Compositions and methods for the synthesis and identification of covalent aptamers
Compositions and methods for preparing and identifying aptamers and aptamer-protein conjugates are provided. The use of a nucleotide analog functionalized with an amine-reactive cross-linker facilitates covalent cross-linking of the aptamer to a protein, in particular site-specific cross-linking to the Fc domain of an antibody.
1. A method for producing one or more aptamers that bind to a target protein comprising
(a) contacting a target protein with an aptamer library comprising a plurality of aptamers each having at least one nucleotide analog functionalized with an amine-reactive cross-linker;
(b) selecting from the aptamer library one or more aptamers that covalently bind, and exhibit specific binding affinity for, the target protein; and
(c) introducing a triazole-modified nucleotide into the sequence of the one or more aptamers that covalently bind, and exhibit specific binding affinity for, the target protein, wherein the triazole-modified nucleotide is introduced into the one or more aptamers by:
(i) incorporating a phosphoramidite comprising the structure of Formula I:
wherein R 1 is H, O-methyl, dimethyl, fluoro, amino, C-allyl, arabinofluoro, methylene, difluoromethylene or a protecting group; and R 2 is a protecting group,
(ii) deprotecting the aptamers, and
(iii) activating the deprotected aptamers to form an amine-reactive cross-linker.
2. A phosphoramidite comprising the structure of Formula I:
wherein R 1 is H, O-methyl, dimethyl, fluoro, amino, C-allyl, arabinofluoro, methylene, difluoromethylene or a protecting group; and R 2 is a protecting group.
3. A kit comprising the phosphoramidite of claim 2 .
4. The method of claim 1 , wherein each aptamer of the plurality of aptamers is bound to an unmodified double-stranded DNA copy of the aptamer.
5. The method of claim 1 , wherein the at least one nucleotide analog is 5-ethynyldeoxyuridine, 5-ethynyluridine, or 2′-modified 5-ethynyluridine.
6. The method of claim 1 , wherein the amine-reactive cross-linker is an N-hydroxysuccinimide ester or imidoester.