Single-molecule peptide sequencing using guanidinylating agents

Abstract

The present disclosure provides reagents and methods useful for single-molecule sequencing of proteins through use of a sequencing reagent of Formula I, IV, IV', IV", IV-A, IV-B, or V-C. The reagents and methods described herein provide for high-throughput single molecule and high efficiency protein and peptide sequencing in mild conditions allowing for high resolution investigation of complex biological systems.

Description

This specification discloses reagents and methods for single-molecule protein sequencing using guanidinolation reagents. The reagents and methods described herein provide high-throughput and high-efficiency single-molecule protein and peptide sequencing under mild conditions, enabling high-resolution studies of complex biological systems.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A sequencing reagent of Formula I:(Formula I), or a stereoisomer, tautomer, or salt thereof, wherein:A comprises a reactive group configured to form a covalent bond with an N-terminal amino acid of a peptide, wherein said reactive group comprises a guanidinylating agent;B comprises a capture-binding moiety; andL1comprises a linker coupled to A and B.

2. The sequencing reagent of claim 1, wherein said reactive group comprises the structure ofFormula I-B:or a stereoisomer, tautomer, or salt thereof, wherein:R2are each independently hydrogen, R4, OH, OR4, NH2, or -NHR4;R4is C1-6 alkyl or C1-6 alkoxy, each of which is optionally substituted with halo, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, oxo, aryl, and 5-6-membered heteroaryl, wherein the aryl and heteroaryl are optionally substituted with one or two members selected from halo, -OH, C1-4 alkyl, C1-4 alkoxy, C1-4 haloalkyl, NO2, CN, COOR", and C0N(R")2, where each R" is independently H or C1-3 alkyl; ring A is a 5-6-membered heteroaryl ring comprising up to three N atoms as ring members and is optionally fused to an additional aryl or 5-6-membered heteroaryl ring, and wherein said aryl and 5-6-membered heteroaryl ring are each optionally substituted with one or two groups selected from C1-4 alkyl, C1-4 alkoxy, -OH, halo, C1-4 haloalkyl, NO2, COOR, CONR2, -SO2Ra, -NR2, phenyl, and 5-6 membered heteroaryl; wherein each R is independently selected from hydrogen and C1-3 alkyl optionally substituted with OH, ORa, -NH2, -NHRa, or -NRa2; andeach Rais C1-3 alkyl, optionally substituted with OH, oxo, C1-2 alkoxy, or CN; wherein two R, or two R", or two Raon the same N can optionally be taken together to form a 4-7 membered heterocyclic ring, optionally containing an additional heteroatom selected from N, O and S as a ring member, and optionally substituted with one or two groups selected from halo, C1-2 alkyl, OH, oxo, C1-2 alkoxy, and CN.

3. The sequencing reagent of claim 2, wherein each R2are independently hydrogen or R4.

4. The sequencing reagent of claim 2 or 3, wherein R4is C1-6 alkyl or C1-6 alkoxy, each optionally substituted with one or more of C1-6 alkyl, oxo, and aryl.

5. The sequencing reagent of any one of claims 2-4, wherein Ring A is pyrazole or triazole, each optionally fused with aryl.

6. The sequencing reagent of any one of claims 2-5, wherein Ring A is pyrazole, triazole, or7. The sequencing reagent of any one of claims 2-6, wherein said reactive group comprising the structure Formula I-B is:stereoisomer, tautomer, or salt thereof.

8. The sequencing reagent of any one of claims 1-7, wherein said capture-binding moiety comprises a polymer.

9. The sequencing reagent of claim 8, wherein said polymer comprises polyethylene glycol (PEG).

10. The sequencing reagent of claim 9, wherein said PEG comprises between 1 and 20 monomers.

11. The sequencing reagent of claim 8, wherein said polymer comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

12. The sequencing reagent of any one of claims 8-11, wherein said polymer is covalently linked to a substrate.

13. The sequencing reagent of any one of claims 1 to 12, wherein said capture-binding moiety comprises a click chemistry moiety.

14. The sequencing reagent of any one of claims 1-13, wherein said capture-binding moiety comprises an azide or an alkyne.

15. The sequence reagent of any one of claims 1-14, wherein said capture-binding moiety is configured to couple to a capture-moiety, wherein said capture moiety comprises a thiol group.

16. The sequencing reagent of any one of claims 1-7, wherein said sequencing reagent comprises:whereinindicates orientation of said capture-binding moiety relative to said reactive group.

17. The sequencing reagent of claim 15, wherein said capture-binding moiety comprises a thiol group, and wherein said capture-binding moiety is configured to couple to a surface that comprises an acrylate group.

18. The sequencing reagent of any one of claims 1-17, wherein said reactive group comprises Formula II- A:(Formula II- A) wherein:R3is hydrogen or substituted or unsubstituted Ci-6 alkyl, or heteroalkyl;R8and R9are independently hydrogen or NO2; and whereinindicates orientation of said reactive group relative to said capture-binding moiety.

