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US-12618099-B2 - Compositions and methods for analyte detection

US12618099B2US 12618099 B2US12618099 B2US 12618099B2US-12618099-B2

Abstract

Methods of analyzing nucleic acids of a cell are provided.

Inventors

  • George M. Church
  • Jehyuk Lee
  • Daniel Levner
  • Michael Super

Assignees

  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Dates

Publication Date
20260505
Application Date
20241104

Claims (20)

  1. 1 . A system, comprising: (a) a cell or tissue sample immobilized on a solid support, wherein said cell or tissue sample comprises an analyte at a spatial location of said cell or tissue sample; (b) a detection reagent comprising: (i) a probe configured to bind to said analyte, and (ii) a plurality of predetermined subsequences; (c) a plurality of decoder probes, wherein a decoder probe of said plurality of decoder probes comprises a sequence complementary to a predetermined subsequence of said plurality of predetermined subsequences, and wherein said decoder probe is associated with an optically detectable label; (d) an optical detection subsystem configured to detect an optical signal signature from said optically detectable label at said spatial location in said cell or tissue sample; and (e) a computer having software or an algorithm that: (i) performs a comparison of temporal readouts of optical signal signatures detected by said optical detection subsystem in a plurality of temporally sequential readout cycles with an optical code assigned to said plurality of predetermined subsequences, and (ii) identifies said analyte based on agreement between a temporal order of optical signal signatures of said optical signal signatures and said optical code.
  2. 2 . The system of claim 1 , further comprising a wash buffer.
  3. 3 . The system of claim 2 , wherein said wash buffer comprises a salt, a detergent, or a denaturant.
  4. 4 . The system of claim 2 , wherein said wash buffer comprises formamide or dimethyl sulfoxide (DMSO).
  5. 5 . The system of claim 1 , further comprising a signal removal buffer.
  6. 6 . The system of claim 5 , wherein said signal removal buffer comprises a cleavage reagent, a quenching agent, a bleaching agent, a reducing agent, an oxidation agent, or any combination thereof.
  7. 7 . The system of claim 1 , further comprising a photo-bleaching subsystem configured to photo-bleach said optically detectable label.
  8. 8 . The system of claim 1 , further comprising a buffer for performing a hybridization reaction.
  9. 9 . The system of claim 1 , further comprising a nucleic acid ligase.
  10. 10 . The system of claim 1 , wherein said software or said algorithm determines a location of said optical signal signatures detected in said plurality of temporally sequential readout cycles and corrects a spatial shift in said location during said plurality of temporally sequential readout cycles.
  11. 11 . The system of claim 10 , wherein said software or said algorithm aligns a distinguishable feature detected in readout cycles of said plurality of temporally sequential readout cycles to correct said spatial shift in said location.
  12. 12 . The system of claim 1 , wherein said optical detection subsystem comprises a laser or a light emitting diode.
  13. 13 . The system of claim 1 , wherein said optical detection subsystem comprises a microscope.
  14. 14 . The system of claim 13 , wherein said microscope comprises an inverted fluorescent microscope, an epi-fluorescent microscope, or a confocal microscope.
  15. 15 . The system of claim 1 , wherein said analyte is a nucleic acid analyte and wherein said detection reagent comprises nucleic acid.
  16. 16 . The system of claim 1 , wherein said analyte is a protein analyte and said probe comprises an antibody.
  17. 17 . The system of claim 1 , wherein said optically detectable label comprises a fluorescent label.
  18. 18 . The system of claim 17 , wherein said optically detectable label is directly attached to said decoder probe via a covalent bond.
  19. 19 . The system of claim 17 , wherein said optically detectable label is indirectly attached to said decoder probe via a non-covalent bond.
  20. 20 . The system of claim 17 , wherein said optically detectable label is indirectly attached to said decoder probe via an intermediary molecule.

