said alkylamine is selected from the group consisting of ethylamine, propylamine and butylamine;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (ii) eluting said RNA by passing a suitable buffer through said column;
- and (iii) collecting the eluate from said column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
3 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising the steps of:
- (a) deprotection, wherein said deprotection comprises removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (ii) eluting said RNA by passing a suitable buffer through said column;
- and (iii) collecting the eluate from said column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
3 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising:
- (a) deprotection, wherein said deprotection comprises:
- (i) removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with an alkylamine or a mixture of ammonium hydroxide and alkylamine at a temperature of about 60-70 degrees centigrade for about 5-15 minutes, under conditions suitable for the removal of said exocyclic protecting groups, wherein said alkylamine is selected from the group consisting of ethylamine, propylamine and butylamine;
- and (ii) removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (ii) eluting said RNA by passing a suitable buffer through said column;
- and (iii) collecting the eluate from said column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
5 more elements
A process for deprotecting and purifying chemically synthesized RNA having one or more chemical modifications, comprising the steps of:
- (a) deprotection, wherein said deprotection is a one pot deprotection process comprising:
- (i) removing any exocyclic protecting groups from said RNA molecule by contacting said RNA with anydrous alkylamine at a temperature of about 60-70 degrees centigrade for at least 5 minutes under conditions suitable for the removal of said exocyclic protecting groups;
- and (ii) removing 2′-O-alkylsylyl protecting group from said RNA by contacting said RNA with a solution of anhydrous triethylamine-hydrogen fluoride (aHF-TEA) in triethylamine and N-methylpyrrolidine at a temperature of about 60-70 degrees centigrade for about 0.25-24 hour under conditions suitable for said removal of the 2′-O-alkylsylyl protecting groups;
- and
- (b) purification, wherein said purification comprises:
- (i) loading said RNA on to an anion exchange high-performance liquid chromatography (HPLC) column;
- (ii) eluting said RNA by passing a suitable buffer through said column;
- and (iii) collecting the eluate from said column and recovering said RNA from said eluate, under conditions which allow for the purification of the RNA.
5 more elements
Dependencies are inferred from the claim text.