Protein sequencing via coupling of polymerizable molecules
“The present disclosure provides methods and systems for sequencing proteins. One or more methods disclosed herein may use linkers having an amino acid-reactive group and an additional reactive moiety that may be used to couple a polymerizable molecule. The linker may couple to a polymerizable molecule and an amino acid of a peptide and a capture moiety via the polymerizable molecule, followed by cleavage of the amino acid from the peptide. Further processing and analysis may be conducted using, for example, nanopores or nanogaps. ”
1. A method of sequencing a peptide at single-amino acid resolution, comprising:
(a) providing said peptide, a capture moiety comprising a first nucleic acid molecule, a linker, and a second nucleic acid molecule;
(b) coupling said linker to a terminal amino acid of said peptide;
(c) coupling said linker to said second nucleic acid molecule;
(d) coupling said first nucleic acid molecule of said capture moiety to said second nucleic acid molecule;
(e) cleaving said terminal amino acid from said peptide to yield an amino acid-linker-capture moiety (AALC) complex;
(f) providing an additional linker comprising a third nucleic acid molecule, wherein said additional linker is capable of coupling to an additional amino acid of said peptide;
(g) coupling said additional linker to said additional amino acid, thereby generating an additional amino acid-linker complex;
(h) coupling said additional amino acid-linker complex to said AALC complex;
(i) cleaving said additional amino acid from said peptide, thereby generating a stacked AALC complex;
(j) translocating said stacked AALC complex through a nanopore or a nanogap;
(k) during (j), measuring a current signal, thereby obtaining a measured current signal; and
(l) using said measured current signal to determine an identity of said terminal amino acid and said additional amino acid, thereby sequencing said peptide.
2. The method of claim 1 , wherein said linker comprises a reactive moiety.
3. The method of claim 2 , wherein said second nucleic acid molecule comprises an additional reactive moiety capable of reacting with said reactive moiety of said linker, and wherein said coupling of (c) comprises reacting said reactive moiety with said additional reactive moiety.
4. The method of claim 3 , wherein said second nucleic acid molecule comprises an additional linker comprising said additional reactive moiety, wherein said additional linker comprises a modified nucleobase and said additional reactive moiety comprises a click chemistry moiety.
5. The method of claim 1 , wherein said capture moiety is affixed to a substrate.
6. The method of claim 5 , wherein said substrate is substantially planar.
7. The method of claim 5 , wherein said substrate is a bead.
8. The method of claim 1 , wherein said capture moiety is coupled to said peptide.
9. The method of claim 1 , wherein said first nucleic acid molecule or said second nucleic acid molecule comprises a DNA molecule, an RNA molecule, an XNA molecule, or modified variant thereof.
10. The method of claim 9 , wherein said DNA molecule is single-stranded.
11. The method of claim 1 , wherein said coupling of (d) is performed using a ligase.
12. The method of claim 1 , wherein at least a portion of said first nucleic acid molecule is complementary to at least a portion of said second nucleic acid molecule.
13. The method of claim 12 , wherein said coupling of (d) is performed by hybridizing said at least said portion of said first nucleic acid molecule to said at least said portion of said second nucleic acid molecule.
14. The method of claim 1 , wherein said coupling of (d) is performed using a splint oligonucleotide, wherein said splint oligonucleotide comprises a first sequence complementary to at least a portion of said first nucleic acid molecule and a second sequence complementary to at least a portion of said second nucleic acid molecule.
15. The method of claim 1 , wherein said first nucleic acid molecule of said capture moiety comprises a nucleic acid barcode molecule.
16. The method of claim 15 , wherein said nucleic acid barcode molecule identifies said peptide.
17. The method of claim 1 , wherein said terminal amino acid is an N-terminal amino acid.
18. The method of claim 1 , wherein (b) occurs before (c).
19. The method of claim 1 , wherein (c) occurs before (b).
20. The method of claim 1 , further comprising, repeating (f)-(i) to generate said stacked AALC complex.
21. The method of claim 1 , wherein said translocating of (j) occurs at a temperature below ambient temperature.
22. The method of claim 1 , wherein said second nucleic acid molecule comprises a nucleic acid barcode molecule that comprises temporal information.
23. The method of claim 1 , wherein said capture moiety comprises a cleavable moiety.
24. The method of claim 1 , wherein said linker is a bifunctional linker.
25. The method of claim 1 , wherein said linker comprises an amino acid reactive group and a click chemistry moiety.
26. The method of claim 1 , wherein said cleaving of (e) or (i) is performed using treatment with acid.
27. The method of claim 26 , wherein said acid is a Lewis acid.
28. The method of claim 1 , wherein said peptide is derived from a biological sample.
29. The method of claim 1 , wherein said terminal amino acid comprises a post-translational modification or a non-standard amino acid, and wherein ( 1 ) comprises identifying said post-translational modification or said non-standard amino acid.
30. The method of claim 1 , wherein (a)-(i) are performed in absence of a substrate.