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NZ720849B2 - High-throughput and highly multiplexed imaging with programmable nucleic acid probes - Google Patents

High-throughput and highly multiplexed imaging with programmable nucleic acid probes

Info

Publication number
NZ720849B2
NZ720849B2 NZ720849A NZ72084915A NZ720849B2 NZ 720849 B2 NZ720849 B2 NZ 720849B2 NZ 720849 A NZ720849 A NZ 720849A NZ 72084915 A NZ72084915 A NZ 72084915A NZ 720849 B2 NZ720849 B2 NZ 720849B2
Authority
NZ
New Zealand
Prior art keywords
nucleic acids
labeled imager
specific binding
target
docking
Prior art date
Application number
NZ720849A
Other versions
NZ720849A (en
Inventor
Sarit Agasti
Xi Chen
Ralf Jungmann
Peng Yin
Original Assignee
President And Fellows Of Harvard College
Filing date
Publication date
Application filed by President And Fellows Of Harvard College filed Critical President And Fellows Of Harvard College
Priority claimed from PCT/US2015/020034 external-priority patent/WO2015138653A1/en
Publication of NZ720849A publication Critical patent/NZ720849A/en
Publication of NZ720849B2 publication Critical patent/NZ720849B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/601—Detection means characterised by use of a special device being a microscope, e.g. atomic force microscopy [AFM]

Abstract

The present invention provides, inter alia, methods and a kit for imaging, at high spatial resolution, targets of interest. The method comprising 1. A method comprising (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each of the target-specific binding partners is linked to a docking nucleic acid, and wherein target-specific binding partners of different specificity are linked to different docking nucleic acids; (2) optionally removing unbound target-specific binding partners; (3) contacting the sample with labeled imager nucleic acids having a nucleotide sequence that is complementary to a docking nucleic acid; (4) optionally removing unbound labeled imager nucleic acids; (5) imaging the sample to detect location of bound labeled imager nucleic acids; (6) removing the bound label imager nucleic acids from the docking nucleic acids by enzymatically cleaving, modifying or degrading the labeled imager nucleic acids, and (7) repeating steps (3)-(6), each time with a labeled imager nucleic acid having a unique nucleotide sequence relative to all other labeled imager nucleic acids.

Claims (33)

