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 composition comprising:
a salt precipitate of a polynucleotide comprising at least one multivalent cation counterion;
wherein the polynucleotide has a sequence of 7 or more nucleoside subunits complementary to the RNA component of human telomerase and at least two of the nucleoside subunits are joined by a N3′→P5′ thiophosphoramidate inter-subunit linkage.
2. The composition of claim 1 , wherein the at least one multivalent cation counterion is divalent.
3. The composition of claim 2 , wherein the at least one multivalent cation counterion is selected from the group consisting of magnesium, zinc and calcium.
4. The composition of claim 1 , wherein the at least one multivalent cation counterion is magnesium.
5. The composition of claim 1 , wherein the at least one multivalent cation counterion is trivalent.
6. The composition of claim 5 , wherein the at least one multivalent cation counterion is aluminium.
7. The composition of claim 1 , wherein the polynucleotide comprises 3 mol % or more of the multivalent cation counterion relative to a polyanionic backbone of the polynucleotide.
8. The composition of claim 1 , wherein the polynucleotide comprises 1.0% by weight or more of the multivalent cation counterion relative to the polynucleotide.
9. The composition of claim 1 , wherein the composition is a precipitate.
10. The composition of claim 1 , wherein the polynucleotide comprises a sequence comprising 13 or more nucleoside subunits complementary to the RNA component of human telomerase.
11. The composition of claim 1 , wherein the polynucleotide comprises between 10 and 50 contiguous nucleoside subunits complementary to the RNA component of human telomerase.
12. The composition of claim 1 , wherein the nucleoside subunits complementary to the RNA component of human telomerase are all joined by N3′→P5′ thiophosphoramidate inter-subunit linkages.
13. The composition of claim 1 , wherein the polynucleotide comprises a sequence selected from the group consisting of:
(SEQ ID NO: 4)
GTTAGGGTTAG,
(SEQ ID NO: 3)
TAGGGTTAGACAA
and
(SEQ ID NO: 5)
CAGTTAGGGTTAG.
14. The composition of claim 1 , wherein the polynucleotide is conjugated to a lipid moiety via an optional linker.
15. The composition of claim 1 , wherein the polynucleotide has the structure:
wherein each M x+ is independently hydrogen or a cationic counterion, each x is independently 1, 2 or 3 and n is an integer from 5 to 13.
16. The composition 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 1 to 1000.