[go: up one dir, main page]

WO2025199304A1 - Analyse différentielle multiplexée d'interactions protéine-protéine dans des cellules cancéreuses et des cellules uniques - Google Patents

Analyse différentielle multiplexée d'interactions protéine-protéine dans des cellules cancéreuses et des cellules uniques

Info

Publication number
WO2025199304A1
WO2025199304A1 PCT/US2025/020677 US2025020677W WO2025199304A1 WO 2025199304 A1 WO2025199304 A1 WO 2025199304A1 US 2025020677 W US2025020677 W US 2025020677W WO 2025199304 A1 WO2025199304 A1 WO 2025199304A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
barcode
sample
detection agent
oligonucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/020677
Other languages
English (en)
Inventor
Heng Zhu
Joel S. Bader
Hsi-Chang Shih
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Johns Hopkins University
Original Assignee
Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Johns Hopkins University filed Critical Johns Hopkins University
Publication of WO2025199304A1 publication Critical patent/WO2025199304A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies

Definitions

  • Yeast two-hybrid and related methods involve highly engineered systems that are removed from the cellular context of actual tumor cells.
  • Other cell-based technologies require expression of proteins modified to include tags for affinity purification, proximity labeling, or imaging, which are feasible for applications to model systems but not to tumor specimens obtained from human subjects.
  • Methods that can be multiplexed, that can identify quantitative differences in interaction partners, and that can dissect heterogeneity of human tumors at the level of single tumor cells are lacking. Measuring the state of the transcriptome in bulk through RNA-seq, and dissecting heterogeneity through single-cell RNA-seq have transformed the ability to understand transcriptional changes in cancer, but still do not directly reveal the aberrant interactions that cause these changes.
  • the methods comprise: (i) contacting the sample with at least one first detection agent, wherein each of the at least one first detection agent comprises an antibody, or fragment thereof, to a first protein of interest covalently attached to a first barcode oligonucleotide; (ii) contacting the sample with at least one second detection agent, wherein the second detection agent comprises an antibody, or fragment thereof, to a second protein of interest covalently attached to a second barcode oligonucleotide, wherein the first barcode oligonucleotide and the second barcode oligonucleotide are configured for ligation to each other when in close proximity; (iii) ligating the first barcode oligonucleotide and the second barcode oligonucleotide to form a contiguous oligonucleotide; and (iv) detecting formation of the contiguous oligonucleo
  • the methods comprise: (i) contacting the sample with at least one first detection agent, wherein each of the at least one first detection agent comprises an antibody, or fragment thereof, to a first protein of interest covalently attached to a first barcode oligonucleotide comprising a first cleavage site; (ii) contacting the sample with at least one second detection agent, wherein the second detection agent comprises an antibody, or fragment thereof, to a second protein of interest covalently attached to a second barcode oligonucleotide comprising a second cleavage site, wherein the second cleavage site and the first cleavage site create complementary single strand ends; (iii) contacting the sample with: one or more cleavage agents configured to act on the first cleavage site and second cleavage site, and a ligase; and (iv) detecting formation
  • detecting formation of the contiguous oligonucleotide indicates that the first protein of interest and the second target protein of interest are in close proximity.
  • the method comprises contacting the sample with a plurality of first detection agents, each directed to a different first protein of interest, and/or contacting the sample with a plurality of second detection agents, each directed to a different second protein of interest.
  • step (i) and step (ii) are performed simultaneously; step (i) is performed before step (ii); or step (ii) is performed before step (i).
  • step (i) and/or step (ii) comprises contacting the sample with the first detection agent and/or second detection agent for about 10 minutes to about 48 hours.
  • one of the first detection agent or second detection agent is bound to a solid surface or comprises a functional group configured to bind to a solid surface.
  • the solid surface is a bead or particle.
  • the method further comprises separating or isolating solid surface bound detection agents and binding partners from the sample.
  • the first barcode oligonucleotide and the second barcode oligonucleotide have a length of about 20 to 150 basepairs.
  • the first barcode oligonucleotide and the second barcode oligonucleotide each comprise a barcode and a unique molecular identifier (UMI) flanked by primer binding sites.
  • UMI unique molecular identifier
  • each of the first and second cleavage sites are distal to the barcode and UMI in relationship to the site of attachment to the antibody, or fragment thereof.
  • detecting formation of a contiguous oligonucleotide comprises: isolating contiguous oligonucleotides from the sample; and amplifying and/or sequencing contiguous oligonucleotides. JHU Ref. No. C18037_P18037-02 Atty. Docket No. JHU-42990.601
  • the methods further comprise mapping the contiguous oligonucleotides to the protein interactions based on the barcode and/or UMI.
  • the sample is a biological sample.
  • the systems comprise: at least one first detection agent, wherein each of the at least one first detection agent comprises an antibody, or fragment thereof, to a first protein of interest covalently attached to a first barcode oligonucleotide; and at least one second detection agent, wherein the second detection agent comprises an antibody, or fragment thereof, to a second protein of interest covalently attached to a second barcode oligonucleotide.
  • the first barcode oligonucleotide and the second barcode oligonucleotide are configured for ligation to each other when in close proximity.
  • the first and second barcode oligonucleotides are double stranded.
  • the first barcode oligonucleotide and the second barcode oligonucleotide comprise a barcode and a unique molecular identifier (UMI) flanked by primer binding sites.
