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US-12624476-B2 - Chemically encoded spatially addressed library screening platforms

US12624476B2US 12624476 B2US12624476 B2US 12624476B2US-12624476-B2

Abstract

Provided herein are encoded split pool libraries useful, inter alia, for forming highly diverse and dense arrays of ligand domains for screening and detection of a variety of ligand binder molecules. Also provided herein are methods for forming, decoding, binding to, and selectively cleaving the highly diverse and dense arrays of ligand domains.

Inventors

  • Jacob Berlin
  • Gregory Copeland
  • Kathleen Elison
  • Hurik Muradyan

Assignees

  • CITY OF HOPE

Dates

Publication Date
20260512
Application Date
20210422

Claims (20)

  1. 1 . A method comprising: (a) providing a microparticle comprising a plurality of library molecule attachment points (A) on its surface and a plurality of encoding tag attachment points (B) on its surface, (b) attaching a first library molecule to one or more of the library molecule attachment points (A) through a first covalent linker, (c) attaching a first set of one or more encoding tags identifying said first library molecule to a fraction of said encoding tag attachment points (B) through a second covalent linker, (d) attaching a second library molecule to each first library molecule, (e) attaching a second set of one or more encoding tags identifying said second library molecule to another fraction of said encoding tag attachment points (B) through the second covalent linker, (f) optionally repeating steps (d) and (e) to attach additional library molecules and additional sets of one or more encoding tags identifying said additional library molecules to additional fractions of said encoding tag attachment points (B) through the second covalent linker, thereby forming a ligand domain attached to said microparticle, and (g) immobilizing the microparticle on a solid support, wherein said ligand domain is a small molecule or a peptide; wherein said second covalent linker comprises an acyl moiety or a sulfonamide moiety; and wherein said second covalent linker is cleavable under a condition that does not cleave said first covalent linker.
  2. 2 . The method of claim 1 , further comprising performing a decoding procedure on said encoding tags, thereby identifying the composition of said ligand domain and its location on said solid support.
  3. 3 . The method of claim 2 , further comprising binding a ligand binder to said ligand domain, thereby forming a bound ligand binder.
  4. 4 . The method of claim 3 , wherein said ligand binder comprises a detectable moiety.
  5. 5 . The method of claim 3 , wherein said ligand binder is selected from the group consisting of a protein, a mixture of proteins, a nucleic acid, a mixture of nucleic acids, a small molecule, a mixture of small molecules, an element, a mixture of elements, a synthetic polymer, a mixture of synthetic polymers, a cell lysate, and any combination thereof.
  6. 6 . The method of claim 4 , further comprising detecting a location of said bound ligand binder on said solid support.
  7. 7 . The method of claim 6 , wherein said detecting is detecting by a detection device.
  8. 8 . The method of claim 2 , further comprising cleaving said second covalent linker, thereby forming a cleaved microparticle.
  9. 9 . The method of claim 8 , further comprising binding a ligand binder to said ligand domain of said cleaved microparticle, thereby forming a bound ligand binder.
  10. 10 . The method of claim 9 , wherein said ligand binder comprises a detectable moiety.
  11. 11 . The method of claim 9 , wherein said ligand binder is selected from the group consisting of a protein, a mixture of proteins, a nucleic acid, a mixture of nucleic acids, a small molecule, a mixture of small molecules, an element, a mixture of elements, a synthetic polymer, a mixture of synthetic polymers, a cell lysate, and any combination thereof.
  12. 12 . The method of claim 10 , further comprising detecting a location of said bound ligand binder on said solid support.
  13. 13 . The method of claim 12 , wherein said detecting is detecting by a detection device.
  14. 14 . The method of claim 1 , wherein said second covalent linker is acid labile or alkali labile.
  15. 15 . The method of claim 1 , wherein each of said library molecule attachment points (A) comprises an amino group.
  16. 16 . The method of claim 1 , wherein each of said encoding tag attachment points (B) comprises an azide group.
  17. 17 . The method of claim 1 , wherein any one or more of said library molecules comprises a removable protecting moiety.
  18. 18 . The method of claim 17 , further comprising deprotecting the removable protecting moiety.
  19. 19 . The method of claim 1 , wherein said peptide comprises at least one moiety selected from the group consisting of a naturally occurring amino acid, a non-naturally occurring amino acid, and a mimetic of a naturally occurring amino acid.
