Methods and systems for macromolecule labeling
The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization from one or more cells. Such polynucleotide processing may be useful for a variety of applications, including generation of labeled macromolecules, including major mistocompatability complex (MHC) molecules, dextramers, etc. Labeled macromolecules may be generated using an in vitro transcription reaction. Labeled macromolecules may be generated in one or more partitions.
1. A method comprising:
(a) providing a reaction mixture in a partition comprising a major histocompatibility complex (MHC) molecule and a nucleic acid molecule comprising a sequence encoding a polypeptide, wherein said MHC molecule is a soluble MHC molecule; and
(b) subjecting said reaction mixture to conditions sufficient to (i) generate said polypeptide from said nucleic acid molecule by an in vitro translation reaction, and (ii) couple said polypeptide and said nucleic acid molecule to said MHC molecule, thereby yielding a labeled MHC molecule.
2. The method of claim 1 , wherein said nucleic acid molecule is a ribonucleic acid (RNA) molecule and wherein in (b)(i), said polypeptide is generated from said nucleic acid molecule by said in vitro translation reaction.
3. The method of claim 1 , wherein said nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, wherein prior to (b), said DNA molecule is transcribed to yield a messenger RNA (mRNA) molecule, and wherein in (b)(i), said polypeptide is generated from said mRNA molecule by said in vitro translation reaction.
4. The method of claim 3 , wherein said nucleic acid molecule further comprises a T7 promoter sequence.
5. The method of claim 3 , wherein said nucleic acid molecule further comprises a capture sequence.
6. The method of claim 3 , wherein said MHC molecule is coupled to a carrier.
7. The method of claim 6 , wherein said carrier is a protein or polypeptide.
8. The method of claim 6 , wherein said carrier comprises a polymer.
9. The method of claim 8 , wherein said polymer comprises dextran.
10. The method of claim 6 , wherein said nucleic acid molecule comprises biotin, wherein said carrier comprises streptavidin, and wherein said nucleic acid molecule is coupled to said carrier through a biotin-streptavidin interaction.
11. The method of claim 1 , wherein in (a), said MHC molecule comprises a conditional ligand and wherein (b) comprises a peptide exchange action wherein said conditional ligand is exchanged for said polypeptide.
12. The method of claim 11 , wherein said conditional ligand is a polypeptide comprising a photo-labile amino acid, and wherein said conditional ligand is released from said MHC molecule upon application of a photo-stimulus.
13. The method of claim 11 , wherein said conditional ligand is covalently linked to said MHC molecule, wherein said conditional ligand is a polypeptide comprising a protease cleavage domain, and wherein said conditional ligand is released from said MHC molecule upon cleavage of said protease cleavage domain by a protease.
14. The method of claim 11 , wherein said reaction mixture further comprises a molecular chaperone.
15. The method of claim 14 , wherein said molecular chaperone comprises a luminal domain of transporter associated with antigen processing (TAP)-binding protein related (TAPBPR).
16. The method of claim 1 , wherein in (a), said nucleic acid molecule is attached to a support.
17. The method of claim 16 , wherein said support is a bead.
18. The method of claim 17 , wherein said nucleic acid molecule is releasably attached to said bead.
19. The method of claim 18 , further comprising releasing said nucleic acid molecule from said bead.
20. The method of claim 17 , wherein said bead is a gel bead.
21. The method of claim 20 , wherein said gel bead is degradable upon application of a stimulus.
22. The method of claim 21 , wherein said stimulus is a chemical stimulus.
23. The method of claim 22 , wherein said reaction mixture comprises said chemical stimulus.
24. The method of claim 1 , wherein said partition is an aqueous droplet in an emulsion.
25. The method of claim 1 , wherein said partition is a well.
26. The method of claim 1 , further comprising:
(c) providing a plurality of partitions, including said partition, wherein at least a subset of said plurality of partitions each comprises (i) a plurality of soluble MHC molecules and (ii) a plurality of nucleic acid molecules comprising a sequence encoding a common polypeptide, wherein each partition of said subset of said plurality of partitions comprises a nucleic acid molecule encoding a unique polypeptide; and
(d) subjecting said plurality of partitions to conditions sufficient to, in each of said subset of said plurality of partitions, (i) generate said common polypeptide from said nucleic acid molecule and (ii) couple said common polypeptide and a given nucleic acid molecule of said plurality of nucleic acid molecules to a given MHC molecule of said plurality of soluble MHC molecules to yield a plurality of labeled MHC molecules, including said labeled MHC molecule.
27. The method of claim 26 , wherein at least a subset of said plurality of labeled MHC molecules are coupled to a carrier.
28. The method of claim 27 , wherein said plurality of nucleic acid molecules comprises biotin, wherein said carrier comprises streptavidin, and wherein nucleic acid molecules of said plurality of nucleic acid molecules is coupled to said carrier through a biotin-streptavidin interaction.
29. The method of claim 28 , wherein said carrier comprises dextran.