1. A method comprising (1) contacting a sample being tested for the ce of one or more targets with one or more target-specific binding partners, wherein each of the target-specific binding partners is linked to a docking nucleic acid, and n target-specific binding partners of different icity are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with labeled imager nucleic acids having a nucleotide ce that is complementary to a docking c acid, (4) ally removing unbound labeled imager c acids, (5) imaging the sample to detect location of bound labeled imager nucleic acids, (6) removing the bound label imager nucleic acids from the docking nucleic acids by enzymatically cleaving, modifying or degrading the labeled imager nucleic acids, and (7) repeating steps (3)-(6), each time with a labeled imager nucleic acid having a unique nucleotide sequence relative to all other labeled imager nucleic acids.
2. The method of claim 1, wherein the labeled imager nucleic acids are removed by enzymatically cleaving the labeled imager c acids with (i) at least one nicking enzyme or restriction enzyme, (ii) RNase, (iii) nucleic acid glycosylase, or (iv) an enzyme that cleaves at one or more deoxyuridine base.
3. The method of claim 1, wherein removing the d imager nucleic acids comprises enzymatic cleavage and photochemical modification.
4. The method of claim 1, wherein the labeled imager nucleic acids se a photocleavable moiety that is cleaved by UV exposure.
5. The method of claim 1, wherein removing the labeled imager nucleic acids comprises enzymatic cleavage and chemical modification/degradation.
6. A method comprising (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each of the -specific binding partners is linked to a g nucleic acid, and n target-specific binding partners of different specificity are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with labeled imager nucleic acids having a nucleotide sequence that is complementary to a docking nucleic acid and comprises a moiety cleavable by a nucleic acid -DNA glycosylase, (4) optionally removing unbound labeled imager nucleic acids, (5) imaging the sample to detect bound labeled imager nucleic acids, (6) contacting the d imager nucleic acids with a nucleic NA glycosylase, (7) removing the bound labeled imager nucleic acids from the docking nucleic acids with use of the nucleic acid-DNA glycosylase, and (8) repeating steps ), each time with a labeled imager nucleic acid having a unique nucleotide ce relative to all other labeled imager nucleic acids.
7. The method of claim 6, wherein the nucleic acid-DNA ylase is a uracil- DNA ylase.
8. The method of claim 7, wherein the labeled imager nucleic acids comprise at least one deoxyuridine (dU).
9. A method comprising (1) contacting a sample being tested for the ce of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking nucleic acid, and wherein target-specific binding partners of different specificity are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with labeled imager nucleic acids having a tide sequence that is mentary to a docking nucleic acid, (4) optionally removing unbound labeled imager nucleic acids, (5) g the sample to detect location of bound labeled imager nucleic acids, (6) removing the bound labeled imager nucleic acids from the docking c acids by altering temperature and/or buffer condition, and (7) repeating steps (3)-(6), each time with a labeled imager nucleic acid having a unique nucleotide sequence relative to all other labeled imager nucleic acids.
10. The method of claim 9, n the labeled imager nucleic acids are removed from the docking nucleic acids by altering temperature.
11. The method of claim 10, wherein the labeled imager nucleic acids are removed from the docking nucleic acids by sing temperature.
12. The method of any one of claims 9-11, wherein the labeled imager nucleic acids are removed from the docking nucleic acids by altering buffer ion.
13. The method of any one of claims 9-12, wherein the labeled imager nucleic acids are removed from the docking nucleic acids by decreasing salt concentration.
14. The method of claim 13, wherein the salt is Mg++.
15. The method of any one of claims 9-14, wherein the labeled imager nucleic acids are removed from the docking nucleic acids by introducing or increasing the concentration of a denaturant.
16. The method of claim 15, wherein the denaturant is formamide, urea or DMSO.
17. A method comprising (1) contacting a sample being tested for the presence of one or more targets with one or more target-specific binding partners, n each target-specific binding partner is linked to a docking c acid, and wherein target-specific binding partners of different specificity are linked to different docking nucleic acids, (2) optionally removing unbound target-specific binding partners, (3) contacting the sample with labeled imager c acids having a nucleotide sequence that is complementary to a docking nucleic acid, (4) optionally removing unbound labeled imager nucleic acids, (5) imaging the sample to detect location bound labeled imager nucleic acids, (6) removing or modifying the signal-emitting moieties without removing the imager c acid in its entirety, by chemically or photochemically cleaving the linker between the imager nucleic acid and the signal-emitting moiety, and (7) repeating steps (3)-(6), each time with a labeled imager nucleic acids having a unique nucleotide sequence relative to all other labeled imager nucleic acids.
18. The method of claim 17, wherein step (6) comprises cleaving the chemically cleavable linker between the imager c acid and the signal-emitting moiety by introduction of a ng agent.
19. The method of claim 18, wherein the chemically cleavable linker ses disulfide bonds, and the cleaving agent is a reducing agent.
20. The method of claim 19, wherein the ng agent is Dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).
21. The method of any one of claims 17-20, n (6) comprises cleaving the photocleavable linker n the imager nucleic acid and the signal-emitting moiety by UV exposure.
22. The method of any one of claims 1-21, wherein the sample is contacted with more than one target-specific binding partner in step (1).
23. The method of any one of claims 1-22, wherein the target-specific binding partner is an antibody or an antibody fragment.
24. The method of any one of claims 1-22, wherein the target-specific binding partner is a l or engineered ligand, a small molecule, an aptamer, a peptide or an oligonucleotide.
25. The method of any one of claims 1-24, wherein the labeled imager nucleic acids are labeled cally.
26. The method of any one of claims 1-24, wherein the labeled imager nucleic acids each comprise a distinct label.
27. The method of any one of claims 1-26, wherein the labeled imager nucleic acids are fluorescently labeled imager nucleic acids.
28. The method of any one of claims 1-27, wherein the one or more targets are proteins.
29. The method of any one of claims 1-28, n the sample is a cell, a cell lysate or a tissue lysate.
30. The method of any one of claims 1-29, wherein the sample is imaged in step (5) using confocal or epi-fluorescence microscopy.
31. A kit when used for testing for the presence of one or more targets with one or more target-specific binding partners comprising: (1) one or more target-specific binding partners, wherein each of the targetspecific binding rs is linked to a docking nucleic acid, and wherein target-specific binding partners of different specificity are linked to different docking nucleic acids; (2) labeled imager nucleic acids that bind to a g nucleic acid, wherein the kit comprises at least two unique imager nucleic acid compositions; wherein, the kit r comprises an agent for enzymatically ng, modifying, or degrading the labeled imager nucleic acids; or an agent for chemically cleaving a cleavable linker between the imager nucleic acids and the signal moiety; and (3) optionally one or more buffers.
32. The method of any one of claims 1-30, wherein the imager and docking nucleic acids remain bound to each other for at least 30, 35, 40, 45, 50, 55, or 60 minutes or at least 2 hours.
33. The method of any one of claims 1-30, wherein the imager and docking nucleic acids remain bound to each other for at least 30 to 60 s, 30 to 120 minutes, 40 to 60 minutes, 40 to 120 minutes, or 60 to 120 minutes.
NZ720849A 2015-03-11 High-throughput and highly multiplexed imaging with programmable nucleic acid probes NZ720849B2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201461951461P 2014-03-11 2014-03-11
PCT/US2015/020034 WO2015138653A1 (en) 2014-03-11 2015-03-11 High-throughput and highly multiplexed imaging with programmable nucleic acid probes

Publications (2)

Publication Number Publication Date
NZ720849A NZ720849A (en) 2024-04-26
NZ720849B2 true NZ720849B2 (en) 2024-07-30

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