  • the first barcode oligonucleotide and the second barcode oligonucleotide each contain a cleavage site configured to create complementary single strand ends.
  • the cleavage site is distal to the barcode and UMI in relationship to the site of attachment to the antibody, or fragment thereof.
  • the system comprises a plurality of first detection agents, each directed to a different first protein of interest. In some embodiments, the system comprises a plurality of second detection agents, each directed to a different second protein of interest. In some embodiments, one of the first detection agent or second detection agent is bound to a solid surface or comprises a functional group configured to bind to a solid surface. In some embodiments, the solid surface is a bead or particle. In some embodiments, the system further comprises one or more primers configured to bind to the primer binding sites on the first barcode oligonucleotide and/or the second barcode oligonucleotide. Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
  • FIG.1 is a table of characteristics and advantages for Multiplexed Detection of Protein- Protein Interactions (MuDPPI) as compared to existing methods - yeast two-hybrid (Y2H), immunoprecipitation western blot (IP-WB), affinity purification-mass spectrometry (AP-MS), and JHU Ref. No. C18037_P18037-02 Atty. Docket No. JHU-42990.601 proximity-dependent biotin identification (BioID) - for analyzing interactions.
  • MuDPPI facilitates examination of protein interaction in biological samples with endogenous proteins in native cells, rather than labeled proteins in engineered cells.
  • FIG.2 is a schematic of MuDPPI technology for label-free, differential, and single-cell analysis of protein-protein interactions.
  • FIG.3 shows the antibody generation and validation pipeline.
  • the mAb production pipeline includes antigen production; hybridoma production; primary validation using protein arrays; secondary validation by IP, IB, ChIP-seq, and IHC; and, finally, distribution as a community resource.
  • FIGS.4 shows barcoded mAbs.
  • FIG.4A is a schematic of chemical reactions to tether anchor oligos to mAbs.
  • FIG.4B shows the quality control of anchor oligo-conjugated mAbs.
  • FIG.4A is a schematic of chemical reactions to tether anchor oligos to mAbs.
  • FIG.4B shows the quality control of anchor oligo-conjugated mAbs.
  • FIG.6 shows the profile of PPI changes during mitotic cell cycle. As expected, the CDK6/CycD1 and CDK1/CycB dimers are found most significantly enhanced with respective p- values of ⁇ 0.0001 and ⁇ 0.001 (t-tests based on 2 - ⁇ Ct values).
  • FIG.7 shows the detection of transient PPIs in the EGFR pathway. ⁇ Ct values are plotted at different time points post EGF treatment. Each MuDPPI assay was performed in triplicate.
  • FIG.8 shows PPI detection at the single-cell level. Ct values of two known heterodimers (Jun-JunB & Jun-Fos) and one homodimer (JunB-JunB) were significantly lower than those of the GAPDH controls in both single-cell and 50-cell assays. The p-values denoted by one and two asterisks are ⁇ 0.01 and 0.001, respectively (t-tests of Ct values).
  • FIGS.9A and 9B show MuDPPI analysis of cell cycle in HEK 293T samples down to single cells.
  • DETAILED DESCRIPTION Cancer cells subvert normal developmental and signaling pathways, changing the activities of signal transduction pathways and gene regulatory networks and eliminating native interactions and/or creating new molecular interactions. Because of these dysregulated interactions, there is a need to analyze molecular interactions in cancer cells (e.g., tumor specimens), which may differ from interactions in normal cells.
  • cancer cells e.g., tumor specimens
  • Y2H yeast two-hybrid
  • AP-MS affinity purification-mass spectrometry
  • BioID proximity-dependent biotin identification
  • Y2H campaigns have generated interaction databases for yeast, fruit, and human, the Y2H system expresses human proteins in yeast.
  • IP-WB immunoprecipitation western blot
  • FRET fluorescence resonance energy transfer
  • Methods such as AP-MS and BioID requiring expression of tagged proteins provide questionable quantitative evidence for differential interaction analysis.
  • Heterogeneity is a defining characteristic of cancer. Somatic mutations introduce differences between germline and cancer genomes. Clonal heterogeneity arises as different cancer JHU Ref. No. C18037_P18037-02 Atty.
  • the present disclosure provides a platform technology for Multiplexed Detection of Protein-Protein Interactions (MuDPPI) that allows highly multiplexed detection of protein-protein interactions inside cells, tissues, and single cells and enables entirely new capabilities for quantifying the cancer interactome.
  • the platform utilizes barcoded mAbs and converts the detection of protein- protein interactions to sequencing readouts (e.g., Next Generation sequencing) thereby enabling bulk tissue/cell and single-cell studies.
  • the MuDPPI platform provides label-free, differential analysis of protein-protein interactions in cancer cells and single cells not been achieved with any other existing technology (Fig.1). Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 1. Definitions The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.
  • JHU-42990.601 The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art.
  • Antibody and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (for example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site.
  • Immunoglobulin molecules can be of any type (for example, IgG, IgE, IgM, IgD, IgA, and IgY), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.
  • an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody.” JHU Ref. No. C18037_P18037-02 Atty. Docket No.
  • Antibody fragment refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9): 1126- 1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure.
  • the antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody.
  • antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.
  • an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR).
  • the CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below).
  • a whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N- terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain contains one N-terminal variable (V L ) region and one C-terminal constant (C L ) region.
