Post-selec modification methods
Aptamers that bind PDGF and aptamers that bind VEGF are provided. In addition, aptamer constructs comprising a PDGF aptamer and a VEGF aptamer are provided. Pharmaceutical compositions comprising the aptamers and aptamer constructs are provided, as well as methods of treating conditions using the aptamers and aptamer constructs.
1. A method comprising modifying a first aptamer that is capable of binding a target protein whereby the first aptamer comprises a first modified C-5 position uracil, and wherein the modification to the first aptamer results in a second aptamer having improved binding affinity for the target protein compared to the binding affinity of the first aptamer for the target protein; wherein the modification to the first aptamer is a substitution of the first modified C-5 position uracil with a second modified C-5 position uracil, wherein the first modified C-5 position uracil and the second modified C-5 position uracil are different C-5 position modified uracils, and wherein the first modified C-5 position uracil has a hydrophobic moiety selected from the group consisting of:
wherein, the * denotes the point of attachment of the hydrophobic moiety to the C-5 position of the uracil; and
wherein, R″″ is selected from the group consisting of a branched or linear lower alkyl (C1-C20); a hydroxyl (OH); a halogen (F, Cl, Br, I) and a nitrile (CN).
2. The method of claim 1 , wherein the first aptamer and the second aptamer are DNA aptamers, RNA aptamers or a combination thereof.
3. The method of claim 1 , wherein the binding affinity of the second aptamer for the target protein is about 3-fold, 5-fold, 15-fold, 20-fold, 30-fold better than that binding affinity of the first aptamer for the target protein.
4. The method of claim 1 , wherein the binding affinity of the second aptamer for the target protein is up to 100-fold better than that binding affinity of the first aptamer for the target protein.
5. The method of claim 1 , wherein the modification to the first aptamer that results in the second aptamer is selected from the group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), 5-(N-benzylcarboxyamide)-2′-O-methyluridine, 5-(N-benzylcarboxyamide)-2′-fluorouridine, 5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU), 5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU), 5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU), 5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU), 5-(N-imidizolylethylcarboxyamide)-2′-deoxyuridine (ImdU), 5-(N-isobutylcarboxyamide)-2′-O-methyluridine, 5-(N-isobutylcarboxyamide)-2′-fluorouridine, 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), 5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU), 5-(N-tryptaminocarboxyamide)-2′-O-methyluridine, 5-(N-tryptaminocarboxyamide)-2′-fluorouridine, 5-(N-[1-(3-trimethylamonium) propyl]carboxyamide)-2′-deoxyuridine chloride, 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU), 5-(N-naphthylmethylcarboxyamide)-2′-O-methyluridine, 5-(N-naphthylmethylcarboxyamide)-2′-fluorouridine, 5-(N-[1-(2,3-dihydroxypropyl)]carboxyamide)-2′-deoxyuridine), 5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU), 5-(N-2-naphthylmethylcarboxyamide)-2′-O-methyluridine, 5-(N-2-naphthylmethylcarboxyamide)-2′-fluorouridine, 5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU), 5-(N-1-naphthylethylcarboxyamide)-2′-O-methyluridine, 5-(N-1-naphthylethylcarboxyamide)-2′-fluorouridine, 5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU), 5-(N-2-naphthylethylcarboxyamide-2′-O-methyluridine, 5-(N-2-naphthylethylcarboxyamide)-2′-fluorouridine, 5-(N-3-benzofuranylethylcarboxyamide)-2′-deoxyuridine (BFdU), 5-(N-3-benzofuranylethylcarboxyamide)-2′-O-methyluridine, 5-(N-3-benzofuranylethylcarboxyamide)-2′-fluorouridine, 5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU), 5-(N-3-benzothiophenylethylcarboxyamide)-2′-O-methyluridine, and 5-(N-3-benzothiophenylethylcarboxyamide)-2′-fluorouridine.
6. The method of claim 1 , wherein the modification to the first aptamer that results in the second aptamer is selected from the group consisting of 5-(N-benzylcarboxyamide)-2′-deoxyuridine (BndU), 5-(N-phenethylcarboxyamide)-2′-deoxyuridine (PEdU), 5-(N-thiophenylmethylcarboxyamide)-2′-deoxyuridine (ThdU), 5-(N-isobutylcarboxyamide)-2′-deoxyuridine (iBudU), 5-(N-tyrosylcarboxyamide)-2′-deoxyuridine (TyrdU), 5-(N-3,4-methylenedioxybenzylcarboxyamide)-2′-deoxyuridine (MBndU), 5-(N-4-fluorobenzylcarboxyamide)-2′-deoxyuridine (FBndU), 5-(N-3-phenylpropylcarboxyamide)-2′-deoxyuridine (PPdU), 5-(N-tryptaminocarboxyamide)-2′-deoxyuridine (TrpdU), 5-(N—R-threoninylcarboxyamide)-2′-deoxyuridine (ThrdU), 5-(N-naphthylmethylcarboxyamide)-2′-deoxyuridine (NapdU), 5-(N-2-naphthylmethylcarboxyamide)-2′-deoxyuridine (2NapdU), 5-(N-1-naphthylethylcarboxyamide)-2′-deoxyuridine (NEdU), 5-(N-2-naphthylethylcarboxyamide)-2′-deoxyuridine (2NEdU) and 5-(N-3-benzothiophenylethylcarboxyamide)-2′-deoxyuridine (BTdU).