  20. 20 . The method of claim 19 , wherein said peptide further comprises a portion having non-peptide functionality.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 16/940,499, filed Jul. 28, 2020, which is a continuation of U.S. patent application Ser. No. 15/553,140, filed Aug. 23, 2017, now U.S. Pat. No. 10,760,181, which is a national stage under 35 U.S.C. § 371 of International Application No. PCT/US2016/019426, filed Feb. 24, 2016, wherein International Application No. PCT/US2016/019426 claims the benefit of U.S. Provisional Application No. 62/120,262, filed Feb. 24, 2015. This application is also a continuation of U.S. patent application Ser. No. 16/943,630, filed Jul. 30, 2020, which is a continuation of U.S. patent application Ser. No. 16/555,531, filed Aug. 29, 2019, now U.S. Pat. No. 10,767,278, which is a continuation of U.S. patent application Ser. No. 15/553,140, filed Aug. 23, 2017, now U.S. Pat. No. 10,760,181, which is a national stage under 35 U.S.C. § 371 of International Application No. PCT/US2016/019426, filed Feb. 24, 2016, wherein International Application No. PCT/US2016/019426 claims the benefit of U.S. Provisional Application No. 62/120,262, filed Feb. 24, 2015. The contents of each of the above are herein incorporated by reference in their entireties and for all purposes. REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE The Sequence Listing written in file Seq_Listing_P34713US05.txt, created Apr. 22, 2021, 6197 bytes, machine format IBM-PC, MS Windows operating system, is hereby incorporated by reference. BACKGROUND OF THE INVENTION The number of molecules displayed by arrays used for screening and detection methods is restricted by the synthetic method of the array. In theory, split pool synthesis can generate enormous libraries—limited only by the number of chemical steps and number of unique building blocks utilized per step (i.e. a 5 step library utilizing 100 unique building blocks per step would in theory yield a 1005 or 10 billion member chemical library). However, in practice, encoded split pool strategies face numerous practical constraints. Libraries that are decodable but not screenable or vice versa are not useful. The encoding strategy may be practically limited in the number and type of chemical steps or building blocks used. An encoded split pool library platform which requires large particles for decoding (e.g., by radio frequency tags or mass spectrometry) will normally need to contain fewer library members than a similar library that can be created on smaller particles. If assays are to be performed on a particle, the ligand density on each particle and the surface chemistry environment around each ligand should not interfere with the assay. The serial nature of reported decoding strategies also limits the number of “hits” which can be identified in a cost effective manner in a given screen, and therefore can limit the size of a library that is screened. The present invention addresses these and other problems in the art. BRIEF SUMMARY OF THE INVENTION In one aspect, a microparticle is provided. The microparticle is covalently attached to a ligand domain through a first linker; and a nucleic acid domain through a second linker, wherein the second linker is cleavable and the first linker is not cleavable under a condition that the second linker is cleavable. In another aspect, a solid support attached to a microparticle is provided, wherein the microparticle is covalently attached to (i) a ligand domain through a first linker; and (ii) a cleaved linker moiety. In another aspect, a method of forming a cleaved microparticle is provided. The method includes attaching a microparticle as provided herein including embodiments thereof to a solid support, thereby forming an immobilized microparticle. The second linker of the immobilized microparticle is cleaved, thereby forming a cleaved microparticle. In another aspect, a method of detecting a ligand binder is provided. The method includes (i) attaching a microparticle as provided herein including embodiments thereof to a solid support, thereby forming an immobilized microparticle. (ii) A complementary nucleic acid is bound to the nucleic acid domain of the immobilized microparticle and a location of the nucleic acid domain on the solid support is determined, thereby forming a decoded and mapped microparticle. (iii) The second linker of the decoded and mapped microparticle is cleaved, thereby forming a mapped and cleaved microparticle. (vi) A ligand binder is bound to the ligand domain of the mapped and cleaved microparticle; and (v) a location of the bound ligand binder on the solid support is identified, thereby detecting the ligand binder. In another aspect, a method of detecting a ligand binder is provided. The method includes (i) contacting a ligand binder with a microparticle as provided herein including embodiments thereof thereby forming a bound ligand binder. (ii) A location of the bound ligand binder is i