  • the light chains of antibodies can be assigned to one of two distinct types, either kappa ( ⁇ ) or lambda ( ⁇ ), based upon the amino acid sequences of their constant domains.
  • each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds.
  • the light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain.
  • the remaining constant regions of the heavy chains are aligned with each other.
  • the variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • the V H and V L regions have the same general structure, with each region comprising four framework (FW or FR) regions.
  • the term “framework region,” as used herein, refers to the relatively conserved amino acid sequences within the variable region which are located between the CDRs.
  • FR1, FR2, FR3, and FR4 There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4.
  • the framework regions form the ⁇ sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).
  • the term “close proximity” refers to two targets (e.g., target X and target Y) that are in physical or spatial proximity, either due to direct binding between the two targets or indirectly due to interactions between other molecules, cells, or the like.
  • target X and target Y are on different molecules.
  • target X and target Y are different molecules and are in “close proximity” when they are present in the same complex, bound to the same binding partner (e.g., protein, nucleic acid, small molecule, drug), in a similar location (e.g., on a cell membrane or in the same organelle), or on two associated structures or cells.
  • binding partner e.g., protein, nucleic acid, small molecule, drug
  • a similar location e.g., on a cell membrane or in the same organelle
  • the term “contacting” as used herein refers to bring or put in contact, to be in or come into contact.
  • contact refers to a state or condition of touching or of immediate or local proximity.
  • detecting generally refers to any form of measurement, and includes determining whether an element is present or not. This term includes quantitative and/or qualitative determinations.
  • nucleic acid polynucleotide
  • oligonucleotide are used herein to describe a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions.
  • bases are synonymous with “nucleotides” (or “nucleotide”), the monomer subunit of a polynucleotide.
  • nucleoside and nucleotide are intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles.
  • nucleoside and nucleotide include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like.
  • JHU-42990.601 The term “complementary” refers to specific binding between polynucleotides based on the sequences of the polynucleotides.
  • a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridization assay.
  • polynucleotides are complementary to each other if they follow conventional base- pairing rules, e.g., A pairs with T (or U) and G pairs with C, although regions (e.g., less than 5 nucleotides) of mismatch, insertion, or deleted sequence may be present.
  • base- pairing rules e.g., A pairs with T (or U) and G pairs with C, although regions (e.g., less than 5 nucleotides) of mismatch, insertion, or deleted sequence may be present.
  • protein interaction refers to interactions between different proteins. The interactions may be due to direct binding (covalent or non-covalent) interaction or due to biochemical associations and processes which result in two different proteins to be in close proximity.
  • protein interactions include, but are not limited to, interactions between protein components of a single multi-protein complex, protein binding pairs, two proteins binding the same target, two proteins localizing to a single location within a cell, two proteins on two different cells being in close proximity due to cell-cell interactions, two parts, subunits, or domains of a protein being in close proximity or non-covalently interacting as a result of folding, unfolding, activation, post-translational processing, binding of a target ligand or substrate, and the like.
  • sample is used in its broadest sense. In one sense, it is meant to include a specimen obtained from any source, including biological samples.
  • Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Such examples are not however to be construed as limiting the sample types.
  • a sample is a fluid sample such as a liquid sample.
  • liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, pleural effusions, ascites, thin needle aspirates and spinal fluid). Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples.
  • the methods comprise: (i) contacting the sample with at least one first detection agent, wherein each of the at least one first detection agent comprises an antibody, or fragment thereof, to a first protein of interest covalently attached to a first barcode oligonucleotide; (ii) contacting the sample with at least one second detection agent, wherein the second detection agent comprises an antibody, or fragment thereof, to a second protein of interest covalently attached to a second barcode oligonucleotide, wherein the first barcode oligonucleotide and the second barcode oligonucleotide are configured for ligation to each other when in close proximity; (iii) ligating the first barcode oligonucleotide and the second barcode oligonucleotide to form a contiguous oligonucleotide; and (iv) detecting formation of the contiguous oligonucleotide.
  • the cleavage sites in the first detection agent or second detection agent are the same. In some embodiments, the cleavage sites in the first detection agent or second detection agent are different. In either instance, the single stranded ends created are configured to be complementary to hybridize and form a single contiguous oligonucleotide when treated with a ligase when in close proximity.
  • the barcode oligonucleotides comprise a barcode sequence.
  • the barcode sequence may be any length or sequence.
  • the barcode sequence can be used to specify if the detection agent is a first detection agent or a second detection agent. For example, the barcode sequence can be used to specify if the detection agent is bound or configured to bind to a solid surface, as described elsewhere herein.
  • the barcode oligonucleotides comprise a unique molecular identifier (UMI) sequence.
  • UMI may be any suitable sequence of nucleic acids of any suitable length.
  • the UMI may be a sequence specifically correlated with a specific protein of interest, such that identifying the UMI allows identification of the protein of interest to which the first or second detection agent is targeted.
