Methods of polynucleotide preparation using multivalent cation salt compositions
Aspects of the disclosure include methods for the preparation of a polynucleotide. In some embodiments, the method includes contacting a first polynucleotide composition including: a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and non-target synthetic products and reagents; with a multivalent cation salt to precipitate a polynucleotide salt including at least one multivalent cation counterion; and separating the polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition including the polynucleotide salt. In certain embodiments, the method further includes contacting the polynucleotide salt with a reverse phase chromatography support; and eluting from the chromatography support a third polynucleotide composition including the polynucleotide. Also provided are compositions including a salt of the polynucleotide including at least one multivalent cation counterion.
1. A method of preparing a polynucleotide, the method comprising:
a) contacting a first polynucleotide composition with a multivalent cation salt to precipitate a first polynucleotide salt comprising at least one multivalent cation counterion;
b) separating the first polynucleotide salt from the contacted first polynucleotide composition to produce a second polynucleotide composition comprising the first polynucleotide salt;
wherein the first polynucleotide composition comprises:
(i) a polynucleotide having a sequence of 7 or more nucleoside subunits and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage; and
(ii) soluble non-target synthetic products and reagents;
c) contacting the second polynucleotide composition comprising the first polynucleotide salt from step (b) with a reverse phase chromatography support; and
d) eluting from the reverse phase chromatography support a third polynucleotide composition comprising a second soluble polynucleotide salt.
2. The method of claim 1 , wherein the polynucleotide comprises a sequence comprising 13or more nucleoside subunits complementary to the RNA component of human telomerase.
3. The method of claim 1 , wherein the polynucleotide comprises between 10and 50 contiguous nucleoside subunits complementary to the RNA component of human telomerase.
4. The method of claim 2 , wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages.
5. The method of claim 1 , wherein the polynucleotide comprises a sequence selected from the group consisting of: GTTAGGGTTAG (SEQ ID NO:4), TAGGGTTAGACAA (SEQ ID NO:3) and CAGTTAGGGTTAG (SEQ ID NO: 5 ).
6. The method of claim 1 , wherein the polynucleotide is conjugated to a lipid moiety via an optional linker.
7. The method of claim 1 , wherein the second polynucleotide salt has the structure:
wherein each M x + is independently hydrogen or a cationic counterion, each x is independently 1, 2 or3 and n is an integer from 5 to 13.
8. The method of claim 1 , wherein after the eluting step d) the second polynucleotide salt is a pharmaceutically acceptable salt of the polynucleotide.
9. The method of claim 1 , wherein after the eluting step d) the second polynucleotide salt is a monovalent cation salt of the polynucleotide.
10. The method of claim 9 , wherein the second polynucleotide salt is a sodium salt of the polynucleotide.
11. The method of claim 1 , further comprising, prior to the contacting step a), cleaving the polynucleotide from a solid phase synthesis support to produce the first polynucleotide composition as a crude synthetic preparation of the polynucleotide.
12. The method of claim 1 , wherein prior to the contacting step a) the first polynucleotide composition comprises a monovalent cation salt of the polynucleotide.
13. The method of claim 1 , wherein the contacting step a) comprises loading and eluting the first polynucleotide composition from a cation exchange support.
14. The method of claim 1 , wherein the separating step b) comprises centrifuging the contacted first polynucleotide composition to spin down the first polynucleotide salt precipitate.
15. The method of claim 1 , wherein the separating step b) comprises filtering the first polynucleotide salt from the contacted first polynucleotide composition.
16. The method of claim 1 , wherein the second polynucleotide composition of step b) is loaded directly onto the reverse phase chromatography support.
17. The method of claim 1 , further comprising, prior to the contacting step c), dissolving the second polynucleotide composition in a solvent.
18. The method of claim 1 , wherein the at least one multivalent cation counterion is divalent.
19. The method of claim 18 , wherein the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc and calcium.
20. The method of claim 1 , wherein the at least one multivalent cation counterion is trivalent.
21. The method of claim 20 , wherein the at least one multivalent cation counterion is aluminium.
22. The method of claim 1 , wherein the first polynucleotide salt further comprises a monovalent cation counterion.
23. The method of claim 1 , wherein the polynucleotide is described by Formula (I):
wherein:
each B is independently a purine, a protected purine, a pyrimidine or a protected pyrimidine, or an analog thereof;
each X is independently oxygen or sulfur;
each R 3 is independently hydrogen, fluoro, hydroxyl, an alkoxy, a substituted alkoxy or a protected hydroxyl;
R 6 is amino, hydroxyl, a protected amino, a protected hydroxy, —O-T-Z or —NH-T-Z;
each T is independently an optional linker;
each Z is independently H, a lipid, a carrier, an oligonucleotide, a polymer, a polypeptide, a detectable label, or a tag; and
n is an integer of 7 to 100.
24. The method of claim 1 , wherein the nucleoside subunits are all joined by inter-subunit linkages each independently selected from N3′→P5′ thiophosphoramidate inter-subunit linkage and N3′→P5′ phosphoramidate inter-subunit linkage.