Switchable aptamers
The disclosure relates to a switchable aptamer having a high affinity for a selected target such as a virus, cell or antibody when in the presence of a binding ion and a low affinity for said target in the absence of said binding ion. The switchable aptamer may be isolated from a pool comprising a mixture of aptamers by incubating the pool with the target ligand and a binding ion to form target-aptamer complexes; separating unbound aptamer molecules from the target-aptamer complexes; contacting the target-aptamer complexes with a chelating agent having affinity for the binding ion wherein a switchable aptamer specific to said target is released from the target-aptamer complexes; and isolating the switchable aptamer released in the preceding step.
1. A switchable aptamer which is obtained through a method comprising:
a) incubating a pool comprising a mixture of aptamers with a target ligand and a binding ion to form target-aptamer complexes comprising said target ligand and aptamers specific to said target;
b) separating unbound aptamer molecules from the target-aptamer complexes;
c) contacting the target-aptamer complexes with a chelating agent having affinity for said binding ion wherein a switchable aptamer specific to said target is released from the target-aptamer complexes; and
d) isolating the switchable aptamer released in step c;
wherein the switchable aptamer comprises a nucleotide sequence represented in any one of SEQ ID NOS: 31 through 47 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NOS: 31 through 47.
2. The switchable aptamer of claim 1 wherein said binding ion is calcium or magnesium or a combination thereof.
3. A method for purifying a selected target from a complex mixture, the method comprising the steps of:
a) incubating switchable aptamer with the complex mixture in the presence of a binding ion to produce target-aptamer complexes wherein the switchable aptamer comprises a nucleotide sequence represented in any one of SEQ ID NOS: 31 through 47 or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NOS: 31 through 47;
b) separating the target-aptamer complexes from unbound components of the complex mixture;
c) adding a chelating agent having affinity for the binding ion to the target-aptamer complexes to release the target from the target-aptamer complexes; and
d) separating the released target from said switchable aptamer.
4. The method of claim 3 performed at a maximum temperature of 25° C.
5. The method of claim 3 wherein the binding ion is calcium or magnesium or a combination thereof.
6. The method of claim 3 wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA) or a combination thereof.
7. The method of claim 3 wherein the target is a virus, a cell or an antibody.
8. The method of claim 7 wherein the virus is Vesicular Stomatis Virus (VSV).
9. The method of claim 7 wherein said cell is a eukaryote.
10. The method of claim 9 wherein said cell is receptor positive for one of: a Neuropilin 1 (NRP) receptor, a Leukemia inhibitory factor (LIF) receptor, a Patched 1 (PTCH1) receptor, a Delta-Like Ligand 4 (0114) receptor or a plasminogen activator/urokinase receptor (PLAUR).
11. The method of claim 3 wherein target-aptamer complexes are separated from the unbound components of the complex mixture using a magnetic field or by elution.
12. The method of claim 3 comprising the further step of re-using the separated switchable aptamer to repeat said steps a) through d) on the same or a different complex mixture.
13. The method of claim 3 wherein said switchable aptamer is obtained through a method comprising:
a) incubating said pool with said target ligand and a binding ion to form target-aptamer complexes comprising said target ligand and aptamers specific to said target;
b) separating unbound aptamer molecules from the target-aptamer complexes;
c) contacting the target-aptamer complexes with a chelating agent having affinity for said binding ion wherein a switchable aptamer specific to said target is released from the target-aptamer complexes; and
d) isolating the switchable aptamer released in step c.
14. A switchable aptamer comprising the nucleotide sequence of SEQ ID NO: 43, or a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with SEQ ID NO: 43.