  • UMIs can also be used to account for PCR and sequencing artifacts in subsequent sequencing analysis.
  • the barcode oligonucleotides can vary in length based on the size of the described components. For example, the barcode oligonucleotides can have a length of about 20 to about 150 basepairs.
  • the barcode oligonucleotide is covalently attached to the antibody or fragment thereof.
  • the covalent attachment is via a direct bond between the antibody or the fragment thereof and the barcode oligonucleotide.
  • the barcode oligonucleotide is covalently attached via a linker.
  • General methods of conjugating oligonucleotides to antibodies are known to those skilled in the art.
  • a typical conjugation method includes use of a linker compound that includes two distinct reactive moieties, which react with different types of functional groups (e.g., one group that reacts with an amine, such as an activated ester group, and one group that reacts with a thiol, such as a maleimide group).
  • Such reactive moieties used in conjugation reactions are well-known to those skilled in the art, and include activated esters such as succinimidyl and sulfosuccinimidyl esters and pentafluorophenyl esters, maleimides, azides, alkynes, hydrazines, isocyanates, isothiocyanates, haloacetamides, and the like.
  • activated esters such as succinimidyl and sulfosuccinimidyl esters and pentafluorophenyl esters
  • maleimides such as succinimidyl and sulfosuccinimidyl esters and pentafluorophenyl esters
  • maleimides azides, alkynes, hydrazines
  • isocyanates isothiocyanates
  • haloacetamides haloacetamides
  • the linker can include one or more nucleotides.
  • the linker can comprise an oligonucleotide sequence. Such a sequence may be considered separate from the oligonucleotide sequence to which the nucleic acid component of the signal-generating complex can hybridize.
  • the linker can include additional atoms or groups; for example, if the antibody is reacted with 2-iminothiolane, it is understood that the linker will further include atoms derived from such reaction.
  • the linker comprises an antibody-binding domain.
  • an antibody binding domain comprises Protein A, Protein G, Protein L, CD4, or a fragment thereof.
  • the antibody-binding domain is an engineered antibody-binding domain, such as to include a non-natural amino acid, a photoreactive group, or a crosslinker.
  • the antibody binding domain is operably linked to a photoreactive amino acid group, for example, benzoylphenylalanine (BPA), resulting in a photoreactive antibody binding domain (pAbBD).
  • BPA benzoylphenylalanine
  • pAbBD photoreactive antibody binding domain
  • the antibody-binding domain (AbBD) is operably linked to a photoreactive amino acid which is operably linked to an antibody or a fragment thereof.
  • an antibody is first reacted with the linker compound to provide a functionalized antibody, which is subsequently reacted with the barcode oligonucleotide to provide the detection agent.
  • a barcode oligonucleotide is first reacted with the linker compound to provide a functionalized barcode oligonucleotide, which is subsequently reacted with an antibody to provide the detection agent.
  • an antibody is first conjugated with anchor oligonucleotide.
  • the anchor oligonucleotide comprises a nucleic acid sequence which hybridizes to at least a portion of a single-stranded barcode oligonucleotide.
  • step (i) and step (ii) are performed simultaneously.
  • step (i) is performed before step (ii).
  • step (ii) is performed before step (i).
  • step (i) and/or step (ii) comprises contacting the sample with the first and/or second detection agent for about 10 minutes to about 48 hours.
  • step (i) and/or step (ii) comprises contacting the sample with the first and/or second detection agent at a temperature of about 4 °C to about 25 °C. JHU Ref. No. C18037_P18037-02 Atty. Docket No. JHU-42990.601
  • the methods further comprise separating or isolating detection agents and binding partners from the sample. Separating or isolating the detection agents and their respective binding partners from the remainder of the sample facilitates analysis of those protein interactions formed in step (i) and step (ii).
  • the first detection agent or second detection agent is bound to a solid surface or comprises a functional group configured to bind to a solid surface.
  • detecting the contiguous oligonucleotide comprises isolating contiguous oligonucleotides from the sample and amplifying and/or sequencing the contiguous oligonucleotides. Any methods known in the art for purifying or separating nucleic acid can be used to isolate the contiguous oligonucleotides.
  • the method comprises treating the sample or isolated detection agents and binding partners with a protease followed by DNA extraction.
  • the protease is selected from trypsin, proteinase K, pepsin, pronase, endoproteinase AspN, and endoproteinase GluC.
  • Any suitable amplification method known in the art allowing for sensitive detection of DNA may be used, including by not limited to polymerase chain reaction (PCR), preferably real time PCR. Sequencing can be accomplished using high-throughput systems, some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, e.g., detection of sequence in real time or substantially real time.
  • the methods further comprise mapping the contiguous oligonucleotides to the protein interactions based on the barcode and/or unique molecular identifier (UMI) sequences. Mapping the contiguous oligonucleotides can comprise extracting the barcodes, primer sequences, UMI, and/or linkers from the detection agents, sequencing the contiguous oligonucleotides, and comparing them to the sequence files created during the analysis to identify those sequences which were found in contiguous oligonucleotides. Further the sequences can be assigned to protein pairs through alignment.
  • UMI unique molecular identifier
  • the methods may further comprise counting protein pairs, generating statistics for individual proteins, protein pairs, types of proteins. This data can be used to examine and determine abundance of the individual proteins, protein pairs, types of proteins. Details for the mapping and subsequent analysis is provided in Examples 1, 2, and 6 below. Mapping and subsequent analysis may be completed using computer implemented methods, also provided in the present disclosure. JHU Ref. No. C18037_P18037-02 Atty. Docket No. JHU-42990.601