19. The sequencing reagent of any one of claims 1-18, wherein said reactive group comprises:! B ; wherein ''--" indicates orientation of said reactive group relative to said capture-binding moiety; andR8and R9are independently H or NO2.

20. The sequencing reagent of any one of claims 1-16, wherein said reactive group comprisesFormula III- A:(Formula III- A) wherein R3is hydrogen or substituted or unsubstituted C1-6 alkyl; and R8and R9are independently hydrogen or NO2.

21. The sequencing reagent of any one of claims 1-20, wherein L1comprises a cleavable linker.

22. The sequencing reagent of claim 21, wherein said cleavable linker comprises a disulfide bond, a hydrazone, a PEG linker, a DNA molecule comprising a cleavage site, a peptide that is cleavable by an enzyme, an ester, or a de-click chemistry moiety.

23. The sequencing reagent of any one of claims 1-22, wherein said reactive group is linked by a covalent bond with said N-terminal amino acid of said peptide.

24. The sequencing reagent of claim 23, wherein said capture-binding moiety is linked directly or indirectly to a substrate.

25. The sequencing reagent of any one of claims 1-24, wherein said sequencing reagent has a structure of Formula IV:(Formula IV)or a stereoisomer, tautomer, or salt thereof, wherein:R1comprises one or more leaving groups;R2and R3are independently selected from one or more linkers, hydrogen, or null; orR2and R3are taken together with the atoms from which they are attached to form an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, said aryl, heteroaryl, cycloalkyl, or heterocycloalkyl being optionally substituted with one or more linker-click-chemistry moieties;R4is one or more linkers, hydrogen, or null;R5, R6, and R7are independently click-chemistry moieties or null;= is a single or double bond; wherein, when present, at least one of R5, R6, and R7comprise a click-chemistry moiety.

26. The sequencing reagent of claim 25, wherein the sequencing reagent has the structure of Formula IV’:(Formula IV’) or a stereoisomer, tautomer, or salt thereof, wherein:R1comprises one or more leaving groups;R2and R3are taken together with the atoms from which they are attached to form an aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, said aryl, heteroaryl, cycloalkyl, or heteroalkyl being substituted with one or more linker-click chemistry moieties; andR4is hydrogen.

27. The sequencing reagent of claim 25 or 26, wherein R2and R3are taken together with the atoms from which they are attached to form heterocycloalkyl substituted with one or more linker-click chemistry moieties.

28. The sequencing agent of claim 25, wherein the sequencing reagent has the structure of Formula IV”:(Formula IV”) or a stereoisomer, tautomer, or salt thereof, wherein:R1comprises one or more leaving groups;R2and R3are independently selected from a linker, hydrogen, or null;R4is substituted or unsubstituted Ci-6 alkyl, substituted or unsubstituted Ci-6 heteroalkyl, substituted or unsubstituted Ci-6 alkoxy, hydrogen, or null; and R6, if present, is a click chemistry moiety.

29. The sequencing reagent of claim 25-28, wherein said leaving group comprises an electrophilic group.

30. The sequencing reagent of claim 29, wherein said electrophilic group comprises S, SCH3, SO3H, SO2CF3, or NHTf.

31. The sequencing reagent of claim 29, wherein said electrophilic group comprises SR*, wherein R* comprises hydrogen, R’, OH, OR’, NH2, or NHR’, wherein R’ is a C1-6 alkyl optionally substituted with one or more members selected from halo, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, phenyl, 5-membered heteroaryl, and 6-membered heteroaryl, wherein the phenyl, 5-membered heteroaryl, and 6-membered heteroaryl are optionally substituted with one or two members selected from halo, -OH, C1-3 alkyl, C1-3 alkoxy, C1-3 haloalkyl, NO2, CN, COOR", and CON(R")2, where each R" is independently H or C1-3 alkyl.

32. The sequencing reagent of claim 29, wherein said electrophilic group is substituted or unsubstituted C3-10 aryl or substituted or unsubstituted C3-10 heteroaryl.

33. The sequencing reagent of claim 25, wherein one or more of R2and R5are null.

34. The sequencing reagent of any one of claims 25-33, wherein R1is:wherein R8and R9are independently selected from hydrogen or an electron withdrawing group.

35. The sequencing reagent of any one of claims 25-26 or 29-34, wherein the sequencing reagent has the structure of Formula V-C:(Formula V-C) or a stereoisomer, tautomer, or salt thereof, wherein:R8and R9are independently selected from hydrogen or an electron withdrawing group; andR10is a linker-click chemistry moiety (e.g., CFfc-azide).

36. The sequencing reagent of any one of claims 28-34, wherein said sequencing reagent has the structure of Formula IV- A:(Formula IV- A) or a stereoisomer, tautomer, or salt thereof, wherein:R3is a linker, hydrogen, or null;R4is substituted or unsubstituted Ci-6 alkyl, substituted or unsubstituted Ci-6 heteroalkyl, substituted or unsubstituted Ci-6 alkoxy, hydrogen, or null; and R6, if present, is a click chemistry moiety;R8and R9are independently hydrogen or an electron withdrawing group.

