De novo engineering of site-specific molecules by tethering
Materials and methods for specifically isolating and identifying biomolecules that bind to selected targets.
1. A method for enriching for a molecule that binds to a target molecule, comprising:
(a) contacting the target molecule with a population of candidate binding partners, wherein each candidate binding partner is coupled to a different nucleic acid tag, wherein each candidate binding partner further comprises or is coupled to a first tethering partner, wherein the target molecule is coupled to a second tethering partner, and wherein the first and second tethering partners interact and promote binding between the target molecule and a candidate binding partner that binds to the target molecule; and
(b) separating at least one candidate binding partner bound to the target molecule from candidate binding partners not bound to the target molecule.
2. The method of claim 1 , wherein the population of candidate binding partners comprises polypeptides.
3. The method of claim 1 , wherein the population of candidate binding partners comprises nucleic acids.
4. The method of claim 1 , wherein the population of candidate binding partners comprises aptamers.
5. The method of claim 1 , wherein the population of candidate binding partners comprises small molecules.
6. The method of claim 1 , wherein the nucleic acid tag is a DNA tag.
7. The method of claim 1 , wherein the nucleic acid tag is a DNA tag, and wherein the method further comprises using the sequence of the DNA tag to identify the at least one candidate binding partner.
8. The method of claim 1 , wherein the nucleic acid tag is an mRNA tag.
9. The method of claim 1 , wherein the nucleic acid tag is an mRNA tag, and wherein the method further comprises using reverse transcription of the mRNA tag and sequencing cDNA resulting from the reverse transcription to identify the at least one candidate binding partner.
10. The method of claim 1 , wherein the first tethering partner is a first cysteine residue within each candidate binding partner, the second tethering partner is a second cysteine residue within the target molecule, and wherein the first and second cysteine residues interact to form a disulfide bond when a candidate binding partner has affinity for the target molecule.
11. The method of claim 1 , wherein the first tethering partner is a first biomolecule coupled to the candidate binding partners and the second tethering partner is a second biomolecule coupled to the target molecule, and wherein the first and second tethering partners interact when a candidate binding partner has affinity for the target molecule.
12. The method of claim 1 , wherein the first tethering partner is a first synthetic molecule coupled to the candidate binding partners and the second tethering partner is a second synthetic molecule coupled to the target molecule, and wherein the first and second tethering partners interact when a candidate binding partner has affinity for the target molecule.
13. The method of claim 1 , further comprising using a display technique.
14. The method of claim 13 , wherein the display technique comprises ribosome display.
15. The method of claim 13 , wherein the display technique comprises mRNA display, phage display, covalent DNA display, CIS display, STABLE, or microbead display.
16. The method of claim 1 , wherein the target molecule is a polypeptide.
17. The method of claim 16 , wherein the polypeptide has a length of 10 to 50 amino acids.
18. The method of claim 16 , wherein the polypeptide has a length of 50 to 100 amino acids.
19. The method of claim 1 , further comprising separating target molecules tethered to a candidate binding partner from target molecules not tethered to a target molecule.
20. The method of claim 19 , wherein the target molecules are biotinylated, and wherein the further separating comprises contacting the biotinylated target molecules with streptavidin-conjugated beads.