  • the sample is a biological sample.
  • the biological sample can be derived from various sources.
  • the biological sample is a tissue specimen or is derived from a tissue specimen.
  • the biological sample is a blood sample or is derived from a blood sample.
  • the biological sample is a cytological sample or is derived from a cytological sample.
  • biological sample is cultured cells. Any manner of protein interactions can be detected by the methods disclosed herein. The methods may detect interactions between two different proteins. For example, in some embodiments, the first detection agent binds, directly or indirectly, to a first protein of interest, and the second detection agent binds, directly or indirectly, to a second protein of interest. Close proximity may indicate, for example, that the two different proteins localize to similar structures in the same cell, are within the same multi-protein complex, associate with a common binding partner, or are direct binding partners to each other. The methods are not limited by the proteins of interest.
  • the proteins of interest may include cell cycle proteins, signal-pathway related proteins, disease-related proteins, transcription factors, and the like.
  • the proteins of interest may be cancer-related proteins.
  • the proteins of interest may include transcription factors with known roles in cancer, e.g., BACH1/2, BRD9, CEBP family members, CREB5, ERG, FOS/JUN and related proteins, GATA4, FOX family members, KLFs, MYC and MYC-associated factors, SMAD family members, SOX family members, STAT family members, and multiple zinc fingers, as well as HDACs and other chromatin-associated proteins. 3.
  • Systems Disclosed herein are systems for detecting protein interactions.
  • the systems comprise at least one first detection agent, wherein each of the at least one first detection agent comprises an antibody, or fragment thereof, to a first protein of interest covalently attached to a first barcode oligonucleotide; and at least one second detection agent, wherein the second detection agent comprises an antibody, or fragment thereof, to a second protein of interest covalently attached to a second barcode oligonucleotide.
  • the first barcode oligonucleotide and the second barcode oligonucleotide are configured for ligation to each other when in close proximity.
  • the first barcode oligonucleotide and the second barcode oligonucleotide are fully or partially double stranded.
  • the first barcode oligonucleotide and the second barcode oligonucleotide each contain a cleavage site configured to create complementary single strand ends.
  • the barcode oligonucleotides comprise a barcode and a unique molecular identifier (UMI) flanked by primer binding sites.
  • UMI unique molecular identifier
  • Each of the sequences for the barcode, UMI, and the primer binding sites can be separated by spacer base pairs or can be immediately adjacent to each other. Descriptions provided above to the barcode, UMI and cleavage sites for the disclosed methods are also applicable to the systems disclosed herein.
  • one of the first detection agent or second detection agent is bound to a solid surface or comprises a functional group configured to bind to a solid surface.
  • the solid surface is a bead or particle.
  • the solid surface may be provided separately from the first detection agent or second detection agent, and accordingly, the system may comprise reagents to conjugate the first detection agent or second detection agent to the solid surface.
  • the systems disclosed herein can be used to detect any number of protein interactions simultaneously or sequentially.
  • the system comprises a plurality of first detection agents, each directed to a different first protein of interest.
  • the system comprises a plurality of second detection agents, each directed to a different second protein of interest.
  • the system further comprises one or more primers configured to bind to the primer binding sites on the first barcode oligonucleotide and/or the second barcode oligonucleotide.
  • the systems may further comprise one or more reagents necessary for protease digestion, DNA extraction, nucleic acid amplification (e.g., PCR), and nucleic acid sequencing. Many such reagents are known in the art and commercially available.
  • suitable reagents include conventional reagents employed in nucleic acid amplification reactions, such as, for example, one or more enzymes having polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleotide, or ribonucleotide triphosphates (dNTPs/rNTPs; for example, deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate) blocking agents, labeling agents, and the like.
  • enzyme cofactors such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • NAD nicotinamide adenine dinucleotide
  • salts such as magnesium or nicotinamide adenine dinucleotide (NAD)
  • NAD nicotinamide
  • kits comprising at least one first detection agent and/or and at least one second detection agent, as described herein.
  • kits comprise one or more components to generate at least one first detection agent and/or and at least one second detection agent, as described herein.
  • the kit may include one or more antibodies, functionalized with a linker or an anchor oligonucleotides, a single stranded barcode oligonucleotide, functionalization reagents, DNA synthesis reagents, and the like.
  • the kits can also comprise other components necessary for carrying out the disclosed methods, including primers, cleavage agents, proteases, ligases, DNA purification reagents, as described elsewhere herein.
  • the kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit.
  • the materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents.
  • Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. It is understood that the disclosed kits can be employed in connection with the disclosed methods.
  • the kit may further contain containers or devices for use with the methods or compositions disclosed herein.
  • the kits optionally may provide additional components such as buffers and disposable single-use equipment (e.g., pipettes, cell culture plates, flasks).
  • the kits provided herein are in suitable packaging.
  • Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately. 5.