Description

RELATED APPLICATION DATA This application is a continuation application which claims priority to U.S. patent application Ser. No. 18/614,970, filed on Mar. 25, 2024, which is a continuation application which claims priority to U.S. patent application Ser. No. 18/064,956, filed on Dec. 13, 2022, which is a continuation application which claims priority to U.S. patent application Ser. No. 17/671,803, now U.S. Pat. No. 11,566,277, filed on Feb. 15, 2022, which is a continuation application which claims priority to U.S. patent application Ser. No. 17/238,642, now U.S. Pat. No. 11,293,051, filed on Apr. 23, 2021, which is a continuation application which claims priority to U.S. patent application Ser. No. 16/941,585, now U.S. Pat. No. 11,021,737, filed on Jul. 29, 2020, which is a continuation-in-part application which claims priority to U.S. patent application Ser. No. 16/157,243, now U.S. Pat. No. 11,078,520, filed on Oct. 11, 2018, which is a continuation application that claims priority to U.S. patent application Ser. No. 14/774,282, now U.S. Pat. No. 10,138,509, filed on Sep. 10, 2015, which is a National Stage Application under 35 U.S.C. 371 of PCT Application No. PCT/US2014/018580 designating the United States and filed Feb. 26, 2014; which claims the benefit of U.S. Provisional Application No. 61/777,383 and filed Mar. 12, 2013 each of which are hereby incorporated by reference in their entireties. U.S. patent application Ser. No. 16/941,585, now U.S. Pat. No. 11,021,737, filed on Jul. 29, 2020 is also a continuation-in-part application which also claims priority to U.S. patent application Ser. No. 16/393,215, now U.S. Pat. No. 11,293,054, filed on Apr. 24, 2019, which is a continuation application which claims priority to U.S. patent application Ser. No. 16/255,920, now abandoned, filed on Jan. 24, 2019, which is a continuation application which claims priority to U.S. patent application Ser. No. 14/366,486, now U.S. Pat. No. 10,227,639, filed on Jun. 18, 2014, which is a National Stage Application under 35 U.S.C. 371 of PCT Application No. PCT/US2012/071398 designating the United States and filed Dec. 21, 2012; which claims the benefit of U.S. Provisional Application No. 61/579,265 and filed Dec. 22, 2011, each of which are hereby incorporated by reference in their entireties. STATEMENT OF GOVERNMENT INTERESTS This invention was made with government support under HG005550 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 8, 2022, is named “Sequence_Listing_010498_01457_ST26” and is 2.3 KB in size. FIELD OF THE INVENTION The present invention relates to methods of making a three-dimensional matrix of nucleic acids and amplifying, detecting and sequencing such nucleic acids within the matrix. BACKGROUND OF THE INVENTION Since many gene products such as RNA and proteins are enriched in regions where they function, their location provides an important clue to their function. This property has been used for in situ fluorescent hybridization, immunohistochemistry and tissue-specific reporter assays in numerous areas of biological research. Current methods involve extracting nucleic acid molecules from their native environment or making synthetic nucleic acid molecules, amplifying them in solution and placing them on a flat array surface or beads for gene detecting via hybridization or sequencing, making it impossible to identify the cellular origin of individual nucleic acids. SUMMARY Embodiments of the present invention are directed to methods of making a three dimensional matrix of nucleic acids. Embodiments of the present invention are directed to methods of making a three dimensional matrix including nucleic acids covalently bound into a matrix or into or to a matrix material. The nucleic acids may be co-polymerized with the matrix material or cross-linked to the matrix material or both. According to one aspect, a plurality of nucleic acid sequences of certain length, such as DNA or RNA sequences are part of a three-dimensional copolymer. The nucleic acids may then be amplified and sequenced in situ, i.e. within the matrix. The three-dimensional matrix of nucleic acids provides, in a certain aspect, an information storage medium where the nucleic acids, i.e. a sequence of one or more nucleotides, represent stored information which can be read within the three-dimensional matrix. According to one aspect, nucleic acids such as DNA or RNA sequences of given length are covalently attached to a matrix material to preserve their spatial orientation in the x, y and z axes within the matrix. It is to be understood that the three dimensional matrix may include a matrix material and that the term copolymer, matrix and matrix material may be used interchangeabl