Compositions and methods for screening aptamers
The disclosure is directed to methods and compositions for screening a library of aptamers for aptamers having a binding affinity to a target molecule. The methods and compositions described herein utilize a throughput approach that is able to simultaneously measure binding affinity and link the binding affinity to the identity (e.g., sequence) of the aptamer.
1. A method for screening a library of aptamers for aptamers having binding affinity to a target molecule, comprising:
(a) providing a solid support comprising clusters of sequence-identified adaptor end- linked aptamers,
wherein each cluster comprises a plurality of identical sequence-identified adaptor end-linked aptamers comprising one or more non-natural nucleotide covalently bonded to a binding agent, and optionally extended solid support-linked oligonucleotides comprising complements of the adaptor end-linked aptamers, and wherein the sequence of the adaptor end-linked aptamers in each cluster is unique compared to that of the other clusters;
(d) probing the clusters of adaptor end-linked aptamers linked to the solid support with a target molecule;
(e) washing unbound target molecule from the solid support;
(f) screening the solid support for binding and location of the target molecule; and
(g) correlating the location of binding of the target molecule to the nucleotide sequence of an solid support-linked aptamer at the location of binding of the target molecule, thereby identifying the nucleotide sequence of an aptamer that binds the target molecule.
2. The method of claim 1 , wherein the providing comprises:
(a) hybridizing a plurality of adaptor end-linked aptamers to adaptor-complementary oligonucleotides linked to a solid support;
(b) subjecting the hybridized adaptor end-linked aptamers to nucleotide sequencing by:
(i) extending with a polymerase the hybridized adaptor-complementary oligonucleotide in a template-dependent manner to generate an extended solid support- linked antisense oligonucleotide whose sequence is complementary to the adaptor end- linked aptamer; and
(ii) generating clusters of identical solid support-linked oligonucleotides having sense and antisense strands through bridge amplification;
(iii) selectively cleaving sense oligonucleotides, leaving only antisense oligonucleotides linked to the solid support;
(iv) sequencing by synthesis the antisense oligonucleotides; and
(v) recording the location and nucleotide sequence of the antisense oligonucleotides linked to the solid support;
(c) performing bridge amplification of the antisense oligonucleotides linked to the solid support in the presence of one or more non-natural nucleotide to form clusters of extended solid support-linked sense oligonucleotides and antisense oligonucleotides, thereby introducing a non-natural nucleotide into the sense oligonucleotides wherein the non-natural nucleotide comprises a functional group to form the sequence-identified adaptor end-linked aptamers;
(d) selectively cleaving the antisense oligonucleotides from the solid support, thereby providing a solid support comprising clusters of sequence-identified adaptor end-linked aptamers.
3. The method of claim 2 , wherein the non-natural nucleotide comprises an alkyne or azide functional group.
4. The method of claim 3 , wherein the non-natural nucleotide comprises an alkyne functional group.
5. The method of claim 4 , wherein the non-natural nucleotide is C8-alkyne-dUTP.
6. The method of claim 2 , further comprising following the performing bridge amplification, modifying the non-natural nucleotide by covalently conjugating a binding agent comprising a compatible functional group to the non-natural nucleotide, wherein the compatible functional group in the binding agent and the functional group in the non-natural nucleotide react to form a modified non-natural nucleotide comprising the binding agent.
7. The method of claim 6 , wherein the compatible functional group in the binding agent is an azide.
8. The method of claim 7 , wherein during the modifying the azide undergoes Cu-catalyzed azide-alkyne cycloaddition (CuAAC) with the alkyne in the non-natural nucleotide to form a covalent bond.
9. The method of claim 1 , wherein the binding agent is an amino acid, a sugar, a peptide, or a protein.
10. The method of claim 9 , wherein the sugar is a monosaccharide or a polysaccharide.
11. The method of claim 9 , wherein the peptide is a synthetic peptide.
12. The method of claim 9 , wherein the protein is a synthetic protein.
13. The method of claim 1 , wherein the binding agent is an azide-modified tyrosine.
14. The method of claim 1 , wherein the binding agent is an azide-modified tryptophan.
15. The method of claim 1 , wherein the binding agent is an azide-modified boronic acid.
16. The method of claim 1 , wherein the target molecule is a peptide, a protein, a small molecule, a mixture of cellular membrane fragments, or a microorganism.
17. The method of claim 1 , wherein the target molecule is labeled.
18. The method of claim 1 , wherein the adaptor end-linked aptamers comprise a label and the target molecule induces a conformational change in the adaptor end-linked aptamers upon binding that alters signal from the label.
19. The method of claim 1 , wherein the target molecule is in a complex mixture including non-target molecules.
20. The method of claim 1 , further comprising performing a binding assay that measures binding affinity the adaptor end-linked aptamers to the target molecule.
21. The method of claim 20 , wherein the binding assay is a fluorescence- based binding assay.
22. The method of claim 1 , wherein the solid support comprises a polystyrene surface, a polypropylene surface, a gold surface, a glass surface, or a silicon wafer.
23. The method of claim 1 , wherein the target molecule is insulin.
24. The method of claim 23 , wherein the aptamer has a sequence that has at least 85% identity to the sequence of SEQ ID NO:13.