  • Examples Example 1 Label-free analysis of protein-protein interactions A resource of stringently validated monoclonal mAbs targeting human transcription factors has been created and shared (see Example 2 for additional details). These validated mAbs can be used to detect interactions between unlabeled proteins in native cells (Fig.2). mAbs for hundreds of proteins of interest are used to create two modified sets: Set A, which is biotinylated and barcoded with a specific dsDNA sequence, and Set B, which is barcoded with a different dsDNA sequence but not biotinylated.
  • the Set A barcodes share a single anchor sequence (Fig.2, red line), and the Set B JHU Ref. No. C18037_P18037-02 Atty. Docket No. JHU-42990.601 barcodes share a different anchor sequence (Fig.2, blue line).
  • the Set A mAbs are separately immobilized on streptavidin beads, washed stringently, and pooled, and the Set B mAbs are also pooled. Cell populations of interest are then lysed, incubated overnight at 4° C with aliquots of the Set A and Set B pools, and subjected to three high-salt washes and three low-salt washes.
  • the total barcode length is 88 bp (20nt primer + 11nt barcode + 8nt UMI for Set A and Set B, plus 10nt total linker sequence), and >100nt paired-end reads are used for overlapping coverage of the barcode.
  • FLASH merges the read-pairs prior to paring the barcodes, reducing to UMIs if desired, and counting reads or UMI events for each protein pair and orientation, e.g., with computer implemented software or methods. Standard sequence quality controls ensure that all barcodes are of the proper A-B form expected from Golden Gate Assembly and assess the ability to map reads to barcodes without error.
  • JHU-42990.601 a mixture of 154 mAbs against 10,000 HEK293 cells. After a total of 20 PCR cycles, the amplicons were running at the expected size range (229-243 bp), and 4M reads were obtained with i-Seq. Interaction detection Statistical analyses identify interactions whose barcode counts are significantly over-represented accounting for multiple testing of all 15K+ possible pairs. For simplicity, “read count” is used to refer to raw count or to UMI-reduced events.
  • n 12 denotes the read count for the configuration with protein 1 with a Set A barcode and protein 2 with a set B barcode
  • n21 denote the swapped orientation with protein 2 from Set A and protein 1 from Set B.
  • Let a 1 and a 2 represent the number of reads with protein 1 or protein 2 from Set A
  • b1 and b2 the corresponding counts for proteins 1 or 2 from Set B.
  • T Denoting T as the total number of reads mapping to barcodes, and assuming a simple null hypothesis of random pairing, n12 is approximately Poisson-distributed with an expected value of a 1 b /T.
  • n 12 +n 21 under the null is a Poisson- distributed random variable with expected value (a1b2+a2b1)/T.
  • the cell cycles of HEK293 cells were synchronized by starving the cells in serum-free media for 24 hours, followed by releasing the serum starvation with serum at different time points so that cells entering M, G2, S and G1 phases were collected at the end.
  • CDK-cyclin interactions were quantified using RT-PCR with primer pairs specific for CDK6/Cyclin D1 and CDK1/Cyclin B barcodes. The Ct values of GAPDH-cyclin pairs were subtracted from those of each CDK/cyclin pair to obtain - ⁇ Ct values.
  • ⁇ Ct values were obtained by subtracting Ras-Raf pSer259 Ct values from the Ras-Raf Ct values at each time point post EGF treatment.
  • the ⁇ Ct values equal to log 2 (Ras-Raf/Ras-Raf pSer259 ), were significantly higher after 4 min versus time 0 (Fig.7).
  • JHU-42990.601 analysis barcode counts (whether read counts or UMI counts) will be used as input to deseq2 for difference finding.
  • a 0.05 family-wise error rate will be used to identify interactions that are differentially represented in the low-invasive vs high-invasive cell lines.
  • Example 4 Detecting interactions in single cells The detection limit of MuDPPI was explored using sorted HEK293 cells (Fig.8). Flow cytometry was used to deposit 1 and 50 HEK293 cells into two groups of 16 wells, and the formation of three known protein pairs: Jun-JunB, Jun-Fos, and JunB homodimer, was examined with GAPDH again serving as the negative control.
  • HEK cells were split and allowed to incubate for 2 days in serum-containing media to promote growth and proliferation. Subsequently, the cells underwent serum starvation by switching to serum-free media, inducing a quiescent state for cell cycle synchronization. After serum starvation, cells were selectively released into different cell-cycle phases by reintroducing serum- containing media at specific time points. Key measures of the cell cycle include ploidy and cell size.
  • G1 phase cells have 2N ploidy and prepare for DNA replication.
  • S phase DNA is duplicated to 4N ploidy in preparation for cell division.
  • M phase involves synthesis of proteins and organelles to support mitosis and division into two 2N daughter cells.
  • G0 quiescent phase
  • Accurate sorting of cells based on their cell-cycle phases was achieved through gentle dissociation using TrypLE, followed by washing in HBSS, filtering the sample, and seeding it into 384-well V-bottom plates at varying densities.
  • the staining of cells with a cell cycle reagent allows DNA to bind the dye stoichiometrically, enabling the determination of DNA content.
  • the flow cytometric analysis of cell count versus linear fluorescence generates a histogram of DNA content distribution for the cell cycle.
  • a standard modeling algorithm can be used to determine whether the cells are in the G0/G1 phase, S phase, G2, or polyploidy state. JHU Ref. No.
  • the counts were normalized to 10,000 counts per cell, applied PCA, and plotted the samples along PC1 and PC2.
  • Samples were grouped according to cell cycle phase, G1 vs. S, including samples with only a single cell (FIG.9A). These results demonstrated the ability of MuDPPI to generate high-quality data from single cells. Analysis based on protein pair interaction counts The same data was analyzed using pair interaction counts. Again, cells clustered by cell cycle phase (FIG.9B). A second axis showed variation with the number of cells in the sample.
  • Example 6 MuDPPI computational methods Parsing MuDPPI sequencing library design.
  • the MuDPPI sequencing library is provided as a table of mAbs, usually identified by gene symbol, and nucleotide sequences for the A-side and JHU Ref.
