Methods for encoding and sorting in vitro translated proteins
View Patent ↗Described herein are methods and reagents for encoding and sorting in vitro translated proteins.
1. A method for encoding a plurality of in vitro translated proteins with an addressing element, said method comprising the steps of:
(a) providing a plurality of in vitro translated proteins, wherein said in vitro translated proteins are isolated from a plurality of RNA-protein fusion molecules by degrading the RNA portions of the RNA-protein fusion molecules;
(b) attaching the plurality of in vitro translated proteins from (a) to DNA linkers; and
(c) binding to each of the DNA linkers an encoding molecule comprising a linker-specific alignment element and a linkage element positioned between said addressing element and said linker-specific alignment element, wherein every encoding molecule contains the same addressing element, thereby encoding the plurality of in vitro translated proteins with the same addressing element.
2. The method of claim 1 , wherein said encoding molecule comprises nucleic acids.
3. The method of claim 1 , wherein said encoding molecule comprises nucleic acid analogs.
4. The method of claim 1 , wherein said linkage element comprises polyethylene glycol units.
5. The method of claim 4 , wherein said polyethylene glycol units are hexaethylene oxide.
6. The method of claim 1 , wherein the proteins are bound to the encoding molecules through hybridization of said DNA linkers to the linker-specific alignment elements.
7. The method of claim 1 , wherein affinity tags are attached to the encoding molecules and said method further comprises isolating one or more encoded proteins using the affinity tag.
8. The method of claim 1 , wherein the encoding molecules are functionalized with cross-linking moieties and said method further comprises cross-linking said encoding molecules to the proteins.
9. A method for encoding a plurality of in vitro translated proteins with an addressing element, said method comprising the steps of:
(a) providing a plurality of in vitro translated proteins, which in vitro translated proteins are labeled with one or more reporter tags, wherein said in vitro translated proteins are isolated from a plurality of RNA-protein fusion molecules by degrading the RNA portions of the RNA-protein fusion molecules; and
(b) binding DNA linkers to the proteins, wherein each of said DNA linkers is bound to the same addressing element, thereby encoding said plurality of in vitro translated proteins with the same addressing element.
10. The method of claim 9 , wherein said DNA linkers are functionalized with cross-linking moieties and said method further comprises cross-linking said DNA linkers to the proteins.
11. A method for encoding a plurality of in vitro translated proteins with an addressing element, said method comprising the steps of:
(a) providing a plurality of in vitro translated proteins, wherein said in vitro translated proteins are isolated from a plurality of RNA-protein fusion molecules by degrading the RNA portions of the RNA-protein fusion molecules; and
(b) binding nucleic acid linkers to the proteins, wherein each of said nucleic acid linkers is bound to the same addressing element and the addressing element branches off from each of said nucleic acid linkers, thereby encoding said plurality of in vitro translated proteins with the same addressing element.
12. The method of claim 11 , wherein the addressing elements are bound to the nucleic acid linkers by linkage elements.
13. The method of claim 12 , wherein the linkage elements comprise polyethylene glycol units.
14. The method of claim 13 , wherein said polyethylene glycol units are hexaethylene oxide.
15. The method of claim 11 , wherein said nucleic acid linkers are functionalized with cross-linking moieties and said method further comprises cross-linking said nucleic acid linkers to the proteins.
16. The method of claim 1 , 9 , or 11 , further comprising immobilizing the encoded proteins formed in step (c) of claim 1 , step (b) of claim 9 , or step (b) of claim 11 onto a solid support.
17. The method of claim 16 , wherein the immobilized encoded proteins are selected from a mixture of encoded proteins.
18. The method of claim 16 , wherein said solid support is a glass or silica-based chip.
19. The method of claim 16 , wherein said solid support is a bead.
20. The method of claim 16 , wherein capture probes are attached to said solid support.
21. The method of claim 20 , wherein said capture probes comprise nucleic acids.
22. The method of claim 20 , wherein said capture probes comprise nucleic acid analogs.
23. The method of claim 20 , wherein the encoded proteins are immobilized onto said solid support by hybridization to said capture probes.
24. The method of claim 1 or 11 , wherein the proteins to be encoded are labeled with one or more reporter tags.
25. The method of claim 24 , wherein said reporter tags are fluorophores.
26. The method of claim 7 , wherein the affinity tag is biotin.
27. The method of claim 8 , 10 , or 15 , wherein said cross-linking moieties are chosen from the group consisting of psoralen, azido compounds, and sulfur-containing molecules.
28. The method of claim 16 , wherein said solid support is functionalized with cross-linking moieties and said method further comprises cross-linking the encoded proteins to said solid support.
29. The method of claim 28 , wherein the cross-linking moieties are chosen from the group consisting of psoralen, azido compounds, and sulfur-containing molecules.
30. The method of claim 8 , wherein the 5′ termini of the encoding molecules are functionalized with electrophiles that cross-link regioselectively with a nucleophilic amino acid side chain of the proteins.
31. The method of claim 1 , 9 , or 11 , wherein the plurality of encoded proteins are in vitro translated from RNAs isolated from the same source.
32. A method for encoding an in vitro translated protein, said method comprising the steps of:
(a) providing an in vitro translated protein, wherein said protein is isolated from an RNA-protein fusion by degrading the RNA portion of the RNA-protein fusion; and
(b) binding to the protein an encoding molecule, wherein said encoding molecule comprises nucleic acids or nucleic acid analogs, and further comprises a reporter tag, thereby encoding said protein.
33. The method of claim 32 , wherein said reporter tag is a fluorophore.
34. The method of claim 32 , further comprising immobilizing the encoded protein formed in step (b) onto a solid support.
35. The method of claim 34 , wherein said solid support is a glass or silica-based chip.
36. The method of claim 34 , wherein said solid support is a bead.