Deprotection and purification of oligonucleotides and their derivatives
Method for synthesis, deprotection, and/or purification of nucleic acid molecules, such as oligonucleotides comprising one or more ribonucleotides. Such nucleic acid molecules include siRNA, dsRNA, ribozymes, antisense, and aptamers.
1. A process comprising the steps of:
a) synthesizing a nucleic acid molecule comprising one or more ribonucleotides, 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;
b) contacting said nucleic acid molecule from step (a) with aqueous alkylamine, trialkylamine, or alkylamine and trialkylamine, under conditions suitable for the removal of any 2′-amino protecting groups, exocyclic amino (base) protecting groups and/or phosphate protecting groups, which may be individually present or absent, from said molecule;
c) contacting reaction mixture having said nucleic acid molecule from step (b) with a polar solvent and trialkylamine.hydrogen fluoride under conditions suitable for the removal of a 2′-OH protecting group;
d) loading reaction mixture having said nucleic acid molecule from step (c) onto a media selected from the group consisting of Pharmacia Source Q15, Biorad Macroprep 25Q, Pharmacia Q-sepharose, Perceptive POROS HQ, TOSOHAAS Q-5PW-HR, Q-5PW, and super Q-5PW media in a suitable buffer comprising buffers selected from the group consisting of water, 20% ethanol in about 20 mM sodium phosphate and about 0.1 M NaCl and acetonitrile in about 20 mM sodium phosphate and about 0.1 M NaCl;
e) applying a purification gradient using a suitable elution buffer, analyzing the fractions and allowing for the pure fractions to be pooled and desalted.
2. The process of claim 1 , wherein said nucleic acid molecule comprising one or more ribonucleotides is a siRNA molecule.
3. The process of claim 2 , wherein said siRNA molecule further comprises one or more 2′-deoxy-2′-fluoro nucleotides.
4. The process of claim 1 , wherein said aqueous alkylamine is aqueous methylamine.
5. The process of claim 1 , wherein said aqueous alkylamine is 40% aqueous methylamine.
6. The process of claim 1 , wherein said trialkylamine.trihydrofluoride is triethylamine.trihydrofluoride (TEA.3HF).
7. The process of claim 1 , wherein said 2′-OH protecting group comprises the t-butyldimethylsilyl (TBDMSi) protecting group.
8. The process of claim 1 , wherein said nucleic acid molecule comprises one or more chemical modifications.
9. A process of purifying a nucleic acid molecule, comprising the steps of:
a) loading said nucleic acid molecule onto a media selected from the group consisting of Pharmacia Source Q15, Biorad Macroprep 25Q, Pharmacia Q-sepharose, Perceptive POROS HQ, TOSOHAAS Q-5PW-HR, Q-5PW, and super Q-5PW media in a loading buffer comprising buffers selected from the group consisting of water, 20% ethanol in about 20 mM sodium phosphate and about 0.1 M NaCl and acetonitrile in about 20 mM sodium phosphate and about 0.1 M NaCl;
b) applying a purification gradient using a suitable elution buffer, analyzing the fractions and allowing for the pure fractions to be pooled and desalted.
10. The process of claim 9 , wherein said nucleic acid molecule comprises one or more ribonucleotides.
11. The process of claim 9 , wherein said nucleic acid molecule comprises one or more chemical modifications.
12. The process of claims 1 or 9 , wherein said nucleic acid molecule is a single stranded nucleic acid molecule.
13. The process of claims 1 or 9 , wherein said nucleic acid molecule is a double stranded nucleic acid molecule.
14. The process of claim 13 , wherein said double stranded nucleic acid molecule comprises one or more chemical modifications.
15. The process of claim 13 , wherein said each strand of the double stranded nucleic acid molecule is of length between 19 and 23 nucleotides.
16. The process of claims 8 or 11 , wherein said chemical modification is a sugar modification.
17. The process of claim 16 , wherein said sugar modification is a 2′-sugar modification.
18. The process of claims 8 or 11 , wherein said chemical modification is a base modification.
19. The process of claims 8 or 11 , wherein said chemical modification is a phosphate backbone modification.
20. The process of claim 19 , wherein said phosphate backbone modification is phosphorothioate.
21. The process of claims 8 or 11 , wherein said chemical modification is a terminal end modification.
22. The process of claim 21 , wherein said end modification is at the 5′-end of said nucleic acid molecule.
23. The process of claim 21 , wherein said end modification is at the 3′-end of said nucleic acid molecule.
24. The process of claim 21 , wherein said end modification is at both the 5′- and 3′-end of said nucleic acid molecule.