  • a sample table containing sample names, sample information, and locations of read files is generated based on the aforementioned. Merging reads. If read pairs are overlapping, the reads are merged into a single sequence for improved signal quality and simplified downstream analysis. The software FLASH is used for read merging.97-99% of the read pairs are typically able to be merged. Creating Zhu alignment/map (ZAM) file for sequence reads. The merged reads are analyzed to identify the A-side and B-side barcodes and assign each read to an A-B pair. Also the UMI sequence are provided for UMI-based read counting. Reads with mismatches are discarded. Perfect matching is used for speed. This approach typically yields perfect matches for 60-70% of reads.
  • Imperfect matches could be implemented as an alternative.
  • a pseudo-transcriptome of all possible barcode ligation products could be generated, and then an aligner-mapper such as bowtie2, hisat, or bwa, or a mapper such as salmon could be used to assign reads with mismatches permitted.
  • Counting AB pairs The ZAM file is parsed and for each possible AB pair, the total number of reads (‘total’ mode) and the number of unique UMIs among the total (‘UMI’ mode) is provided. Only reads with both A-side and B-side matches are counted. Generating statistics for proteins. The AB pair counts are used to generate statistics for each protein in the MuDPPI library.
  • Statistics include the number of reads for the protein on the A- side, the number of reads on the B-side, and the sum of A-side and B-side, both for the total mode and the UMI mode of analysis. mAbs with an imbalance, with reads observed on one side but not the other, could be identified based on the statistics. Generating statistics for protein pairs. Protein-level statistics and the total number of observations are used to calculate a null hypothesis for the expected number of observations, a log- ratio of observed to expected observations (with and add-1 estimator to avoid undefined values), and a p-value for enrichment or depletion relative to the null.
  • the null expectation is calculated according to a model in which A-B pairs are randomized, keeping the expected number of A-side and B-side observations for each protein equal to the observed numbers from the protein statistics.
  • the p-values are modeled as a Poisson distribution, although other approaches such as a negative bionomial JHU Ref. No. C18037_P18037-02 Atty. Docket No. JHU-42990.601 distribution with dispersion are possible.
  • Statistics are generated for AB and BA orientations, the combined number of AB and BA orientations, and for total mode and UMI mode analysis. Generating group statistics.
  • the sample table generated above is used to define sets of samples that should be grouped for analysis together.
  • RNA sequencing analysis For example, given samples that are single cells, combine the single cell read counts together into an aggregated data set. This is implemented by first calculating AB counts for each sample individually as above. Then, the data sets from these files are streamed through the methods to calculate protein and protein-pair statistics. Analyzing differential abundance of proteins and interactions.
  • One approach to analyze differential abundance of interactions is to use group statistics to identify protein-pair interactions that are significantly present in one sample or sample group but not in a second sample or sample group. This can be accomplished directly from the statistics generated for each sample or group.
  • An alternative approach is to use count-based comparisons implemented by software for RNA sequencing analysis, for example deseq2 or edgeR. Protein comparisons are possible with standard normalization approaches.
  • raw counts may be used to assess the overall abundance of an interacting pair.
  • the raw approach tests the hypothesis that the overall level of a protein complex has changed.
  • An alternative is to normalize the raw counts by the expected number of observations.
  • the normalized approach tests the hypothesis that protein complex membership has changed.
  • Creating an AnnData object for single-cell or multi-sample analysis Methods to analyze single-cell count matrix data, most relevant being software for single-cell RNA sequencing data, are readily applied to MuDPPI count matrices, whether at the protein or protein-pair level.
  • Data exchange formats provide data sets in a format that can be imported by other software.
  • the Python anndata package was used to export MuDPPI data for downstream analysis as an H5AD format file.
  • the H5AD format is based on the hierarchical data format. Analyzing single-cell MuDPPI proteomics data.
  • the Python scanpy library was used to explore MuDPPI data. Data sets saved as H5AD files are read by scanpy and used to analyze multiple samples that may be generated from bulk sample, from small numbers of cells, or from single cells. Analysis can be based on counts of individual proteins from individual A-side and B- side barcodes. Alternatively, analysis can be based on counts of interactions of protein pairs or on log-ratios of observed to expected interactions of protein pairs.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Pathology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente divulgation concerne des procédés et des systèmes d'identification d'interactions protéiques à partir d'échantillons de masse et de cellule unique. En particulier, la présente divulgation concerne des procédés et un système faisant intervenir un ou plusieurs premiers agents de détection, chacun desdits un ou plusieurs premiers agents de détection comprenant un anticorps, ou un fragment de celui-ci, dirigé contre une première protéine d'intérêt liée de manière covalente à un premier oligonucléotide à code à barres ; un ou plusieurs seconds agents de détection, lesdits un ou plusieurs seconds agents de détection comprenant un anticorps, ou un fragment de celui-ci, dirigé contre une seconde protéine d'intérêt liée de manière covalente à un second oligonucléotide à code à barres, le premier oligonucléotide à code à barres et le second oligonucléotide à code à barres étant conçus pour une ligature l'un à l'autre lorsqu'ils sont à proximité immédiate d'un oligonucléotide contigu.
PCT/US2025/020677 2024-03-22 2025-03-20 Analyse différentielle multiplexée d'interactions protéine-protéine dans des cellules cancéreuses et des cellules uniques Pending WO2025199304A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463568749P 2024-03-22 2024-03-22
US63/568,749 2024-03-22