37. The sequencing reagent of claim 36, wherein R4is unsubstituted Ci-6 alkyl.

38. The sequencing reagent of claim 36 or 37, wherein R4is Ci alkyl.

39. The sequencing reagent of any one of claims 36-38, wherein R3is Ci-Ce alkyl.

40. The sequencing reagent of any one of claims 34-39, wherein said electron withdrawing group comprises a haloalkyl, a halogen, an amide, a carbonyl, or NO2.

41. The sequencing reagent of any one of claims 34-40, wherein said electron withdrawing group comprises NO2.

42. The sequencing reagent of any one of claims 34-41, wherein at least one of R8and R9areNO2.

43. The sequencing reagent of any one of claims 34-42, wherein R8is NO2.

44. The sequencing reagent of any one of claims 34-43, wherein R9is NO2.

45. The sequencing reagent of any one of claims 34-40, wherein said electron withdrawing group is CF3.

46. The sequencing reagent of any one of claims 34-39, wherein said electron withdrawing group is SO3H.

47. The sequencing reagent of any one of claims 34-39, wherein said electron withdrawing group is NR3, wherein R comprises an alkyl.

48. The sequencing reagent of any one of claims 34-39, wherein said electron withdrawing group is NH3.

49. The sequencing reagent of any one of claims 34-39, wherein said electron withdrawing group is CN.

50. The sequencing reagent of any one of claims 34-40, wherein said electron withdrawing group is COCI.

51. The sequencing reagent of any one of claims 36-42, wherein said sequencing reagent of Formula IV-A is52. The sequencing reagent of claim 25, wherein R1is S.

53. The sequencing reagent of claim 25 or 52, wherein said sequencing reagent has a structure of Formula IV-B:(Formula IV-B) wherein:R2, R3, and R4are independently selected from hydrogen, one or more linkers, or null; andR5, R6, and R7are independently click-chemistry moieties or null.

54. The sequencing reagent of any one of claims 25-53, wherein said click chemistry moiety comprises an azide or an alkyne.

55. The sequencing reagent of claim 25, 28, or 53-54, wherein said linker is a cleavable linker.

56. The sequencing reagent of claim 25, 28, or 53-54, wherein said linker comprises Ci-6 alkyl, a disulfide bond, hydrazone, a PEG linker, a DNA molecule comprising a cleavage site, a peptide that is cleavable by an enzyme, an ester, or a de-click chemistry moiety.

57. The sequencing reagent of any one of claims 53-56, wherein R2is hydrogen.

58. The sequencing reagent of any one of claims 53-57, wherein R3is Ci-Ce alkyl.

59. The sequencing reagent of any one of claims 53-58, wherein R6is an azide.

60. The sequencing reagent of any one of claims 53-59, wherein R4is hydrogen.

61. The sequencing agent of any one of claims 25-60, wherein the sequencing reagent is selected from:or a stereoisomer, tautomer, or salt thereof.

62. The sequencing reagent of any one of claims 25, 28, or 53, wherein said linker comprises PEG.

63. A method of using said sequencing reagent of any one of claims 1-62, comprising: a. providing a capture moiety and a polymeric analyte; b. contacting said polymeric analyte with said sequencing reagent, wherein said sequencing reagent binds to a monomer of said polymeric analyte to form a sequencing reagent-monomer complex;c. coupling said capture-binding moiety to said capture moiety; d. cleaving said sequencing reagent-monomer complex from said polymeric analyte, thereby providing a detectable complex; and e. detecting said detectable complex.

64. The method of claim 63, wherein said polymeric analyte comprises a polypeptide.

65. The method of claim 63 or 64, wherein said monomer comprises a terminal amino acid residue.

66. The method of any one of claims 63-65, wherein said capture moiety comprises a DNA molecule.

67. The method of any one of claims 63-66, wherein detecting said detectable complex comprises contacting said detectable complex with a binding agent.

68. The method of claim 67, wherein said binding agent comprises an antibody, nanobody, single chain variable fragment (scFv), or aptamer.

69. The method of claim 67 or 68, wherein said binding agent comprises a polymerizable molecule.

70. The method of claim 69, wherein said polymerizable molecule comprises a nucleic acid molecule.

71. The method of any one of claims 63-70, further comprising repeating (b)-(e), thereby sequencing said polymeric analyte.

72. The method of any one of claims 63-71, wherein, in (a), said polymeric analyte is coupled to a substrate.

73. The method of any one of claims 63-72, wherein (d) is performed chemically or enzymatically.

74. The method of any one of claims 63-73, wherein (b) occurs before (c).

75. The method of any one of claims 63-73, wherein (b) occurs subsequent to (c).

76. The method of any one of claims 63-75, wherein (e) is performed using a nanopore sequencer.

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