Publications (1)

Publication Number Publication Date
WO2025199304A1 true WO2025199304A1 (fr) 2025-09-25

Family

ID=97140233

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/020677 Pending WO2025199304A1 (fr) 2024-03-22 2025-03-20 Analyse différentielle multiplexée d'interactions protéine-protéine dans des cellules cancéreuses et des cellules uniques

Country Status (1)

Country Link
WO (1) WO2025199304A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127556A1 (fr) * 2016-01-20 2017-07-27 Cdi Laboratories, Inc. Méthodes et compositions pour identifier, quantifier, et caractériser des analytes cibles et des fragments de liaison
WO2023023484A1 (fr) * 2021-08-16 2023-02-23 10X Genomics, Inc. Sondes comprenant une région de code-barres divisée et procédés d'utilisation
US20250101492A1 (en) * 2023-09-25 2025-03-27 The Johns Hopkins University Mapping dna binding

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017127556A1 (fr) * 2016-01-20 2017-07-27 Cdi Laboratories, Inc. Méthodes et compositions pour identifier, quantifier, et caractériser des analytes cibles et des fragments de liaison
WO2023023484A1 (fr) * 2021-08-16 2023-02-23 10X Genomics, Inc. Sondes comprenant une région de code-barres divisée et procédés d'utilisation
US20250101492A1 (en) * 2023-09-25 2025-03-27 The Johns Hopkins University Mapping dna binding

Similar Documents

Publication Publication Date Title
US12071656B2 (en) Methods and compositions for identifying or quantifying targets in a biological sample
WO2016011364A1 (fr) Procédés et compositions pour identifier, quantifier, et caractériser des analytes cibles et des fragments de liaison
JP7038209B2 (ja) エピタコ電気泳動を使用する試料分析のための装置
JP2021512631A (ja) 遺伝子およびタンパク質の発現を検出する生体分子プローブおよびその検出方法
US20120183969A1 (en) Immunodiversity Assessment Method and Its Use
US9562259B2 (en) Method of analyzing chromosomal inversions
AU2012228424A1 (en) A method of analyzing chromosomal translocations and a system therefore
CN110117595B (zh) 一种特异性结合pdl1的核酸适配体及其应用
EP2281883A1 (fr) Procédé de détermination de la méthylation de l'adn
US20110262912A1 (en) Method for measuring dna methylation
WO2017127556A1 (fr) Méthodes et compositions pour identifier, quantifier, et caractériser des analytes cibles et des fragments de liaison
US20060234253A1 (en) Method for detecting a target substance by using a nucleic acid probe
EP2143806B1 (fr) Procédé de mesure de méthylation d'adn
CN113166795A (zh) 用于检测特定核酸的方法
US20160202249A1 (en) Method for measuring modified nucleobase using absorbent polynucleotide and kit for same
WO2025199304A1 (fr) Analyse différentielle multiplexée d'interactions protéine-protéine dans des cellules cancéreuses et des cellules uniques
EP3037531A1 (fr) Procédé et kit pour mesurer une base d'acide nucléique modifiée à l'aide d'une sonde d'acide nucléique hétérogène
Abdulhay et al. Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme
US10107802B2 (en) Method for measuring modified nucleobase using solid phase probe, and kit for same
Iatrou et al. N-Glycosylation of the Ig receptors shapes the antigen reactivity in chronic lymphocytic leukemia subset# 201
EP4547862A1 (fr) Détection de biomolécules dans des cellules uniques
CN113403382B (zh) Ube2f在诊治股骨头坏死中的应用
CN113999912A (zh) 用于预测酪氨酸激酶抑制剂耐药的标志物
CN106978479A (zh) 一种基于高通量测序检测t细胞白血病微小残留病的方法
US10526608B2 (en) In vitro selection of formaldehyde cross-linking aptamers

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25774345

Country of ref document: EP

Kind code of ref document: A1